US011344501B2

( 12 ) Pimentel
UnitedetStates
al .
Patent ( 10) Patent No.: US 11,344,501 B2
(45) Date of Patent : * May 31 , 2022
( 54 ) ANTI- METHANOGENIC COMPOSITIONS ( 58 ) Field of Classification Search
AND USES THEREOF CPC A61K 47/02 ; A61K 47/12 ; A61K 47/32 ;
A61K 47/38
( 71 ) Applicant: Cedars - Sinai Medical Center , Los See application file for complete search history .
Angeles, CA (US )
( 72 ) Inventors: Mark Pimentel , Los Angeles, CA (US ); ( 56 ) References Cited
Ruchi Mathur , Los Angeles, CA (US ) ;
Steve Kanzer , Boca Raton , FL (US ); U.S. PATENT DOCUMENTS
Vince Wacher , Rockville, MD (US) 225,202 A 3/1880 Wylie
5,232,946 A 8/1993 Hurnaus et al .
( 73 ) Assignee : Cedars - Sinai Medical Center , Los (Continued )
Angeles, CA (US )
( * ) Notice : Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS
patent is extended or adjusted under 35 AU 2003273141 8/2009
U.S.C. 154 (b ) by 37 days. AU 2014239164 9/2015
This patent is subject to a terminal dis (Continued )
claimer.
OTHER PUBLICATIONS
( 21 ) Appl . No.: 16 /859,082
Saha, World J Gastroenterol. Jun . 14 , 2014 ; 20 ( 22 ) : 6759-6773 ]
(22 ) Filed : Apr. 27, 2020 ( Year: 2014 ) . *
( Continued )
( 65 ) Prior Publication Data
US 2020/0323781 A1 Oct. 15 , 2020 Primary Examiner San Ming R Hui
Related U.S. Application Data (74 ) Attorney, Agent, or Firm — Nixon Peabody LLP ;
Linda B. Huber
( 63 ) Continuation of application No. 16/ 405,667 , filed on
May 7 , 2019 , now Pat. No. 10,668,159 , which is a
(Continued ) ( 57 ) ABSTRACT
(51 ) Int. Ci. The present invention relates to , in part, methods and
A61K 9/20 ( 2006.01 ) compositions for the treatment of methanogen -associated
A61K 4738 ( 2006.01 ) disorders such as , for example , Irritable Bowel Syndrome
(Continued ) (IBS ) . Particularly, modified - release formulations compris
(52) U.S. Cl. ing at least one antimethanogenic statin are provided which
CPC A61K 9/2054 ( 2013.01 ) ; A61K 9/2846
release the antimethanogenic statin in the intestines.
(2013.01 ) ; A61K 9/2886 ( 2013.01 ) ;
(Continued ) 22 Claims , 18 Drawing Sheets
Stomach Share the
2

weni bundi
• Capsule containing & * Capsule dissolves in
Wand QUODENUM
excise ang- orded
heads
Enteric coated
capsu?a to avoid the
stomata
 US 11,344,501 B2
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Related U.S. Application Data FOREIGN PATENT DOCUMENTS

continuation of application No. 15 / 940,063 , filed on AU 2015301596 2/2017
Mar. 29 , 2018 , now Pat . No. 10,328,151 , which is a BR 11 2015 022825-9 A2 11/2017
continuation of application No. 14/ 826,115 , filed on BR 11 2017 002761-5 A2 12/2017
Aug. 13 , 2015 , now Pat. No. 9,956,292 . CA 2486585 12/2003
CA 2903493 9/2014
CA 2955666 2/2016
( 60 ) Provisional application No. 62 / 036,948 , filed on Aug. CN 1681522 10/2005
13 , 2014 , provisional application No. 62 /043,649, 102497786 6/2012
6/2013
filed on Aug. 29 , 2014 , provisional application No. CN
103142552 A
103230593 8/2013
62/043,789, filed on Aug. 29 , 2014 , provisional CN 105208861 12/2015
application No. 62 / 141,355 , filed on Apr. 1 , 2015 . CN 106687107 5/2017
EP 1 505 989 2/2005
(51 ) Int. Cl. EP 1 609 852 12/2005
A61K 31351 ( 2006.01) EP 2251 017 11/2010
A61K 47/12 EP 2 967 060 1/2016
( 2006.01 ) EP 3179983 6/2017
A61K 9/28 (2006.01 ) EP 3277274 2/2018
A61K 9/48 ( 2006.01) GB 423083 1/1935
A61K 31/00 ( 2006.01 ) GB 2 338 244 12/1999
( 52 ) U.S. Ci . HK 1214752 8/2016
CPC HK 1238153 4/2018
A61K 9/4808 (2013.01 ) ; A61K 31/00 JP 60-133852 7/1985
(2013.01 ) ; A61K 31/351 ( 2013.01 ) ; A61K JP 03-275630 12/1991
47/12 (2013.01 ) ; A61K 47/38 (2013.01 ) JP
JP
08-310960
2005-526861
11/1996
9/2005
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JP 2017-528516 A 9/2017
U.S. PATENT DOCUMENTS JP 2017528516 9/2017
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Synthetic Biologics , Synthetic Biologies ' Novel Irritable Bowel from the Internet : URL : https://www.youtube.com/watch?v=
Syndrome with Constipation ( IBS - C ) Program Featured in Ameri lxzseth6Z8, Retrieved on Oct. 3 , 2018 , 1 Page .
can College of Gastroenterology Poster, 2015 , 3 Pages . Morgavi et al . , Fungal Secondary Metabolites from Monascus spp .
clinicaltrials.gov ., A Study of the Effect of SYN -010 on Subjects Reduce Rumen Methane Production in vitro and in vivo . , 2013 , J.
with IBS - C udy Results, 2017 , Retrieved from the Internet: Anim . Sci . , vol . 91 ( 2 ) , pp . 848-860 .
URL : https://clinicaltrials.gov/ct2/show/results/NCT02495623?sect
X70156 # outcomel, Retrieved on Jul. 19 , 2018 , 7 Pages . * cited by examiner
U.S. Patent May 31 , 2022 Sheet 1 of 18 US 11,344,501 B2

Ascendigwoln
Cetum
leum oo

FIG
1A
. Jejunm .
6

indC•iaspoluves
Stomach 1PH.4-2.07 DUOENM
IRc*Coanptsiunleg acSiorncldeos c(op-playteonedr)drug-lcioracdleds theatocvposiudle
cEnotaeridc stomach
U.S. Patent May 31 , 2022 Sheet 2 of 18 US 11,344,501 B2

AscendigColn 5.6-.8
32
Ger

leurs Oo
*****
8
3
tocpd(oe)irsnpiletcreosinrys
dfrorpH•Tieprlfmenodasmn-etblcp-doerlaydtumegsdr i
r
at
/
R
d*l
e m
o a
eci
s n
u e
a
m l
g
IB
.
FIG

**

On

Stomach 1PH.4-2.0
indCc•ioansptilunvesg
dpiSoTflOMyeArmCHnt-l,drug-coadted
beads
U.S. Patent May 31 , 2022 Sheet 3 of 18 US 11,344,501 B2

AscendigColn 5.6-.8 tw-

or
(
*-cw•Caiptshuilne
aibptslhueiltneD*
c) c(oriafpseurlnets
dh)tiafbevlrenttorpH-deipmned -ntrpelofiase cf•Uosmboeinfatruilon
therapis ltM•ualbtilaeytr*Odetaurymienrs risneltiasel •Ilcannayer rdetlmaisne proetis
?

CA

23
leum
2
.
FIG
6.61

Duodenm 89-65
Stomach 1PH4-20
U.S. Patent May 31 , 2022 Sheet 4 of 18 US 11,344,501 B2

8 ?
AscendigColn 6.6
5
8
:
C
ODE 2.623
B

leum
3A
.
FIG
6. -74 inrwobi,sCvanetmcgusirvolawnbtdhy C•rlagt(f,pHiu)snediolmpemzandsbelnt
byd*Lagtofopfleruaimtymbielndrty
sappH-c•Cetohnmlryesictulhdrves boncRtodparoeiflgtnsuredilatcy torl,olagt*Adueafpirltyeumarseg

Duodenm 0. 0 incdreorluaosgne
Gltrinudseprolaeumcsgetr

Stomach 1PH.4-20
88
08
U.S. Patent May 31 , 2022 Sheet 5 of 18 US 11,344,501 B2

AscendigCOTN56-.83
Cecum 56- .9
Heum 2013 tr,csf•Piohnemloiuasngthlrowues
stortoe•Waxpivlaoetmduisergn
3B
.
FIG Jejunm 42
6-7
6

theandiconlton

Duodenm 6.6
5.9
I 1 1
todCiaspoluvesrwaxpel uasge
Stomach 1PH.4-2.07
9000 wax
a
in
drug
plug )
c*Coanptsiulneg 6000
PEG
e.g.
(
U.S. Patent May 31 , 2022 Sheet 6 of 18 US 11,344,501 B2

AscendigColn 5.6-.8 ceSntoalerins

We u 5.8-6.9
Sta in P2ulse
Heum 3-7.8
:
7
Sta in
Jejunm 6.-74
4
.
FIG

Absorptin
Duodenum 5.0-6.
lR(ICR)eloceasle
Dual
Pulse
R(
DR
ICR
+
)
elease

Stomach 1PH.4-2.0 ??????????

a Ooo ooo

?
U.S. Patent May 31 , 2022 Sheet 7 of 18 US 11,344,501 B2

7-12h AscendigColn 5.6 .8 B*,maetchenorginsH2Oa*Hbisogrhptlivye*Lowfreefluid v•HiscogsihtyLpurmeisnuarle*Ldiosmpestreadl

Cecum 162 9 1 COL N
l eum 7.3.18 L

LIL
(
LI
( SCRLovast inlpoevucasltndie disopleurtin etocpornlitorny

Jejunm 6-7A
L

5
.
FIG

Duodenm 5.9-6. X

om 1PH4-20 *

HPMC
c(
size
)apsule
1 lm(L)oivnatsbalteins
and
DR
(
coated
-
5.5
pH
a
Oc(
-ICR
)poHa7t.e0d
:contain g
U.S. Patent May 31 , 2022 Sheet 8 of 18 US 11,344,501 B2

%
83
2mg1-23 4.mg5- .0 1mg.4-12.815mg.9-17.8
LB-h(9-18%)yadcrotxoyancied
COL N )h(Byadcrtoelyisal

%
83 mg 140ma14.0mg 19.5
mg 5.5
25 /
mg

BILE 14
%
thei(mg)rleDatonsavdmtpciretansoiaedgtn ,c.Fortlitobelowisanboshleuarotipnmesoacdnlintheandcofvpa31%l.duobfstleriupsoathneidgl
6
.
FIG

%
21
ISNMTEALINE % 25
69 mg 5.5mg 0.0mg5.5mg

A(31%T)BSORTPAIOLN 32mg 70mg 0.0mg7.0mg

LB-h(10%)yadcrotxoyanceid
%
10
STOMACH % 40mg
100

14C
[
]
40mg DR
0.0mg0.0mg0.0mg )(Acidhydrolysi
mg
7 mg
ICR14 T21
mgotal
IRLovast in SYN-010
U.S. Patent May 31 , 2022 Sheet 9 of 18 US 11,344,501 B2

min
60
45
30
,
Samples
20
15
10
5
at
)
mL
( vHipms0tFyhPn.lrtioaL4eurglCo5ehd min
60
45
,
30
Samples
20
15
10
5
at
)
mL
( vHiums0tFyhlPn.rtioeaL4nurglCo5ehd
cm5xICR1DRpionmlbtaiunlestd
swc1HinaiPpnskrtMzuelhCr
min
120
mL
at
)
5
(
Sample
Ft0umsihyl.roei4ung5dhe
vHiPnaLtlCo
FIG
.
7

°C3s;prpm757a.0de40l.d5e 500
(
HCl
1.2
pH
)SDS
ml min
120
;
)
L
/
g
0.625
( Tmtop-wirnatrsbmfletrds /winnewvbeusflelr (500ml)bpH5.9Naphuofspheatre Add0NaOH(400mL).05M
SDS)(Noad it onal bPH7.2Nap(865mL)huofspheatre 60minSDS(10.75g/L):
2AUpSarPtus 20
(
SDS
60
:
)
L
/
g
min
U.S. Patent May 31 , 2022 Sheet 10 of 18 US 11,344,501 B2

260
220240
7.2
pH
2 200
* NmL04A.a0dO5HM 180
1

5w .w9pH
8
.
FIG
.
8
100140160120 Time
(
min
)

80
cHPMC
in1x(size)oampbsinueld
MHw Cw.I01 60
cplus
-
5.5
pH
(
DR
x
1
)mioniatabeldtm)
cinoattbletds
-
7.0
pH
(
ICR
x
5
sawinrtkehr eTxrpilmcantes
,75Type2arpmp ar tus 40
20
0

50 45 40 35 30 25 20 15 10 5 0

) mg ( Dissolved Lovastatin
U.S. Patent May 31 , 2022
9 Sheet 11 of 18 US 11,344,501 B2

70

60

50

40
)Dose(mgAltoprev®
FIG.9A 30

20

40 20 07 09 091 -180
) ppm ( CH4 Breath in Baseline from Change
U.S. Patent May 31 , 2022 Sheet 12 of 18 US 11,344,501 B2

70

60

50

40

A?ltoprev
)
mg
(
Dose
FIG
.
9B
30

20

10

%
40 %
20 %
0
-120%
CHA Breath in Baseline from Change %
U.S. Patent May 31 , 2022 Sheet 13 of 18 US 11,344,501 B2

180

160

140

120

100
FIG
9C
.
BCH4(ppm)aseline
80

60

40 A(
30
)
mg
ltoprev®
A(
Omg
15
)
ltoprev® A&(
)ltoprev®
mg
60
20

40 20 -20 -40 -60 -10 -120 -140 -160 -180

-80
) ppm ( CH4 Breath in Baseline from Change
U.S. Patent May 31 , 2022 Sheet 14 of 18 US 11,344,501 B2

180

160 A(
o
15
)
mg
ltoprev®
A)ltoprev® A&
mg
30
( mg
60
(
)
ltoprev®
140

120

100
BCH4(ppma)seline
9D
.
FIG 80

60

40

20

%
40 %
20 %
0 %
-20 -40
% %
-60 %
-80 -100%With %
120
CHA Breath in Baseline from Change %
U.S. Patent May 31 , 2022 Sheet 15 of 18 US 11,344,501 B2

10A
.
FIG FIG.10B
1
U.S. Patent May 31 , 2022 Sheet 16 of 18 US 11,344,501 B2

Col n

11
.
FIG
U.S. Patent May 31 , 2022 Sheet 17 of 18 US 11,344,501 B2

36

32

ICR
(
7.0
PH
m6x
)
mg
42
initablets
minitablets
DR
5.5
PH
x
6
wytm
)
mg
42
( x
1initablets 1
mm
5
+
DR
m(
ICR
42
)
mg MwIR
xt(
40
)
mg
eiavybaoclnerot® Alatboplrevt®
x
1
tways
XR
)
mg
40
(
28
24
20

Time
(h
)

FIG.12A 16
Mean(n=5)LPinDogsoaelvcsteoalmtnisae
8
-
12

30.0 25.0 20.0 15.0 10.0 5.0 0.0

04
) mL / ng ( lactone Lovastatin
U.S. Patent May 31 , 2022 Sheet 18 of 18 US 11,344,501 B2

36

32

initablets 1
minitablets whether
DR
5.5
pH
6x
netim
)
mg
42
( x
5
+
DR
m(
ICR
42
)
mg
initablets
evacor® myfam
AXR
x
1t(
)
mg
40
latobplrevt®
7.0
pH
x
6
m42
)
ICR
(mg
MIR
x
1
in
money
tablet
40
(
)
mg 28
24

Time
(
h
)
16
20
12B
.
FIG inDogsLPß-HMean(n=5)yoeldvraosxetyalmcisand

* 8

12
8
4
-

30.0 25.0 5.0 20.0 15.0 10.0 0.0

) mL/ng( hydroxyacid- B Lovastatin
 US 11,344,501 B2
1 2
ANTI -METHANOGENIC COMPOSITIONS There remains a need for safe and effective approaches for
a
AND USES THEREOF the long term suppression of enteric methanogenesis and / or
excessive methane production in the treatment of diseases
CROSS - REFERENCE TO RELATED such as IBS .
APPLICATIONS 5
SUMMARY OF THE INVENTION
This application is aa continuation of U.S. patent applica Accordingly, the present invention provides, inter alia ,
tion Ser. No. 16 / 405,667 filed May 7 , 2019 which is a improved methods and formulations for the treatment of
continuation of U.S. patent application Ser. No. 15 / 940,063
filed Mar.29, 2018, now U.S. Pat. No. 10,328,151 , which is 10 various methanogen -associated disorders. and
In one
usesaspect ,the
a continuation of U.S. patent application Ser. No. 14/826 , present invention relates to compositions of modi
fied - release formulations which comprise at least one anti
115 filed Aug. 13 , 2015 , now U.S. Pat . No. 9,956,292 , which methanogenic agent, including, for example, statin hydroxy
claims the benefit of U.S. Provisional Patent Application acid molecules that, without wishing to be bound by theory,
Nos .62/036,948 , filed Aug. 13 , 2014 , 62/043,649, filed Aug. 15 are typically effective inhibitors of 3 -hydroxy -3-methyl
29 , 2014 , 62 /043,789 , filed Aug. 29 , 2014 , and 62 / 141,355 , glutaryl-coenzyme A (HMG - CoA) reductase and statin lac
filed Apr. 1 , 2015 , the entire contents of which are herein tones that, without wishing to be bound by theory, are
incorporated by reference . typically ineffective HMG - CoA reductase inhibitors ( collec
FIELD OF THE INVENTION
tively “ antimethanogenic statins ” ). In various embodiments,
20 the formulations and methods described herein eradicate or
reduce methane production, which is causative of, or cor
The present invention relates to , in part, methods and relative with various methanogen - associated disorders,
compositions for the treatment of methanogen -associated including, for example, IBS (e.g. IBS - C ) , diabetes and
disorders such as , for example, Irritable Bowel Syndrome obesity . In various embodiments, the formulations and meth
( IBS ) . 25 ods described herein target the gastrointestinal (GI ) tract and
therefore provide for specific delivery to a site of methano
BACKGROUND gen colonization and /or methane production and / or accu
mulation while avoiding or reducing systemic exposure to
The human microbiome plays an important role in both antimethanogenic statins and minimizing their systemic
health and disease . While the majority of microorganisms 30 effects. As such, the present invention provides for effective
inhabiting the gastrointestinal system of humans and ani- treatments that avoid side effects associated with chronic
mals have a beneficiary role in , for example, aiding diges- systemic statin administration (e.g. muscle pain , abnormali
tion of important nutrients, it is known that a minority of ties in liver enzyme tests , etc. ) . Further, in some embodi
otherwise previously considered “ commensal” organisms 35 ments, the present invention surprisingly treats bowel-dis
play a role in the pathogenesis of various diseases . orders despite reports linking statin use to , for example,
Irritable Bowel Syndrome ( IBS ) affects an estimated 30 constipation ( see , e.g. , Fernandes et al . Possible association
million people in the United States alone . IBS is aa functional between statin use and bowel dysmotility. BMJ Case
gastrointestinal (GI ) disorder that results in abdominal pain Reports 2012 ; 10.1136 / bcr.10.2011.4918 and Merck Global
Medical Information . Professional Information Response
and / or discomfort, along with changes in bowel habits. IBS 40 UK11-010274
is classified into four subtypes based on a person's stool rated by reference , the contents of which are hereby incorpo
in their entireties ). Further, in some
consistency: constipation - associated IBS ( IBS - C ) ; diarrhea embodiments, the present invention surprisingly treats dia
associated IBS (IBS - D ) ; mixed ( or alternating) IBS (IBS - M betes despite reports linking statin use to this disorder ( see ,
or IBS -A) ; and unsubtyped ( or unspecified ) IBS ( IBS -U) . e.g. Naci et al . , Comparative tolerability and harms of
Recent studies have suggested that certain methane pro- 45 individual statins : a study -level network meta -analysis of
ducing microorganisms inhabiting the gut known as metha 246 955 participants from 135 randomized , controlled trials .
nogens may play a causative role in constipation . Specifi- Circ Cardiovasc Qual Outcomes 6 ( 4 ) : 390-9 , the contents
cally, studies suggest a link between intestinal methane of which are hereby incorporated by reference in their
( CHA) production and constipation in IBS - C as well as entirety ).
chronic idiopathic constipation (CIC ) . Methane (CH4 ) pro- 50 In some embodiments , the modified - release formulations
duction in humans is due to methanogenic archaea in the release at least 60 % of the anti-methanogenic agent, such as
intestine . These organisms serve a critical biological func- anti-methanogenic statins , after the stomach and into one or
tion by removing the by -products of bacterial fermentation more regions of the intestinal tract. In certain embodiments,
of polysaccharides, notably hydrogen gas (Hz ) and short- the formulation releases the antimethanogenic statin in the
chain fatty acids ( SCFAs ). The dominant methanogen inhab- 55 small intestine, including one or more of the duodenum ,
iting the human gut is the archaea, Methanobrevibacter jejunum , and ileum . In other embodiments, the formulation
smithii (M. smithii ). In vitro susceptibility testing has dem- releases the anti-methanogenic statin in the large intestine
onstrated that methanogens such as M. smithii are highly (e.g. , one or more of the cecum , ascending, transverse ,
resistant to most classes of antibiotics. Further, complete descending or sigmoid portions of the colon, and rectum ).
eradication of intestinal methanogens via a single course of 60 In various embodiments, the antimethanogenic statin is
therapy is unlikely using broad spectrum antibiotics, leading selected from atorvastatin , cerivastatin , dalvastatin, eptasta
to methanogen recolonization and methanogenesis returning tin, fluindostatin , fluvastatin , lovastatin , mevastatin , pitavas
to pathogenic levels . Continuous use of antibiotics is asso- tatin, pravastatin , rosuvastatin , simvastatin , velostatin , and
ciated with various side effects and increased risk of devel- pharmaceutically acceptable salts , stereoisomers, or prodrug
oping antibiotic resistance . Further still , long - term use of 65 derivatives thereof. In some embodiments, the anti-metha
antibiotics may disrupt the otherwise potentially beneficial nogenic statin is selected from lovastatin, pravastatin , and
bacterial intestinal microbiome and gastrointestinal flora . simvastatin . In one embodiment, the statin is pravastatin and
 US 11,344,501 B2
3 4
pharmaceutically acceptable salts , stereoisomers , or prodrug anion transporting polypeptide ( OATP ) transporter, such as
derivatives thereof. In another embodiment, the antimetha- one or more of green tea extract, epicatechin gallate ( ECG)
nogenic statin is lovastatin and pharmaceutically acceptable and epigallocatechin gallate ( EGCG) . In some embodi
salts , stereoisomers, or prodrug derivatives thereof. In one ments, the OATP inhibitor is released prior to release of the
embodiment, the antimethanogenic statin is simvastatin and 5 statin . The formulations of the present invention may also
pharmaceutically acceptable salts , stereoisomers , or prodrug further include an additional therapeutic agent such as , by
derivatives thereof. In some embodiments, the antimetho- way of non - limiting example , a prokinetic agent.
genic statin is in either the lactone or ß -hydroxyacid form . In one aspect , the present invention provides for methods
In some embodiments, the antimethanogenic statin is the of inhibiting or reducing methanogenesis and /or methane
lactone form of lovastatin . 10 accumulation by administering the formulations described
In various embodiments, the modified - release formula- herein to a subject in need thereof. In some embodiments ,
tion is administered orally to a subject in need thereof. In one the subject suffers from IBS , such as IBS - C . In other
embodiment, the formulation may be in the form of a embodiments, the subject suffers from obesity. In yet
capsule or a tablet. In an embodiment, the formulation another embodiment, the subject suffers from diabetes. In
comprises a modified -release coating that is substantially 15 various aspects , the present invention provides for methods
stable in gastric fluid. In another embodiment, the modified- of treating or preventing a methanogen - associated disorder
release coating may be degraded by a microbial enzyme optionally selected from one or more of IBS , such as IBS - C ,
present in the gut flora . In a further embodiment, the diabetes, and obesity by administering the formulations
modified - release coating may have a solubility and / or sta- described herein to a subject in need thereof.
bility that is pH dependent. In other embodiments , the 20 In another aspect , the present invention also provides for
modified - release coating may have a time -dependent ero- methods of treating constipation by administering the for
sion profile. mulations described herein to a subject in need thereof. A
In various embodiments, the modified -release formula- further aspect of the invention provides methods for treating
tion comprises a first dose of at least one anti -methanogenic (e.g. reducing or eliminating) enteric methane production by
statin and a second dose of at least one antimethanogenic 25 administering the formulations described herein to a subject
statin (e.g. the first and second doses may be the same or in need thereof.
different antimethanogenic statin at a given dose , or the first
and second doses may be the same antimethanogenic statin DESCRIPTION OF THE FIGURES
at the same or different doses ) . In various embodiments, the
first dose and the second dose are released at different times 30 FIGS . 1A - 1B depict some embodiments of a modified
and / or at different pHs and in different regions of the release formulation in the form of encapsulated beads which
gastrointestinal tract . In some embodiments, the first and / or releases a first statin dose at the duodenum and a second
second dose of at least one antimethanogenic statin is statin dose at the ileum .
encapsulated in a core particle. A modified -release coating FIG . 2 depicts embodiments of modified - release formu
may be disposed over the core particle to form a modified- 35 lations as multi- layer capsules or tablets for statin delivery
release particle . In certain embodiments, the formulation to the intestines ( an illustrative commercial material is
comprises a plurality of modified -release particles. In an shown, related materials are known in the art ).
illustrative embodiment, the formulation maybe in the form FIGS . 3A - 3B depict embodiments of modified -release
of a capsule. In another embodiment, the first and second formulations for colonic delivery ( an illustrative commercial
dose of at least one antimethanogenic statin is encapsulated 40 material is shown , related materials are known in the art).
in a layer. A modified -release coating may be disposed over FIG . 4 depicts various embodiments of modified - release
the layer to form a modified - release layer. In certain embodi- formulations in the form of capsules that delivers either one
ments, the formulation comprises a plurality of modified- or two doses of statin to the intestines.
release layers . In an illustrative embodiment, the formula- FIG . 5 depicts the release profile of lovastatin from the
tion maybe in the form of aa multilayer tablet . 45 SYN -010 (21 mg ) formulation.
In some embodiments, the first dose and second dose of FIG . 6 shows the estimated lovastatin lactone levels in the
antimethanogenic statins are released at different times and gastrointestinal tract after oral administration .
or at different pHs . In illustrative embodiments, the first dose FIG . 7 depicts the dissolution methodology utilized to
may release the antimethanogenic statin at the duodenum evaluate lovastatin release from enteric - coated mini- tablets
while the second dose may release the antimethanogenic 50 at different pH values .
statin at the ileum . In other embodiments, the first dose may FIG . 8 depicts the dissolution profile of the SYN -010 ( 42
release the antimethanogenic statin at the small intestine mg ) capsule in a Type 2 apparatus at different pH values .
while the second dose may release the antimethanogenic FIGS . 9A - 9D show the results of aa clinical chart review .
statin at the large intestine. FIG . 9A shows an absolute change and FIG . 9B shows
The formulations of the present invention may further 55 percentage change, from baseline in breath methane versus
include a pharmaceutically acceptable excipient. In some ALTOPREV dose ( 15 , 30 or 60 mg q.d. ) . FIG . 9C shows an
embodiments, the formulation may further include an agent absolute change and FIG . 9D shows percentage change,
which prevents or reduces lactone ring - opening , such as an from baseline in breath methane versus baseline breath
esterase inhibitor ( e.g. grapefruit juice ; including flavonoid methane ( ppm ) in patients treated with ALTOPREV ( 15 , 30
components such as , for example, naringenin , kaempferol, 60 or 60 mg q.d.).
morin, galangin, and quercetin ; flavoring ester mixtures in , FIG . 10A shows that 7 weeks of high fat diet augmented
for example , strawberry juice (e.g. phenyl benzoate , propyl stool M. smithii colonization in rats. FIG . 10B shows that the
paraben , phenethyl isobutyrate , bacampicillin, talampicillin, high fat diet also reduced stool wet weight in the rats.
p - tolyl benzoate, ethyl paraben , diethyl phthalate, octyl FIG . 11 shows that after lovastatin administration, ileal
acetate , and pivampicillin ) and /or a paraoxonase inhibitor 65 ratio of M. smithii to total bacteria was reduced .
( e.g. PON1 or PON3 inhibitor ). In some embodiments, the FIGS . 12A - 12B show mean ( n = 5 ) plasma concentration
formulation may further include an inhibitor of the organic time profiles for lovastatin lactone and lovastatin B -hydroxy
 US 11,344,501 B2
5 6
acid, respectively, after oral administration of different lov- wishing to be bound by theory, an effective inhibitor of
astatin formulations to beagle dogs. HMG - CoA reductase or a statin lactone which typically is ,
without wishing to be bound by theory , an ineffective
DETAILED DESCRIPTION OF THE HMG - CoA inhibitor. In some aspects , the anti-methano
INVENTION 5 genic agent is referred to as an “ antimethanogenic statin ” or
" statin .
The present invention is based , in part, on the surprising In one aspect , the present invention provides modified
discovery of formulations and methods that are useful in release formulations comprising at least one antimethano
effectively treating or preventing methanogen - associated genic statin , wherein the formulation releases at least 60 % of
disorders while avoiding side effects . The present invention 10 the antimethanogenic stain after the stomach into one or
provides, inter alia , modified -release formulations compris- more regions of the intestinal tract.
ing one or more anti-methanogenic statins which is useful in , Illustrative statins useful for the invention include, but are
for example, the treatment of methanogen -associated disor- not limited to , atorvastatin , cerivastatin , dalvastatin , eptasta
ders such as , for example, IBS (including , for example, tin, fluindostatin , fluvastatin , lovastatin , mevastatin , pitavas
IBS - C ) . 15 tatin, pravastatin , rosuvastatin , simvastatin , velostatin , and
As used herein , “ antimethanogenic statin ” or “ statin ” pharmaceutically acceptable esters, prodrugs, salts , solvates ,
refers to a class of compounds that is known in the art as enantiomers , stereoisomers, active metabolites , co - crystals,
inhibitors of HMG - CoA reductase used as lipid lowering and other physiologically functional derivatives thereof. In
agents. However, the prior use of the statin compounds does one embodiment, the statin is pravastatin . In another
not necessarily imply a mechanism of action in the treatment 20 embodiment, the statin is lovastatin . In yet another embodi
of methanogenesis. That is , in some embodiments, the statin ment, the statin is simvastatin . In some embodiments, the
may inhibit the enzyme HMG - CoA reductase while in others statin is in either the lactone or hydroxyacid form . In some
it may have another manner of causing an effect. For embodiments, the antimethanogenic statin is the lactone
example , the statin may target a methanogenic enzyme, such form of one or more of atorvastatin , cerivastatin , dalvastatin ,
as , for example, one or more of adh alcohol dehydrogenase ; 25 eptastatin, fluindostatin , fluvastatin, lovastatin, mevastatin ,
fdh formate dehydrogenase; fno F420 -dependent NADP pitavastatin, pravastatin, rosuvastatin , simvastatin , velosta
oxidoreductase; fir formyl -MF :H4MPT formyltransferase; tin . In some embodiments, the antimethanogenic statin is the
fwd formyl -MF dehydrogenase; hmd methylene -H4MPT hydroxyacid form of one or more of atorvastatin , cerivas
dehydrogenase; mch methenyl -H4MPT cyclohydrolase; mtd tatin, dalvastatin , eptastatin , fluindostatin, fluvastatin , lov
F420 -dependent methylene -H4MPT dehydrogenase; mer 30 astatin , mevastatin , pitavastatin , pravastatin , rosuvastatin ,
F420 -dependent methylene - H4MPT reductase ; mtr methyl- simvastatin , velostatin .
H4MPT :COM -methyltransferase ; mcr methyl -COM In some embodiments , the antimethanogenic statin is the
reductase ; and the mtaB methanol : cobalamin methyltrans- lactone form of one or more of lov in , simvastatin, and
ferase ( 7 ) heterodisulfide reductase system . In some embodi- mevastatin . In some embodiments, the antimethanogenic
ments , the statin does not substantially inhibit the enzyme 35 statin
HMG - CoA reductase .
is the lactone form of lovastatin .
In various embodiments, the antimethanogenic statin ( e.g.
Systemic statin usage has been associated with adverse lovastatin ) is substantially in the lactone form at the site of
side effects such as elevation in hepatic enzyme levels and delivery by the present formulations . For example , in some
muscle problems ( e.g. , myalgias, rhabdomyolysis, and embodiments, the amount of GI tract - delivered antimetha
severe myopathy ). Further, systemic statin usage has been 40 nogenic statin ( e.g. lovastatin) which is in the lactone form
linked to digestive disorders in some patients. The modified is more than about 95 % , or more than about 90 % , or more
release formulations of the present invention minimize than about 85 % , or more than about 80 % , or more than about
absorption of the administered antimethanogenic statin from 75 % , or more than about 70% , or more than about 65 % , or
the intestine into the systemic circulation and reduce side more than about 60% , or more than about 55 % , or more than
effects , or disease exacerbating effects, associated with the 45 about 50 % , or more than about 25 % .
statin . Additionally, not all patients with IBS - C or CIC will In various embodiments, the modified -release formula
require lipid lowering therapy, so statin systemic absorption tions of the present invention are designed for immediate
from the modified release formulations of the present inven- release (e.g. upon ingestion ). In various embodiments, the
tion will ideally be insufficient to provide a clinically- modified -release formulations may have sustained -release
meaningful reduction in total cholesterol (total - C ) , or low- 50 profiles, i.e. slow release of the active ingredient ( s) in the
density lipoprotein cholesterol ( LDL - C ) , or apolipoprotein body (e.g. , GI tract) over an extended period of time . In
B ( Apo B ) , or triglycerides ( TG) , or a clinically -meaningful various embodiments, the modified - release formulations
increase in high -density lipoprotein cholesterol ( HDL - C ) may have a delayed - release profile, i.e. not immediately
( for example, a reduction of less than 5 % in serum LDL - C release the active ingredient( s) upon ingestion ; rather, post
levels at 6 weeks ) . 55 ponement of the release of the active ingredient( s) until the
Modified Release Profile composition is lower in the gastrointestinal tract; for
In one aspect , the present invention provides modified example , for release in the small intestine ( e.g. , one or more
release formulations comprising at least one anti -methano- of duodenum , jejunum , ileum ) or the large intestine ( e.g. ,
genic agent, wherein the formulation releases at least about one or more of cecum , ascending, transverse , descending or
60 % of the anti-methanogenic agent, such as anti -methano- 60 sigmoid portions of the colon , and rectum ). For example, a
genic statins , after the stomach and into one or more regions composition can be enteric coated to delay release of the
of the intestinal tract. active ingredient (s ) until it reaches the small intestine or
In various embodiments , the anti -methanogenic agent is large intestine . In some embodiments, there is not a sub
an agent that can inhibit the production of methane, inhibit stantial amount of the active ingredient ( s) of the present
methanogenesis, or inhibit the growth and /or proliferation of 65 formulations in the stool.
methanogens. In some aspects , the anti-methanogenic agent In various embodiments , the modified -release formula
is a statin hydroxyacid molecule which typically is , without tion of the present invention releases ( optionally as a first
 US 11,344,501 B2
7 8
release ) at least 5 % , 10% , 15 % , 20% , 25 % , 30 % , 35 % , 40 % , In a further embodiment, the formulation releases (option
a
45 % , 50% , 55 % , or 60 % of the antimethanogenic statin after ally as a first release ) the antimethanogenic statin in the
the stomach into one or more regions of the intestine. For ileum and / or the ileocecal junction. In various embodiments,
example, the modified -release formulation releases at least the modified - release formulation of the present invention
60 % , at least 61 % , at least 62 % , at least 63 % , at least 64 % , 5 releases at least 5 % , 10% , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % ,
at least 65 % , at least 66 % , at least 67 % , at least 68 % , at least 45 % , 50 % , 55 % , or 60 % of the antimethanogenic statin in
69 % , at least 70 % , at least 71 % , at least 72 % , at least 73 % , the ileum and / or the ileocecal junction. For example , the
at least 74 % , at least 75 % , at least 76 % , at least 77 % , at least modified - release formulation releases at least 60 % , at least
78 % , at least 79 % , at least 80 % , at least 81 % , at least 82 % , 61 % , at least 62 % , at least 63 % , at least 64 % , at least 65 % ,
at least 83 %, at least 84%, at least 85%, at least 86 % ,atleast 10 70atleast
% , at66least
% ,at71least
% , at67least
%, at72least
% , at68least
%, at73least
% , at69least
%, at74least
%,
87 % , at least 88 % , at least 89 % , at least 90 % , at least 91 % , at least 75 % , at least 76 % , at least 77 % , at least 78 % , at least
at least 92 % , at least 93 % , at least 94 % , at least 95 % , at least 79 % , at least 80% , at least 81 % , at least 82 % , at least 83 % ,
96 % , at least 97 % , at least 98 % , at least 99 % , or 100 % of the at least 84 % , at least 85 % , at least 86 % , at least 87 % , at least
antimethanogenic statin in the intestine. 15 88 % , at least 89 % , at least 90 % , at least 91 % , at least 92 % ,
In various embodiments , the modified -release formula at least 93 % , at least 94 % , at least 95 % , at least 96 % , at least
tion releases (optionally as a first release ) the antimethano 97 % , at least 98 % , at least 99 % , or 100 % of the antimetha
genic statin in the small intestine . In various embodiments, nogenic statin in the ileum and / or the ileocecal junction .
the modified - release formulation of the present invention In other embodiments, the modified -release formulation
releases at least 5 % , 10 % , 15 % , 20% , 25 % , 30 % , 35 % , 40 % , 20 releases (optionally as a first release) the antimethanogenic
45 % , 50 % , 55 % , or 60 % of the antimethanogenic statin in statin in the large intestine. In various embodiments , the
the small intestine . For example , the modified -release for- modified - release formulation of the present invention
mulation releases at least 60 % , at least 61 % , at least 62 % , at releases at least 5 % , 10% , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % ,
least 63 % , at least 64 % , at least 65 % , at least 66 % , at least 45 % , 50 % , 55 % , or 60 % of the antimethanogenic statin in
67 % , at least 68 % , at least 69 % , at least 70 % , at least 71 % , 25 the large intestine. For example, the modified - release for
at least 72 % , at least 73 % , at least 74 % , at least 75 % , at least mulation releases at least 60% , at least 61 % , at least 62 % , at
76 % , at least 77 % , at least 78 % , at least 79 % , at least 80 % , least 63 % , at least 64 % , at least 65 % , at least 66 % , at least
at least 81 % , at least 82 % , at least 83 % , at least 84 % , at least 67 % , at least 68 % , at least 69 % , at least 70 % , at least 71 % ,
85 % , at least 86 % , at least 87 % , at least 88 % , at least 89 % , at least 72 % , at least 73 % , at least 74 % , at least 75 % , at least
at least 90 % , at least 91 % , at least 92 % , at least 93 % , at least 30 76 % , at least 77 % , at least 78 % , at least 79 % , at least 80 % ,
94 % , at least 95 % , at least 96 % , at least 97 % , at least 98 % , at least 81 % , at least 82 % , at least 83 % , at least 84 % , at least
at least 99 % , or 100 % of the antimethanogenic statin in the 85 % , at least 86 % , at least 87 % , at least 88 % , at least 89 % ,
small intestine. at least 90 % , at least 91 % , at least 92 % , at least % , at least
In one embodiment, the formulation releases ( optionally 94 % , at least 95 % , at least 96 % , at least 97 % , at least 98 % ,
as a first release ) the antimethanogenic statin in the duode- 35 at least 99 % , or 100 % of the antimethanogenic statin in the
num . In various embodiments , the modified - release formu- large intestine.
lation of the present invention releases at least 5 % , 10 % , In an embodiment, the modified -release formulation
15 % , 20 % , 25 % , 30% , 35 % , 40 % , 45 % , 50 % , 55 % , or 60 % releases (optionally as a first release) the antimethanogenic
of the antimethanogenic statin in the duodenum . For statin in the cecum . In various embodiments, the modified
example, the modified - release formulation releases at least 40 release formulation of the present invention releases at least
60 % , at least 61 % , at least 62 % , at least 63 % , at least 64 % , 5 % , 10 % , 15 % , 20% , 25 % , 30 % , 35 % , 40 % , 45 % , 50% ,
at least 65 % , at least 66 % , at least 67 % , at least 68 % , at least 55 % , or 60 % of the antimethanogenic statin in the cecum .
69 % , at least 70 % , at least 71 % , at least 72 % , at least 73 % , For example, the modified -release formulation releases at
at least 74 % , at least 75 % , at least 76 % , at least 77 % , at least least 60% , at least 61 % , at least 62 % , at least 63 % , at least
78 % , at least 79 % , at least 80 % , at least 81 % , at least 82 % , 45 64 % , at least 65 % , at least 66 % , at least 67 % , at least 68 % ,
at least 83 % , at least 84 % , at least 85 % , at least 86 % , at least at least 69 % , at least 70 % , at least 71 % , at least 72 % , at least
87 % , at least 88 % , at least 89 % , at least 90 % , at least 91 % , 73 % , at least 74 % , at least 75 % , at least 76 % , at least 77 % ,
at least 92 % , at least 93 % , at least 94 % , at least 95 % , at least at least 78 % , at least 79 % , at least 80 % , at least 81 % , at least
96 % , at least 97 % , at least 98 % , at least 99 % , or 100 % of the 82 % , at least 83 % , at least 84 % , at least 85 % , at least 86 % ,
antimethanogenic statin in the duodenum . 50 at least 87 % , at least 88 % , at least 89 % , at least 90% , at least
In another embodiment, the formulation releases (option- 91 % , at least 92 % , at least 93 % , at least 94 % , at least 95 % ,
ally as a first release) the antimethanogenic statin in the at least 96 % , at least 97 % , at least 98 % , at least 99 % , or
jejunum . In various embodiments, the modified - release for- 100 % of the antimethanogenic statin in the cecum .
mulation of the present invention releases at least 5 % , 10 % , In another embodiment, the modified - release formulation
15 % , 20 % , 25 % , 30% , 35 % , 40 % , 45 % , 50 % , 55 % , or 60 % 55 releases (optionally as a first release ) the antimethanogenic
of the antimethanogenic statin in the jejunum . For example , statin in the ascending colon . In various embodiments , the
the modified - release formulation releases at least 60% , at modified - release formulation of the present invention
least 61 % , at least 62 % , at least 63 % , at least 64 % , at least releases at least 5 % , 10% , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % ,
65 % , at least 66 % , at least 67 % , at least 68 % , at least 69 % , 45 % , 50 % , 55 % , or 60 % of the antimethanogenic statin in
at least 70 % , at least 71 % , at least 72 % , at least 73 % , at least 60 the ascending colon . For example, the modified -release
74 % , at least 75 % , at least 76 % , at least 77 % , at least 78 % , formulation releases at least 60 % , at least 61 % , at least 62 % ,
at least 79 % , at least 80% , at least 81 % , at least 82 % , at least at least 63 % , at least 64 % , at least 65 % , at least 66 % , at least
83 % , at least 84 % , at least 85 % , at least 86 % , at least 87 % , 67 % , at least 68 % , at least 69 % , at least 70 % , at least 71 % ,
at least 88 % , at least 89 % , at least 90 % , at least 91 % , at least at least 72 % , at least 73 % , at least 74 % , at least 75 % , at least
92 % , at least 93 % , at least 94 % , at least 95 % , at least 96 % , 65 76 % , at least 77 % , at least 78 % , at least 79 % , at least 80 % ,
at least 97 % , at least 98 % , at least 99 % , or 100 % of the at least 81 % , at least 82 % , at least 83 % , at least 84 % , at least
antimethanogenic statin in the jejunum . 85 % , at least 86 % , at least 87 % , at least 88 % , at least 89 % ,
 US 11,344,501 B2
9 10
at least 90 % , at least 91 % , at least 92 % , at least 93 % , at least example, in some embodiments, the modified -release for
94 % , at least 95 % , at least 96 % , at least 97 % , at least 98 % , mulation is substantially stable in an acidic environment and
at least 99 % , or 100 % of the antimethanogenic statin in the substantially unstable (e.g. , dissolves rapidly or is physically
ascending colon. unstable ) in a near neutral to alkaline environment. In some
In yet another embodiment, the antimethanogenic statin is 5 embodiments, stability is indicative of not substantially
released ( optionally as a first release) in the transverse colon . releasing while instability is indicative of substantially
In various embodiments , the modified - release formulation releasing. For example , in some embodiments, the modified
of the present invention releases at least 5 % , 10 % , 15 % , release formulation is substantially stable at a pH of about
20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , or 60% of the 7.0 or less , or about 6.5 or less , or about 6.0 or less , or about
antimethanogenic statin in the transverse colon . For 10 5.5 or less , or about 5.0 or less , or about 4.5 or less , or about
example , the modified - release formulation releases at least 4.0 or less , or about 3.5 or less , or about 3.0 or less , or about
60 % , at least 61 % , at least 62 % , at least 63 % , at least 64 % , 2.5 or less , or about 2.0 or less , or about 1.5 or less , or about
at least 65 % , at least 66 % , at least 67 % , at least 68 % , at least 1.0 or less . In some embodiments, the present formulations
69 % , at least 70 % , at least 71 % , at least 72 % , at least 73 % , are stable in lower pH areas and therefore do not substan
at least 74 % , at least 75 % , at least 76 % , at least 77 % , at least 15 tially release in , for example, the stomach . In some embodi
78 % , at least 79 % , at least 80 % , at least 81 % , at least 82 % , ments, modified - release formulation is substantially stable at
at least 83 % , at least 84 % , at least 85 % , at least 86 % , at least a pH of about 1 to about 4 or lower and substantially
87 % , at least 88 % , at least 89 % , at least 90 % , at least 91 % , unstable at pH values that are greater. In these embodiments,
at least 92 % , at least 93 % , at least 94 % , at least 95 % , at least the modified -release formulation is not substantially
96 % , at least 97 % , at least 98 % , at least 99 % , or 100 % of the 20 released in the stomach . In these embodiments, the modi
antimethanogenic statin in the transverse colon . fied -release formulation is substantially released in the small
In a further embodiment, the antimethanogenic statin is intestine ( e.g. one or more of the duodenum , jejunum , and
released (optionally as a first release ) in the descending ileum ) and /or large intestine (e.g. one or more of the cecum ,
colon . In various embodiments , the modified - release formu- ascending colon , transverse colon, descending colon , and
lation of the present invention releases at least 5 % , 10 % , 25 sigmoid colon) . In some embodiments, modified - release
15 % , 20 % , 25 % , 30% , 35 % , 40 % , 45 % , 50 % , 55 % , or 60 % formulation is substantially stable at a pH of about 4 to about
of the antimethanogenic statin in the descending colon . For 5 or lower and consequentially is substantially unstable at
example , the modified -release formulation releases at least pH values that are greater and therefore is not substantially
60 % , at least 61 % , at least 62 % , at least 63 % , at least 64 % , released in the stomach and /or small intestine (e.g. one or
at least 65 % , at least 66 % , at least 67 % , at least 68 % , at least 30 more of the duodenum , jejunum , and ileum ). In these
69 % , at least 70 % , at least 71 % , at least 72 % , at least 73 % , embodiments, the modified -release formulation is substan
at least 74 % , at least 75 % , at least 76 % , at least 77 % , at least tially released in the large intestine ( e.g. one or more of the
78 % , at least 79 % , at least 80 % , least 81 % , at least 82 % , cecum , ascending colon, transverse colon , descending
at least 83 % , at least 84 % , at least 85 % , at least 86 % , at least colon , and sigmoid colon) . In various embodiments , the pH
87 % , at least 88 % , at least 89 % , at least 90 % , at least 91 % , 35 values recited herein may be adjusted as known in the art to
at least 92 % , at least 93 % , at least 94 % , at least 95 % , at least account for the state of the subject, e.g. whether in aa fasting
96 % , at least 97 % , at least 98 % , at least 99 % , or 100 % of the or postprandial state .
antimethanogenic statin in the descending colon . In some embodiments, the modified - release formulation
In another embodiment, the antimethanogenic statin is is substantially stable in gastric fluid and substantially
released (optionally as a first release ) in the sigmoid colon . 40 unstable in intestinal fluid and, accordingly, is substantially
In various embodiments, the modified - release formulation released in the small intestine (e.g. one or more of the
of the present invention releases at least 5 % , 10 % , 15 % , duodenum , jejunum , and ileum ) and / or large intestine (e.g.
20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , or 60% of the one or more of the cecum , ascending colon, transverse
antimethanogenic statin in the sigmoid colon . For example, colon , descending colon, and sigmoid colon ) .
the modified - release formulation releases at least 60% , at 45 In some embodiments, the modified - release formulation
least 61 % , at least 62 % , at least 63 % , at least 64 % , at least is stable in gastric fluid or stable in acidic environments.
65 % , at least 66 % , at least 67 % , at least 68 % , at least 69 % , These modified - release formulations release about 30% or
at least 70 % , at least 71 % , at least 72 % , at least 73 % , at least less by weight of the antimethanogenic statin and / or addi
74 % , at least 75 % , at least 76 % , at least 77 % , at least 78 % , tional therapeutic agent in the modified - release formulation
at least 79 % , at least 80% , at least 81 % , at least 82 % , at least 50 in gastric fluid with a pH of about 4 to about 5 or less , or
83 % , at least 84 % , at least 85 % , at least 86 % , at least 87 % , simulated gastric fluid with a pH of about 4 to about 5 or
at least 88 % , at least 89 % , at least 90 % , at least 91 % , at least less , in about 15 , or about 30 , or about 45 , or about 60 , or
92 % , at least 93 % , at least 94 % , at least 95 % , at least 96 % , about 90 minutes. Modified - release formulations of the of
at least 97 % , at least 98 % , at least 99 % , or 100 % of the the invention may release from about 0 % to about 30 % ,
antimethanogenic statin in the sigmoid colon . 55 from about 0 % to about 25 % , from about 0 % to about 20 % ,
In certain embodiments, the modified - release formulation from about 0 % to about 15 % , from about 0 % to about 10 % ,
does not substantially release the antimethanogenic statin in about 5 % to about 30% , from about 5 % to about 25 % , from
the stomach . about 5 % to about 20% , from about 5 % to about 15 % , from
In some embodiments , the modified -release formulation about 5 % to about 10 % by weight of the antimethanogenic
is a HPMC capsule filled with enteric - coated mini- tablets 60 statin and / or additional therapeutic agent in the modified
from which lovastatin is released at different intestinal pH release formulation in gastric fluid with a pH of 4-5 , or less
values . The mini - tablets are designed to pass through the or simulated gastric fluid with a pH of 4-5 or less , in about
stomach unchanged then release a small amount of lovas- 15 , or about 30 , or about 45 , or about 60 , or about 90
tatin into the duodenum and the majority of the lovastatin minutes. Modified - release formulations of the invention
dose into the ileocecal junction and colon . 65 may release about 1 % , about 2 % , about 3 % , about 4 % , about
In certain embodiments, the modified - release formulation 5 % , about 6 % , about 7 % , about 8 % , about 9 % , or about 10 %
releases the antimethanogenic statin at a specific pH . For by weight of the total antimethanogenic statin and /or addi
 US 11,344,501 B2
11 12
tional therapeutic agent in the modified - release formulation enzymes in the intestine, such as galactomannans. Also , the
in gastric fluid with a pH of 5 or less , or simulated gastric stability of the modified - release formulation can be depen
fluid with a pH of 5 or less , in about 15 , or about 30 , or about dent on enzyme stability in the presence of a microbial
45 , or about 60 , or about 90 minutes. enzyme present in the gut flora.
In some embodiments , the modified - release formulation 5 In some embodiments, a dual pulse formulation is pro
is unstable in intestinal fluid . These modified - release for- vided . In various embodiments, the present invention pro
mulations release about 70 % or more by weight of the vides for modified - release formulations that release multiple
antimethanogenic statin and / or additional therapeutic agent doses of the antimethanogenic statin, at different locations
in the modified - release formulation in intestinal fluid or along the intestines, at different times , and / or at different pH .
simulated intestinal fluid in about 15 , or about 30 , or about 10 In an illustrative embodiment, the modified - release formu
45 , or about 60 , or about 90 minutes. In some embodiments, lation comprises a first dose of the antimethanogenic statin
the modified - release formulation is unstable in near neutral and a second dose of the antimethanogenic statin , wherein
to alkaline environments . These modified - release formula- the first dose and the second dose are released at different
tions release about 70 % or more by weight of the antime- locations along the intestines , at different times , and / or at
thanogenic statin and / or additional therapeutic agent in the 15 different pH . For example , the first dose is released at the
modified - release formulation in intestinal fluid with a pH of duodenum , and the second dose is released at the ileocecal
about 4-5 or greater, or simulated intestinal fluid with a pH junction and / or colon . In another example , the first dose is
of about 4-5 or greater, in about 15 , or about 30 , or about 45 , released at the jejunum , and the second dose is released at
or about 60 , or about 90 minutes . A modified -release for- the ileum . In other embodiments, the first dose is released at
mulation that is unstable in near neutral or alkaline envi- 20 a location along the small intestine ( e.g. , the duodenum ),
ronments may release 70 % or more by weight of antime- while the second dose is released along the large intestine
thanogenic statin and/ or additional therapeutic agent in the (e.g. , the ascending colon ). In various embodiments, the
modified - release formulation in aa fluid having a pH greater modified - release formulation may release at least one dose ,
than about 5 (e.g. , a fluid having a pH of from about 5 to at least two doses , at least three doses , at least four doses , at
about 14 , from about 6 to about 14 , from about 7 to about 25 least five doses , at least six doses , at least seven doses , or at
14 , from about 8 to about 14 , from about 9 to about 14 , from least eight doses of the antimethanogenic statin at different
about 10 to about 14 , or from about 11 to about 14 ) in from locations along the intestines, at different times , and /or at
about 5 minutes to about 90 minutes, or from about 10 different pH . Each individual dose may comprise the same
minutes to about 90 minutes, or from about 15 minutes to statin or may comprise different statins . For example, the
about 90 minutes , or from about 20 minutes to about 90 30 modified -release formulation may release multiple doses,
minutes, or from about 25 minutes to about 90 minutes, or with the first dose being released at the duodenum and the
from about 30 minutes to about 90 minutes, or from about second and /or additional dose being released at the ileocecal
5 minutes to about 60 minutes, or from about 10 minutes junction and /or colon .
about 60 minutes, or from about 15 minutes to about 60 In some embodiments, the dual pulse formulation is an
minutes, or from about 20 minutes to about 60 minutes , or 35 enteric - coated capsule comprising beads or mini - tablets that
from about 25 minutes to about 90 minutes, or from about comprise an antimethanogenic statin and optionally an addi
30 minutes to about 60 minutes. tional therapeutic agent. In some embodiments, the enteric
In one embodiment, the modified - release formulation coated capsule dissolves in a first area of GI tract to release
may remain essentially intact , or may be essentially the beads or mini - tablets and / or a first population of beads
insoluble , in gastric fluid . The stability of the delayed- 40 or mini - tablets releases in aa second area of the GI tract and
release coating can be pH dependent. Delayed -release coat- ( that is not the same as the first area of the GI tract) and a
ings that are pH dependent will be substantially stable in second population of beads or mini - tablets releases in aa third
acidic environments (pH of about 5 or less ) , and substan- area of the GI tract and ( that is not the same as the first or
tially unstable in near neutral to alkaline environments (pH second areas of the GI tract ). In some embodiments, the
greater than about 5 ) . For example , the delayed -release 45 dose /release ratio ( e.g. how much agent is released in
coating may essentially disintegrate or dissolve in near various locations) can be tuned as needed . In some embodi
neutral to alkaline environments such as are found in the ments, the enteric -coated capsule dissolves in the duodenum
small intestine (e.g. one or more of the duodenum , jejunum , to release the beads or mini - tablets and / or aa first population
and ileum) and / or large intestine (e.g. one or more of the of beads or mini- tablets releases in the duodenum and / or a
cecum, ascending colon, transverse colon, descending 50 second population of beads or mini- tablets releases in the
colon, and sigmoid colon) . ileocecal junction ( see , e.g. FIGS . 1-4 ).
Examples of simulated gastric fluid and simulated intes- In alternative embodiments, the dual pulse formulation is
tinal fluid include, but are not limited to , those disclosed in a water - soluble capsule comprising enteric - coated beads or
the 2005 Pharmacopeia 23NF /28USP in Test Solutions at mini -tablets that comprise an antimethanogenic statin and
page 2858 and / or other simulated gastric fluids and simu- 55 optionally an additional therapeutic agent. Illustrative water
lated intestinal fluids known to those of skill in the art, for soluble capsules include , but are not limited to , gelatin and
example , simulated gastric fluid and / or intestinal fluid pre- hydroxypropyl methylcellulose (HPMC ) capsules. In some
pared without enzymes. embodiments, the water - soluble capsule dissolves in a first
Alternatively , the stability of the modified - release formu- area of GI tract to release the beads or mini - tablets and / or a
lation can be enzyme-dependent . Delayed -release coatings 60 first population of beads or mini-tablets releases in a second
that are enzyme dependent will be substantially stable in area of the GI tract and ( that is not the same as the first area
fluid that does not contain a particular enzyme and substan- of the GI tract) and a second population of beads or
tially unstable in fluid containing the enzyme. The delayed- mini -tablets releases in aa third area of the GI tract and ( that
release coating will essentially disintegrate or dissolve in is not the same as the first or second areas of the GI tract ).
fluid containing the appropriate enzyme. Enzyme- dependent 65 In some embodiments, the water - soluble capsule dissolves
control can be brought about, for example, by using mate- in the stomach to release the beads or mini - tablets and / or a
rials which release the active ingredient only on exposure to first population of beads or mini - tablets releases in the
 US 11,344,501 B2
13 14
duodenum and / or a second population of beads or mini- glycol sorbitan fatty acid esters , polyethylene glycol alkyl
tablets releases in the ileocecal junction and / or colon . ethers, sugar esters, polyethylene glycol alkyl phenols, poly
Modified Release Formulation and Dosage Forms oxyethylene- olyoxypropylene block copolymers, sorbitan
The modified - release formulation of the present invention fatty acid esters, lower alcohol fatty acid esters , ionic
may further comprise a pharmaceutically acceptable carrier 5 surfactants, and mixtures thereof. In some embodiments,
or excipient. As one skilled in the art will recognize, the compositions of the invention may comprise one or more
formulations can be in any suitable form appropriate for the surfactants including, but not limited to , sodium lauryl
desired use and route of administration . Examples of suit sulfate , polysorbate 20 , polysorbate 40 , polysorbate 60 ,
able dosage forms include , for example , oral and parenteral polysorbate 80 , and triethyl citrate .
dosage forms. 10
The modified -release formulation can also contain phar
Suitable dosage forms for oral use include , for example, maceutically acceptable plasticizers to obtain the desired
solid dosage forms such as tablets , dispersible powders, mechanical properties
granules, and capsules . In one embodiment, the modified plasticizers include , butsuchareasnotflexibility and hardness . Such
limited to , triacetin , citric
release formulation is in the form of a tablet. In another
embodiment, the modified -release formulation is in the form 15 acid esters , phthalic acid esters , dibutyl sebacate , cetyl
of a capsule . In yet another embodiment, the modified alcohol , polyethylene glycols, polysorbates or other plasti
release formulation is in the form of a soft - gel capsule . In a cizers.
further embodiment, the modified - release formulation is in The modified - release formulation can also include one or
the form of a gelatin capsule . In a further embodiment, the more application solvents. Some of the more common
modified - release formulation is in the form of a hydroxy- 20 solvents that can be used to apply, for example, a delayed
propyl methylcellulose ( HPMC ) capsule. release coating composition include isopropyl alcohol ,
In such dosage forms, the active compound is mixed with acetone , methylene chloride and the like .
at least one inert, pharmaceutically acceptable excipient or The modified -release formulation can also include one or
carrier such as sodium citrate , dicalcium phosphate, etc. , more disintegrants. Illustrative disintegrants that may be
and / or a ) fillers, diluents, or extenders such as starches, 25 utilized include , but are not limited to crospovidones such as
lactose , sucrose , glucose , mannitol, silicic acid , microcrys- Kollidon® CL , Kollidon® CL - F, Kollidon® CL - SF, or
talline cellulose ( e.g. , Avicel PH102 ) , and Bakers Special Kollidon® CL - M ,
Sugar, etc. , b ) binders such as , for example, carboxymeth- The modified -release formulation can also include one or
ylcellulose, alginates, gelatin , polyvinylpyrrolidone , more alkaline materials . Alkaline material suitable for use in
sucrose, acacia , polyvinyl alcohol, polyvinylpyrrolidone, 30 compositions of the invention include, but are not limited to ,
methylcellulose, hydroxypropyl cellulose , hydroxymethyl sodium , potassium , calcium , magnesium and aluminum salts
cellulose , and copovidones such as Kollidon® VA64, and of acids such as phosphoric acid , carbonic acid , citric acid
Kollidon® VA64 Fine , etc. , c ) humectants such as glycerol, and other aluminum /magnesium compounds. In addition the
etc. , d) disintegrating agents such as agar - agar, calcium alkaline material may be selected from antacid materials
carbonate, potato or tapioca starch , alginic acid , certain 35 such as aluminum hydroxides, calcium hydroxides, magne
silicates , sodium carbonate , cross - linked polymers such as sium hydroxides and magnesium oxide .
crospovidone ( cross - linked polyvinylpyrrolidone ), croscar- The solid oral dosage forms can be prepared by any
mellose sodium (cross - linked sodium carboxymethylcellu- conventional method known in the art, for example granu
lose) , sodium starch glycolate, etc. , e ) solution retarding lation (e.g. , wet or dry granulation) of the active compound
agents such as paraffin , etc. , f) absorption accelerators such 40 (e.g. , statins ) with one or more suitable excipients. Alterna
as quaternary ammonium compounds, etc. , g ) wetting agents tively, the active compound can be layered onto an inert core
such as , for example, cetyl alcohol and glycerol monoste- ( e.g. , a nonpareil /sugar sphere or silica sphere) using con
arate, etc. , h) absorbents such as kaolin and bentonite clay, ventional methods such as fluidized bed or pan coating , or
etc. , i ) lubricants such as talc , calcium stearate, magnesium extruded and spheronized using methods known in the art,
stearate, solid polyethylene glycols , sodium lauryl sulfate , 45 into active compound - containing beads . Such beads can
glyceryl behenate, etc. , j ) antioxidants such as propyl gal- then be incorporated into tablets or capsules using conven
late , butylated hydroxyanisole (BHA) , butylated hydroxy- tional methods.
toluene ( BHT ) , ethylenediaminetetraacetic acid (also known Liquid dosage forms for oral administration include phar
as Edetic Acid or EDTA ) etc. , k) viscosity and dispersion maceutically acceptable emulsions, solutions , suspensions,
agents such as silicon dioxide or silica , and mixtures of such 50 syrups and elixirs . In addition to the active compounds, the
excipients. One of skill in the art will recognize that par- liquid dosage forms may contain inert diluents commonly
ticular excipients may have two or more functions in the oral used in the art such as , for example, water or other solvents,
dosage form . In the case of an oral dosage form , for solubilizing agents and emulsifiers such as ethyl alcohol ,
example , a capsule or a tablet , the dosage form may also isopropyl alcohol, ethyl carbonate , ethyl acetate , benzyl
comprise buffering agents . 55 alcohol , benzyl benzoate , propylene glycol , 1,3 -butylene
The modified release formulation can additionally include glycol, dimethyl formamide, oils ( in particular, cottonseed,
a surface active agent. Surface active agents suitable for use groundnut, corn , germ , olive , castor, and sesame oils ) ,
in the present invention include, but are not limited to , any glycerol, tetrahydrofurfuryl alcohol , polyethylene glycols
pharmaceutically acceptable, non -toxic surfactant. Classes and fatty acid esters of sorbitan, etc., and mixtures thereof.
of surfactants suitable for use in the compositions of the 60 Besides inert diluents, the oral compositions can also
invention include , but are not limited to polyethoxylated include adjuvants such as sweetening, flavoring, and per
fatty acids , PEG - fatty acid diesters, PEG - fatty acid mono- fuming agents.
and di - ester mixtures , polyethylene glycol glycerol fatty Suspensions, in addition to the active compounds, may
acid esters, alcohol - oil transesterification products , contain suspending agents such as , for example, ethoxylated
polyglycerized fatty acids , propylene glycol fatty acid esters , 65 isostearyl alcohols , polyoxyethylene sorbitol and sorbitan
mixtures of propylene glycol esters - glycerol esters , mono- esters, microcrystalline cellulose , aluminum metahydroxide,
and diglycerides , sterol and sterol derivatives, polyethylene bentonite, agar - agar, tragacanth , etc., and mixtures thereof.
 US 11,344,501 B2
15 16
The formulations comprising the therapeutic agents of the water, independent of the pH of the solution. Suitable
present invention may conveniently be presented in unit polymers include , but are not limited to , cellulose ethers,
dosage forms and may be prepared by any of the methods cellulose esters, or cellulose ether - esters, i.e. , a cellulose
well known in the art of pharmacy . Such methods generally derivative in which some of the hydroxy groups on the
include the step of bringing the therapeutic agents into 5 cellulose skeleton are substituted with alkyl groups and
association with a carrier, which constitutes one or more some are modified with alkanoyl groups . Examples include
accessory ingredients. Typically, the formulations are pre- ethyl cellulose , acetyl cellulose , nitrocellulose , and the like.
pared by uniformly and intimately bringing the therapeutic Other examples of insoluble polymers include , but are not
agent into association with a liquid carrier, a finely divided limited to , lacquer, and acrylic and / or methacrylic ester
solid carrier, or both, and then , if necessary, shaping the 10 polymers , polymers or copolymers of acrylate or methacry
product into dosage forms of the desired formulation (e.g., late having a low quaternary ammonium content, or mixture
wet or dry granulation, powder blends , etc., followed by thereof and the like . Other examples of insoluble polymers
tableting using conventional methods known in the art). include EUDRAGIT RS® , EUDRAGIT RL® , and
In various embodiments, the modified - release formula- EUDRAGIT NE® . Insoluble polymers useful in the present
tion of the present invention may utilize one or more 15 invention include polyvinyl esters , polyvinyl acetals, poly
modified -release coatings such as delayed -release coatings acrylic acid esters, butadiene styrene copolymers , and the
to provide for effective, delayed yet substantial delivery of like. In one embodiment, colonic delivery is achieved by use
the antimethanogenic statin to the GI tract together with , of a slowly - eroding wax plug (e.g. , various PEGS , including
optionally, other therapeutic agents. for example , PEG6000 ).
In one embodiment, the delayed - release coating includes 20 In aa further embodiment, the delayed -release coating may
an enteric agent that is substantially stable in acidic envi- be degraded by a microbial enzyme present in the gut flora .
ronments and substantially unstable in near neutral to alka- In one embodiment, the delayed -release coating may be
line environments . In an embodiment, the delayed -release degraded by a bacteria present in the small intestine. In
coating contains an enteric agent that is substantially stable another embodiment , the delayed -release coating may be
in gastric fluid . The enteric agent can be selected from , for 25 degraded by a bacteria present in the large intestine .
example , solutions or dispersions of methacrylic acid copo- The present invention provides for modified - release for
lymers , cellulose acetate phthalate, hydroxypropylmethyl mulations that release multiple doses of the antimethano
cellulose phthalate, polyvinyl acetate phthalate , carboxym- genic statin along the gastrointestinal tract. The overall
ethylethylcellulose, and EUDRAGIT® - type polymer (poly release profile of such a formulation may be adjusted by
(methacrylic acid , methylmethacrylate ), hydroxypropyl 30 utilizing, for example, multiple particle types or multiple
methylcellulose acetate succinate, cellulose acetate trimel- layers. In one embodiment, the first dose of the antimetha
litate , shellac or other suitable enteric coating polymers. The nogenic statin may be formulated for release in , for example,
EUDRAGIT® -type polymers include, for example, the duodenum , whereas the second dose is formulated for
EUDRAGIT® FS 30D , L 30 D - 55 , L 100-55 , L 100 , L 12.5 , delayed release in, for example
e , the ileum . In another
L 12.5 P , RL 30 D , RL PO , RL 100 , RL 12.5 , RS 30 D , RS 35 embodiment, the first dose of the antimethanogenic statin
PO , RS 100 , RS 12.5 , NE 30 D , NE 40 D , NM 30 D , S 100 , may be formulated for release in, for example , the small
S 12.5 , and S 12.5 P. Similar polymers include Kollicoat® intestines, whereas the second dose is formulated for
MAE 30 DP and Kollicoat® MAE 100 P. In some embodi- delayed release in , for example, the large intestines. Alter
ments, one or more of EUDRAGIT® FS 30D , L 30 D - 55 , L natively, multiple doses are released at different locations
100-55 , L 100 , L 12.5 , L 12.5 P RL 30 D , RL PO , RL 100 , 40 alone the intestine.
RL 12.5 , RS 30 D , RS PO , RS 100 , RS 12.5 , NE 30 D , NE In one embodiment, one or more doses of the antimetha
40 D , NM 30 D , S 100 , S 12.5 S 12.5 P , Kollicoat® MAE nogenic statin may be encapsulated in a core particle, for
30 DP and Kollicoat® MAE 100 P is used . In various example, in the form of a microbead or a mini - tablet. For
embodiments, the enteric agent may be a combination of the example, the first dose of the antimethanogenic statin may
foregoing solutions or dispersions. In certain embodiments, 45 be encapsulated in a core particle coated with a modified
one or more coating system additives are used with the release coating designed for release at a first location along
enteric agent. For example, one or more PlasACRYLTM the intestinal tract, and the second dose of the antimetha
additives may be used as an anti - tacking agent coating nogenic statin may be encapsulated in a core particle coated
additive . Illustrative PlasACRYLTM additives include , but with a modified - release coating designed for release at a
are not limited to PlasACRYLTM HTP20 and PlasACRYLTM 50 second location along the intestinal tract. In various embodi
T20 . In an embodiment, Plas ACRYLTM HTP20 is formu- ments, the formulation may comprise a plurality of such
lated with EUDRAGIT® L 30 D - 55 coatings. In another modified -release particles. For example , the formulation
embodiment, PlasACRYLTM T20 is formulated with may be in the form of capsules comprising multiple micro
EUDRAGIT® FS 30 D coatings . beads or multiple mini - tablets. For example , the formulation
In another embodiment, the delayed -release coating may 55 may be in the form of capsules such as , for example, gelatin
degrade as a function of time when in aqueous solution and hydroxypropyl methylcellulose ( HPMC ) capsules com
without regard to the pH and / or presence of enzymes in the prising multiple enteric - coated microbeads or mini - tablets .
solution . Such a coating may comprise a water insoluble In such an embodiment, a combination of microbeads or
polymer. Its solubility in aqueous solution is therefore mini -tablets may be utilized in which each microbead or
independent of the pH . The term “ pH independent ” as used 60 mini -tablet is designed to release at a specific time point or
herein means that the water permeability of the polymer and location . In an alternative embodiment, the formulation is
its ability to release pharmaceutical ingredients is not a formulated as a capsule within a capsule , with each capsule
function of pH and / or is only very slightly dependent on pH . having different time- or pH -dependent release properties.
Such coatings may be used to prepare, for example , sus- In some embodiments, the formulation may comprise
tained release formulations. Suitable water insoluble poly- 65 multiple microbeads or multiple mini - tablets at specific
mers include pharmaceutically acceptable non -toxic poly- ratios so as to release specified amount of the active ingre
mers that are substantially insoluble in aqueous media, e.g. , dients at specific time points or locations . For example , the
 US 11,344,501 B2
17 18
formulation may comprise specific ratios of mini- tablets that fate , tartrate , thiocyanate, toluenesulfonate , undecanoate,
release at a first location (e.g. , the duodenum ) or a first pH valerate salts , and the like . Representative alkali or alkaline
( e.g. , pH of about 5.5 ) and mini- tablets that release at a earth metal salts include sodium , lithium , potassium , cal
second location (e.g. , the ileocecal junction or colon) or a cium , magnesium , and the like , as well as nontoxic ammo
second pH ( e.g. , pH of about 7.0 ) . In some embodiments, the 5 nium , quaternary ammonium , and amine cations, including ,
ratio is about 1:10 to about 10 : 1 . For example, the formu- but not limited to ammonium , tetramethylammonium , tet
lation may comprise mini - tablets that release at a first pH raethylammonium , methylamine, dimethylamine, trimethyl
( e.g. pH of about 5.5 ) and at a second pH ( e.g. , pH of about amine , triethylamine, ethylamine, and the like .
7.0 ) at a ratio of 1 : 1 , 1 : 2 , 1 : 3 , 1 : 4 , 1 : 5 , 1 : 6 , 1 : 7 , 1 : 8 , 1 : 9 , 1:10 ,
2 In various embodiments, the formulation comprises at
10 : 1 , 9 : 1 , 8 : 1 , 7 : 1 , 6 : 1 , 5 : 1 , 4 : 1 , 3 : 1 , or 2 : 1 . In one embodi- 10 least one microbead or mini - tablet. In some embodiments ,
ment, the formulation may comprise mini- tablets that each microbead or mini -tablet comprises about 5-20 % by
release at a first pH (e.g. pH of about 5.5 ) and at a second weight the antimethanogenic statin (which is, in some
pH (e.g. , pH of about 7.0) at a ratio of 1 : 2 . In another embodiments, lovastatin , and in further embodiments , lov
embodiment, the formulation may comprise mini - tablets astatin lactone ) . For example, the antimethanogenic statin
that release at a first pH ( e.g. pH of about 5.5 ) and at a 15 may be present at about 5 % , about 6 % , about 7 % , about 8 % ,
second pH (e.g. , pH of about 7.0 ) at a ratio of 1 : 5 . about 9 % , about 10% , about 11 % , about 12 % , about 13 % ,
In another embodiment, one or more doses of the anti- about 14 % , about 15 % , about 16 % , about 17 % , about 18 % ,
methanogenic statin may be encapsulated in a layer. For about 19 % , or about 20% by weight. In some embodiments ,
example, the first dose of the antimethanogenic statin may each microbead or mini - tablet may further comprise about
be encapsulated in a layer coated with a modified -release 20 50-70% by weight tablet diluent (e.g. , about 50 % , about
coating designed for release at a first location along the 51 % , about 52 % , about 53 % , about 54 % , about 55 % , about
intestinal tract, and the second dose of the antimethanogenic 56 % , about 5'7 % , about 58 % , about 59 % , about 60 % , about
statin may be encapsulated in a layer coated with a modified- 61 % , about 62 % , about 63 % , about 64 % , or about 65 % , or
release coating designed for release at second location about 66 % , about 67 % , or about 68 % , or about 69 % , or
along the intestinal tract. The formulation may comprise a 25 about 70% ) . In some embodiments, each microbead or
plurality of such modified - release layers. For example , the mini -tablet may further comprise about 1-10 % by weight
formulation is in the form of multi -layered tablet or a tablet binder ( e.g. , about 1 % , about 2 % , about 3 % , about
multi- layered capsule or capsules within capsules. Each 4 % , about 5 % , about 6 % , about 7 % , about 8 % , about 9 % , or
layer may have different time- or pH -dependent release about 10% ) . In some embodiments, each microbead or
properties. 30 mini -tablet may further comprise about 0.1-3.0% by weight
In the above embodiments, the coated particles or layers viscosity and dispersion agent (e.g. , about 0.1 % , about
with the delayed -release coating may be further covered 0.2 % , about 0.3 % , about 0.4 % , about 0.5 % , about 0.6 % ,
with an overcoat layer. The overcoat layer can be applied as about 0.7 % , about 0.8 % , about 0.9 % , about 1 % , about 1.1 % ,
described for the other coating compositions. The overcoat about 1.2 % , about 1.3 % , about 1.4 % , about 1.5 % , about
materials are pharmaceutically acceptable compounds such 35 1.6 % , about 1.7 % , about 1.8 % , about 1.9 % , about 2 % , about
as sugar, polyethylene glycol , polyvinylpyrrolidone, poly- 2.1 % , about 2.2 % , about 2.3 % , about 2.4 % , about 2.5 % ,
vinyl alcohol , polyvinyl acetate , hydroxypropyl cellulose , about 2.6 % , about 2.7 % , about 2.8 % , about 2.9 % , or about
methylcellulose , ethylcellulose, hydroxypropyl methylcel- 3.0 % ) . In some embodiments , each microbead or mini -tablet
lulose , carboxymethylcellulose sodium and others, used may further comprise about 0.1-3.0 % by weight lubricant,
alone or in mixtures. The overcoat materials can prevent 40 for example, to facilitate tableting (e.g. , about 0.1 % , about
potential agglomeration of particles coated with the delayed- 0.2 % , about 0.3 % , about 0.4 % , about 0.5 % , about 0.6 % ,
release coating, protect the delayed - release coating from about 0.7 % , about 0.8 % , about 0.9 % , about 1 % , about 1.1 % ,
cracking during the compaction process or enhance the about 1.2 % , about 1.3 % , about 1.4 % , about 1.5 % , about
tableting process . 1.6 % , about 1.7 % , about 1.8 % , about 1.9 % , about 2 % , about
Furthermore, in various embodiments, the agents 45 2.1 % , about 2.2 % , about 2.3 % , about 2.4 % , about 2.5 % ,
described herein may be in the form of a pharmaceutically about 2.6 % , about 2.7 % , about 2.8 % , about 2.9 % , or about
acceptable salt , namely those salts which are suitable for use 3.0 % ) . In some embodiments, each microbead or mini -tablet
in contact with the tissues of humans and other animals may further comprise about 1-10 % by weight tablet disin
without undue toxicity, irritation , allergic response and the tegrant ( e.g. , about 1 % , about 2 % , about 3 % , about 4 % ,
like , and are commensurate with a reasonable benefit / risk 50 about 5 % , about 6 % , about 7 % , about 8 % , about 9 % , or
ratio . Pharmaceutically acceptable salts are well known in about 10% ) . In some embodiments, each microbead or
the art. The salts can be prepared in situ during the final mini -tablet may further comprise about 10-20 % by weight
isolation and purification of the therapeutic agents , or sepa- an enteric polymer that dissolves at a pH of either about 5.5
rately by reacting the free base function with a suitable acid or about 7.0 (e.g. , about 10 % , about 11 % , about 12 % , about
9

or a free acid functionality with an appropriate alkaline 55 13 % , about 14 % , about 15 % , about 16% , about 17 % , about
moiety. Representative acid addition salts include acetate , 18 % , about 19 % , or about 20 % ) .
adipate, alginate , ascorbate , aspartate , benzenesulfonate,
9 In various embodiments, the formulation comprises one
benzoate, bisulfate , borate , butyrate, camphorate, campher- or more of, or two or more of, or three or more of, or four
sulfonate, citrate , cyclopentanepropionate , digluconate , or more of, or five or more of, or all of an antimethanogenic
dodecylsulfate, ethanesulfonate , fumarate , glucoheptonate, 60 statin ( which is , in some embodiments, lovastatin , and in
glycerophosphate , hemisulfate , heptonate , hexanoate , hyd- further embodiments , lovastatin lactone) , the antimethano
robromide, hydrochloride, hydroiodide, 2-hydroxyethane- genic statin (which is , in some embodiments, lovastatin , and
sulfonate , lactobionate , lactate , laurate, lauryl sulfate , in further embodiments , lovastatin lactone) optionally being
malate , maleate, malonate, methanesulfonate, 2 -naphthale- in two doses ; microcrystalline cellulose ( e.g. Avicel PH102 ) ;
nesulfonate , nicotinate, nitrate, oleate , oxalate , palmitate , 65 copovidone (e.g. Kollidon VA64 Fine ) ; silicon dioxide (e.g.
pamoate, pectinate , persulfate , 3 -phenylpropionate, phos- Aerosil 200 ) ; magnesium stearate; crospovidone (e.g. Kol
phate , picrate, pivalate , propionate , stearate, succinate , sul- lidon CL or Kollidon CL - F ) ; where the first dose of at least
 US 11,344,501 B2
19 20
one antimethanogenic statin is encapsulated by an enteric in further embodiments, lovastatin lactone) ; about 60 % by
polymer that dissolves at a pH of about 5.5 (e.g. weight microcrystalline cellulose ( e.g. Avicel PH102 ); about
EUDRAGIT L 30 D - 55 + PlasACRYL HTP20) ; and the sec- 6 % by weight copovidone (e.g. Kollidon VA64 Fine ) ; about
ond dose of at least one antimethanogenic statin is encap- 2 % by weight silicon dioxide ( e.g. Aerosil 200 ) ; about 1 %
sulated by an enteric polymer that dissolves a at pH of about 5 by weight magnesium stearate ; about 5 % by weight
7.0 (e.g. EUDRAGIT FS 30 D + PlasACRYL T20 and / or crospovidone (e.g. Kollidon CL or Kollidon CL - F ) ; and
EUDRAGIT® S 100 ) . about 15 % by weight an enteric polymer that dissolves at a
In various embodiments, the formulation comprises at pH of about 5.5 ( e.g. EUDRAGIT L 30 D -55 + PlasACRYL
least one microbead or mini-tablet. Each microbead or HTP20 ) or about 7.0 (e.g. EUDRAGIT FS 30 D + Pla
mini - tablet comprises about 5-20 % by weight of the anti- 10 SACRYL T20 and / or EUDRAGIT® S 100 ) .
methanogenic statin (which is , in some embodiments , lov- In some embodiments , the formulation comprises at least
astatin , and in further embodiments , lovastatin lactone ). For one microbead or mini- tablet with each microbead or mini
example, the antimethanogenic statin may be present at tablet comprising about 12.2 % by weight lovastatin lactone ;
about 5 % , about 6 % , about 7 % , about 8 % , about 9 % , about about 60.9 % by weight microcrystalline cellulose ( Avicel
10 % , about 11 % , about 12 % , about 13 % , about 14 % , about 15 PH102 ) ; about 6.1 % by weight copovidone (Kollidon VA64
15 % , about 16 % , about 17 % , about 18 % , about 19 % , or Fine) ; about 1.7 % by weight silicon dioxide (Aerosil 200) ;
about 20 % by weight. In some embodiments, each micro- about 0.9 % by weight magnesium stearate; about 5.2 % by
bead or mini-tablet may further comprise about 50-70 % by weight crospovidone (Kollidon CL - F ) ; and either about
weight microcrystalline cellulose (e.g. Avicel PH102 ) . For 13.0% by weight of EUDRAGIT L 30 D -55 + PlasACRYL
example, the microcrystalline cellulose may be present at 20 HTP20 coating ( which dissolves at a pH of about 5.5 ) or
about 50 % , about 51 % , about 52 % , about 53 % , about 54 % , 13 % by weight of EUDRAGIT FS 30 D + PlasACRYL T20
about 55 % , about 56 % , about 57 % , about 58 % , about 59 % , coating ( which dissolves at a pH of about 7.0) .
about 60 % , about 61 % , about 62 % , about 63 % , about 64 % , In various embodiments, the present formulation com
or about 65 % , or about 66 % , about 67 % , or about 68 % , or prise a mini -tablet enteric coating thickness, e.g.
about 69 % , or about 70% by weight. In some embodiments , 25 EUDRAGIT, e.g. EUDRAGIT L 30 D - 55 or EUDRAGIT
each microbead or mini - tablet may further comprise about FS 30 D , of greater than about 10 % , about 13 % , about 15 % ,
1-10 % by weight copovidone (e.g. Kollidon VA64 Fine) . or about 17 % , or about 20 % , or about 25 % .
For example, the copovidone may be present at about 1 % , In various embodiments, the formulation of the present
about 2 % , about 3 % , about 4 % , about 5 % , about 6 % , about invention may comprise at least one mini - tablet that releases
7 % , about 8 % , about 9 % , or about 10% by weight. In some 30 at a first pH ( e.g. pH of about 5.5 ) and at least one mini -tablet
embodiments, each microbead or mini - tablet may further that releases at a second pH (e.g. , pH of about 7.0 ) at a ratio
comprise about 0.1-3.0 % by weight silicon dioxide (e.g. of 1 : 2 . In such embodiments, the formulation may comprise
Aerosil 200 ) . For example , the silicon dioxide may be about 5-20 % by weight the antimethanogenic statin (which
present at about 0.1 % , about 0.2 % , about 0.3 % , about 0.4 % , is , in some embodiments, lovastatin , and in further embodi
about 0.5 % , about 0.6 % , about 0.7 % , about 0.8 % , about 35 ments , lovastatin lactone) . For example, the antimethano
0.9 % , about 1 % , about 1.1 % , about 1.2 % , about 1.3 % , about genic statin may be present at about 5 % , about 6 % , about
1.4 % , about 1.5 % , about 1.6 % , about 1.7 % , about 1.8 % , 7 % , about 8 % , about 9 % , about 10 % , about 11 % , about
about 1.9 % , about 2 % , about 2.1 % , about 2.2 % , about 2.3 % , 12 % , about 13 % , about 14 % , about 15 % , about 16 % , about
about 2.4 % , about 2.5 % , about 2.6 % , about 2.7 % , about 17 % , about 18 % , about 19 % , or about 20 % by weight of the
2.8 % , about 2.9 % , or about 3.0 % by weight. In some 40 entire formulation . In some embodiments, the formulation
embodiments, each microbead or mini - tablet may further may further comprise about 30-60 % by weight tablet diluent
comprise about 0.1-3.0 % by weight magnesium stearate ( for (e.g. , about 30% , about 31 % , about 32 % , about 33 % , about
example, about 0.1 % , about 0.2 % , about 0.3 % , about 0.4 % , 34 % , about 35 % , about 36 % , about 37 % , about 38 % , about
about 0.5 % , about 0.6 % , about 0.7 % , about 0.8 % , about 39 % , about 40 % , about 41 % , about 42 % , about 43 % , about
0.9 % , about 1 % , about 1.1 % , about 1.2 % , about 1.3 % , about 45 44 % , about 45 % , about 46 % , about 47 % , about 48 % , about
1.4 % , about 1.5 % , about 1.6 % , about 1.7 % , about 1.8 % , 49 % , about 50 % , about 51 % , about 52 % , about 53 % , about
about 1.9 % , about 2 % , about 2.1 % , about 2.2 % , about 2.3 % , 54 % , about 55 % , about 56 % , about 57 % , about 58 % , about
about 2.4 % , about 2.5 % , about 2.6 % , about 2.7 % , about 59 % , or about 60 % ) . In some embodiments, the formulation
2.8 % , about 2.9 % , or about 3.0 % ) . In some embodiments, may further comprise about 1-10 % by weight tablet binder
each microbead or mini - tablet may further comprise about 50 (e.g. , about 1 % , about 2 % , about 3 % , about 4 % , about 5 % ,
1-10 % by weight crospovidone ( e.g. Kollidon CL or Kolli- about 6 % , about 7 % , about 8 % , about 9 % , or about 10 % ) .
don CL - F ) . For example , the crospovidone may be present In some embodiments, the formulation may further com
at about 1 % , about 2 % , about 3 % , about 4 % , about 5 % , prise about 0.1-3.0 % by weight viscosity and dispersion
about 6 % , about 7 % , about 8 % , about 9 % , or about 10 % by agent ( e.g. , about 0.1 % , about 0.2 % , about 0.3 % , about
weight. In some embodiments, each microbead or mini- 55 0.4 % , about 0.5 % , about 0.6 % , about 0.7 % , about 0.8 % ,
tablet may further comprise about 10-20 % by weight an about 0.9 % , about 1 % , about 1.1 % , about 1.2 % , about 1.3 % ,
enteric polymer that dissolves at a pH of about 5.5 (e.g. about 1.4 % , about 1.5 % , about 1.6 % , about 1.7 % , about
EUDRAGIT L 30 D -55 + PlasACRYL HTP20 ) or about 7.0 1.8 % , about 1.9 % , about 2 % , about 2.1 % , about 2.2 % , about
( e.g. EUDRAGIT FS 30 D + PlasACRYL T20 and / or 2.3 % , about 2.4 % , about 2.5 % , about 2.6 % , about 2.7 % ,
EUDRAGIT® S 100 ) . For example, the enteric polymer 60 about 2.8 % , about 2.9 % , or about 3.0 % ) . In some embodi
may be about 10% , about 11 % , about 12 % , about 13 % , ments, the formulation may further comprise about 0.1-3.0 %
about 14 % , about 15 % , about 16 % , about 17 % , about 18 % , by weight lubricant, for example, to facilitate tableting ( e.g. ,
about 19 % , or about 20 % by weight. about 0.1 % , about 0.2 % , about 0.3 % , about 0.4 % , about
In some embodiments, the formulation comprises at least 0.5 % , about 0.6 % , about 0.7 % , about 0.8 % , about 0.9 % ,
one microbead or mini - tablet with each microbead or mini- 65 about 1 % , about 1.1 % , about 1.2 % , about 1.3 % , about 1.4 % ,
tablet comprising about 12 % by weight the antimethano- about 1.5 % , about 1.6 % , about 1.7 % , about 1.8 % , about
genic statin (which is , in some embodiments, lovastatin , and 1.9 % , about 2 % , about 2.1 % , about 2.2 % , about 2.3 % , about
 US 11,344,501 B2
21 22
2.4 % , about 2.5 % , about 2.6 % , about 2.7 % , about 2.8 % , releases at a second pH (e.g. , pH of about 7.0 ) at a ratio of
about 2.9 % , or about 3.0 % ) . In some embodiments, the 1 : 5 . In such embodiments, the formulation may comprise
formulation may further comprise about 1-10% by weight about 5-20 % by weight the antimethanogenic statin ( which
tablet disintegrant (e.g. , about 1 % , about 2 % , about 3 % , is , in some embodiments, lovastatin , and in further embodi
about 4 % , about 5 % , about 6 % , about 7 % , about 8 % , about 5 ments, lovastatin lactone) . For example, the antimethano
9 % , or about 10 % ) . In some embodiments , the formulation genic statin may be present at about 5 % , about 6 % , about
may further comprise about 0.5-10 % by weight an enteric 7 % , about 8 % , about 9 % , about 10 % , about 11 % , about
polymer that dissolves at a pH of about 5.5 (e.g. 12 % , about 13 % , about 14 % , about 15 % , about 16 % , about
EUDRAGIT L 30 D - 55 + PlasACRYL HTP20 ) . For example, 17 % , about 18 % , about 19 % , or about 20 % by weight of the
the enteric polymer that dissolves at a pH of about 5.5 may 10 entire formulation . In some embodiments, the formulation
be present in the formulation at about 0.5 % , about 0.6 % , may further comprise about 30-60 % by weight tablet diluent
about 0.7 % , about 0.8 % , about 0.9 % , about 1 % , about 2 % , (e.g. , about 30% , about 31 % , about 32 % , about 33 % , about
about 3 % , about 4 % , about 5 % , about 6 % , about 7 % , about 34 % , about 35 % , about 36 % , about 37 % , about 38 % , about
8 % , about 9 % , or about 10 % by weight. In some embodi- 39 % , about 40 % , about 41 % , about 42 % , about 43 % , about
ments , the formulation may further comprise about 1-15 % 15 44 % , about 45 % , about 46 % , about 47 % , about 48 % , about
by weight an enteric polymer that dissolves at a pH of about 49 % , about 50 % , about 51 % , about 52 % , about 53 % , about
7.0 . (e.g. EUDRAGIT FS 30 D + PlasACRYL T20 and / or 54 % , about 55 % , about 56 % , about 57 % , about 58 % , about
EUDRAGIT® S 100 ) . For example , the enteric polymer that 59 % , or about 60 % ) . In some embodiments, the formulation
dissolves at a pH of about 7.0 may be present in the may further comprise about 1-10 % by weight tablet binder
formulation at about 1 % , about 2 % , about 3 % , about 4 % , 20 (e.g. , about 1 % , about 2 % , about 3 % , about 4 % , about 5 % ,
about 5 % , about 6 % , about 7 % , about 8 % , about 9 % , about about 6 % , about 7 % , about 8 % , about 9 % , or about 10 % ) .
10 % , about 11 % , about 12 % , about 13 % , about 14 % , or In some embodiments, the formulation may further com
about 15 % by weight. In such embodiments, the antimetha- prise about 0.1-3.0 % by weight viscosity and dispersion
nogenic statin (which is , in some embodiments, lovastatin , agent ( e.g. , about 0.1 % , about 0.2 % , about 0.3 % , about
and in further embodiments, lovastatin lactone) may be 25 0.4 % , about 0.5 % , about 0.6 % , about 0.7 % , about 0.8 % ,
released in two doses . The first dose of antimethanogenic about 0.9 % , about 1 % , about 1.1 % , about 1.2 % , about 1.3 % ,
statin is encapsulated by the enteric polymer that dissolves about 1.4 % , about 1.5 % , about 1.6 % , about 1.7 % , about
at a pH of about 5.5 ; and the second dose of antimethano- 1.8 % , about 1.9 % , about 2 % , about 2.1 % , about 2.2 % , about
genic statin is encapsulated by the enteric polymer that 2.3 % , about 2.4 % , about 2.5 % , about 2.6 % , about 2.7 % ,
dissolves a at pH of about 7.0 . 30 about 2.8 % , about 2.9 % , or about 3.0 % ) . In some embodi
For example, the formulation may comprise at least one ments, the formulation may further comprise about 0.1-3.0 %
mini - tablet that releases at a first pH (e.g. pH of about 5.5 ) by weight lubricant, for example, to facilitate tableting ( e.g. ,
and at least one mini - tablet that releases a second pH (e.g. , about 0.1 % , about 0.2 % , about 0.3 % , about 0.4 % , about
pH of about 7.0 ) at a ratio of 1 : 2 . The formulation may 0.5 % , about 0.6 % , about 0.7 % , about 0.8 % , about 0.9 % ,
comprise about 9 % by weight the antimethanogenic statin 35 about 1 % , about 1.1 % , about 1.2 % , about 1.3 % , about 1.4 % ,
(which is , in some embodiments, lovastatin , and in further about 1.5 % , about 1.6 % , about 1.7 % , about 1.8 % , about
embodiments, lovastatin lactone ) ; about 42 % by weight 1.9 % , about 2 % , about 2.1 % , about 2.2 % , about 2.3 % , about
microcrystalline cellulose ( e.g. Avicel PH102 ) ; about 4 % by 2.4 % , about 2.5 % , about 2.6 % , about 2.7 % , about 2.8 % ,
weight copovidone (e.g. Kollidon VA64 Fine) ; about 1 % by about 2.9 % , or about 3.0 % ) . In some embodiments, the
weight silicon dioxide (e.g. Aerosil 200) ; about 0.5 % by 40 formulation may further comprise about 1-10% by weight
weight magnesium stearate ; about 4 % by weight crospovi- tablet disintegrant (e.g. , about 1 % , about 2 % , about 3 % ,
done ( e.g. Kollidon CL or Kollidon CL - F ) ; about 3 % by about 4 % , about 5 % , about 6 % , about 7 % , about 8 % , about
weight an enteric polymer that dissolves at a pH of about 5.5 9 % , or about 10 % ) . In some embodiments, the formulation
( e.g. EUDRAGIT L 30 D - 55 + PlasACRYL HTP20) ; and may further comprise about 0.5-10 % by weight an enteric
about 6 % by weight an enteric polymer that dissolves at a 45 polymer that dissolves at a pH of about 5.5 (e.g.
pH of about 7.0 ( e.g. EUDRAGIT FS 30 D + PlasACRYL EUDRAGIT L 30 D -55 + PlasACRYL HTP20 . For example,
T20 and / or EUDRAGIT® S 100) . the enteric polymer that dissolves at a pH of about 5.5 may
In another example, the formulation may comprise at least be present in the formulation at about 0.5 % , about 0.6 % ,
one mini - tablet that releases at a first pH (e.g. pH of about about 0.7 % , about 0.8 % , about 0.9 % , about 1 % , about 2 % ,
5.5 ) and at least one mini -tablet that releases at aa second pH 50 about 3 % , about 4 % , about 5 % , about 6 % , about 7 % , about
( e.g. , pH of about 7.0) at a ratio of 1 : 2 . The formulation may 8 % , about 9 % , or about 10% by weight. In some embodi
comprise about 8.5 % by weight the antimethanogenic statin ments , the formulation may further comprise about 1-15 %
( which is , in some embodiments , lovastatin , and in further by weight an enteric polymer that dissolves at a pH of about
embodiments, lovastatin lactone) ; about 42.4 % by weight 7.0 (e.g. EUDRAGIT FS 30 D + PlasACRYL T20 and / or
microcrystalline cellulose (e.g. Avicel PH102 ) ; about 4.2 % 55 EUDRAGIT® S 100. For example , the enteric polymer that
by weight copovidone ( e.g. Kollidon VA64 Fine) ; about dissolves at a pH of about 7.0 may be present in the
1.2 % by weight silicon dioxide (e.g. Aerosil 200 ) ; about formulation at about 1 % , about 2 % , about 3 % , about 4 % ,
0.6 % by weight magnesium stearate; about 3.6 % by weight about 5 % , about 6 % , about 7 % , about 8 % , about 9 % , about
crospovidone (e.g. Kollidon CL or Kollidon CL - F ) ; about 10% , about 11 % , about 12 % , about 13 % , about 14 % , or
3 % by weight an enteric polymer that dissolves at a pH of 60 about 15 % by weight. In such embodiments , the antimetha
about 5.5 (e.g. EUDRAGIT L 30 D -55 + PlasACRYL nogenic statin ( which is , in some embodiments, lovastatin ,
HTP20 ) ; and about 6.1 % by weight an enteric polymer that and in further embodiments, lovastatin lactone) may be
dissolves at a pH of about 7.0 (e.g. EUDRAGIT FS 30 released in two doses . The first dose of antimethanogenic
D + Plas ACRYL T20 and / or EUDRAGIT® S 100) . statin is encapsulated by the enteric polymer that dissolves
In another embodiment, the formulation of the present 65 at a pH of about 5.5 ; and the second dose of antimethano
invention may at least one mini - tablet that releases at a first genic statin is encapsulated by the enteric polymer that
pH (e.g. pH of about 5.5 ) and at least one mini -tablet that dissolves a at pH of about 7.0 .
 US 11,344,501 B2
23 24
For example, the formulation may comprise at least one Administration and Dosage
mini - tablet that releases at a first pH (e.g. pH of about 5.5 ) It will be appreciated that the actual dose of the antime
and at least one mini -tablets that release at a second pH ( e.g. , thanogenic statin to be administered according to the present
invention will vary according to the particular compound ,
pH of about 7.0 ) at a ratio of 1 : 5 . The formulation may
comprise about 10% by weight the antimethanogenic statins the
Manyparticular
factors dosage
that mayformmodify
, and the
the action
mode ofof administration
the antimetha.
( which is , in some embodiments, lovastatin , and in further nogenic statin ( e.g. , body weight, gender, diet , time of
embodiments, lovastatin lactone ) ; about 50 % by weight administration , route of administration, rate of excretion,
microcrystalline cellulose ( e.g. Avicel PH102 ) ; about 5 % by condition of the subject, drug combinations, genetic dispo
weight copovidone ( e.g. Kollidon VA64 Fine); about 1 % by 10 sition and reaction sensitivities ) can be taken into account by
weight silicon dioxide (e.g. Aerosil 200) ; about 0.5 % by those skilled in the art . Administration can be carried out
weight magnesium stearate ; about 4 % by weight crospovi- continuously or in one or more discrete doses within the
done ( e.g. Kollidon CL or Kollidon CL - F ) ; about 2 % by maximum tolerated dose . Optimal administration rates for a
weight an enteric polymer that dissolves at a pH of about 5.5 given set of conditions can be ascertained by those skilled in
( e.g. EUDRAGIT L 30 D - 55 + PlasACRYL HTP20) ; and 15
the art using conventional dosage administration tests .
about 9 % by weight an enteric polymer that dissolves at a Individual doses of the antimethanogenic statin can be
pH of about 7.0 . (e.g. EUDRAGIT FS 30 D + PlasACRYL administered in unit dosage forms ( e.g., tablets or capsules )
T20 and / or EUDRAGIT® S 100) . containing, for example, from about 0.01 mg to about 100
mg , from about 0.1 mg to about 100 mg , from about 0.1 mg
In another example, the formulation may comprise at least to about 90 mg , from about 0.1 mg to about 80 mg , from
one mini -tablet that releases at a first pH (e.g. pH of about 20 about 0.1 mg to about70 mg, from about 0.1 mg to about 60
5.5 ) and at least one mini -tablet that releases at a second pH mg , from about 0.1 mg to about 50 mg , from about 0.1 mg
( e.g. , pH of about7.0) at a ratio of 1 : 5 . The formulation may to about 40 mg active ingredient, from about 0.1 mg to about
comprise about 10 % by weight the antimethanogenic statin 30 mg , from about 0.1 mg to about 20 mg , from about 0.1
( which is , in some embodiments, lovastatin , and in further mg to about 10 mg , from about 0.1 mg to about 5 mg , from
embodiments, lovastatin lactone ) ; about 50 % by weight 25 about 0.1 mg to about 3 mg , from about 0.1 mg to about 1
microcrystalline cellulose ( e.g. Avicel PH102 ) ; about 5 % by mg per unit dosage form , or from about 5 mg to about 80 mg
weight copovidone (e.g. Kollidon VA64 Fine ) ; about 1.4 % per unit dosage form . For example , a unit dosage form can
by weight silicon dioxide (e.g. Aerosil 200) ; about 0.7 % by be about 0.01 mg , about 0.02 mg , about 0.03 mg , about 0.04
weight magnesium stearate; about 4.3 % by weight crospovi mg , about 0.05 mg , about 0.06 mg , about 0.07 mg , about
done ( e.g. Kollidon CL or Kollidon CL - F ) ; about 1.8 % by 30 0.08 mg , about 0.09 mg , about 0.1 mg , about 0.2 mg, about
0.3 mg , about 0.4 mg , about 0.5 mg , about 0.6 mg , about 0.7
weight an enteric polymer that dissolves at a pH of about 5.5 mg , about 0.8 mg , about 0.9 mg , about 1 mg , about 2 mg ,
( e.g. EUDRAGIT L 30 D - 55 + PlasACRYL HTP20) ; and about 3 mg , about 4 mg , about 5 mg , about 6 mg , about 7
about 8.9 % by weight an enteric polymer that dissolves at a mg , about 8 mg , about 9 mg , about 10 mg , about 11 mg ,
pH of about 7.0 . (e.g. EUDRAGIT FS 30 D + PlasACRYL 35 about 12 mg , about 13 mg , about 14 mg , about 15 mg , about
T20 and / or EUDRAGIT® S 100) . 16 mg , about 17 mg , about 18 mg , about 19 mg , about 20
The therapeutic agents or their pharmaceutically accept mg , about 21 mg , about 22 mg , about 23 mg , about 24 mg ,
able salts which are used in accordance with the present about 25 mg , about 26 mg , about 27 mg , about 28 mg , about
invention may exhibit stereoisomerism by virtue of the 29 mg , about 30 mg , about 31 mg , about 32 mg , about 33
presence ofone or more asymmetric or chiral centers in the 40 mg , about 34 mg , about 35 mg , about 36 mg , about 37 mg ,
compounds. The present invention contemplates the various about 38 mg , about 39 mg , about 40 mg , about 41 mg , about
stereoisomers and mixtures thereof. Desired enantiomers 42 mg , about 43 mg , about 44 mg , about 45 mg , about 46
can be obtained by chiral synthesis from commercially mg , about 47 mg , about 48 mg , about 49 mg , about 50 mg ,
available chiral starting materials by methods well known in about 51 mg , about 52 mg , about 53 mg , about 54 mg , about
the art, or may be obtained from mixtures of the enantiomers 45 55 mg , about 56 mg , about 57 mg , about 58 mg , about 59
by resolution using known techniques. mg , about 60 mg , about 61 mg , about 62 mg , about 63 mg ,
Solvate as used herein refers to a pharmaceutically about 64 mg , about 65 mg , about 66 mg , about 67 mg , about
acceptable solvate form of a specified therapeutic agent that 68 mg , about 69 mg , about 70 mg , about 71 mg , about 72
retains the biological effectiveness of such agent. Examples mg , about 73 mg , about 74 mg , about 75 mg , about 76 mg ,
of solvates include therapeutic agents of the invention in 50 about 77 mg , about 78 mg , about 79 mg , about 80 mg , about
combination with , for example , water, isopropanol, ethanol, 81 mg, about 82 mg , about 83 mg , about 84 mg , about 85
methanol, DMSO , ethyl acetate , acetic acid , or etha- mg , about 86 mg , about 87 mg , about 88 mg , about 89 mg ,
nolamine . about 90 mg , about 91 mg , about 92 mg , about 93 mg , about
Prodrug, as used herein refers to a therapeutic agent that 94 mg , about 95 mg , about 96 mg , about 97 mg , about 98
is converted under physiological conditions or by solvolysis 55 mg , about 99 mg , or about 100 mg , inclusive of all values
or metabolically (e.g. , in vivo ) to a specified agent that is and ranges therebetween . In an embodiment, individual dose
pharmaceutically active . of the antimethanogenic statin is administered in an unit
Active metabolite , as used herein refers to a pharmaco- dosage form containing 21 mg of the active ingredient. In
logically active product produced through metabolism in the another embodiment, individual dose of the antimethano
body of a specified therapeutic agent. 60 genic statin is administered in an unit dosage form contain
Co -crystal as used herein refers to a physical association ing 42 mg of the active ingredient.
of two or more molecules which owe their stability through In one embodiment, the antimethanogenic statin is admin
non -covalent interaction . One or more components of this istered at an amount of from about 0.01 mg to about 100 mg
molecular complex provide a stable framework in the crys- daily, an amount of from about 0.1 mg to about 100 mg
talline lattice . In certain instances , the guest molecules are 65 daily, from about 0.1 mg to about 95 mg daily, from about
incorporated in the crystalline lattice as anhydrates or sol- 0.1 mg to about 90 mg daily, from about 0.1 mg to about 85
vates . mg daily, from about 0.1 mg to about 80 mg daily, from
 US 11,344,501 B2
25 26
about 0.1 mg to about 75 mg daily, from about 0.1 mg to range of 0.01 mg/kg to about 6 mg/kg of body weight, in a
about 70 mg daily, from about 0.1 mg to about 65 mg daily, range of about 0.05 mg /kg to about 5 mg/kg of body weight,
from about 0.1 mg to about 60 mg daily, from about 0.1 mg in a range of about 0.05 mg /kg to about 4 mg/kg of body
to about 55 mg daily, from about 0.1 mg to about 50 mg weight, in a range of about 0.05 mg/kg to about 3 mg/kg of
daily, from about 0.1 mg to about 45 mg daily , from about 5 body weight, in a range of about 0.05 mg /kg to about 2
0.1 mg to about 40 mg daily, from about 0.1 mg to about 35 mg/kg of body weight, in a range of about 0.05 mg/kg to
mg daily, from about 0.1 mg to about 30 mg daily , from about 1.5 mg /kg of body weight, or in a range of about 0.05
about 0.1 mg to about 25 mg daily, from about 0.1 mg to mg/kg to about 1 mg/kg of body weight.
about 20 mg daily, from about 0.1 mg to about 15 mg daily, In accordance with certain embodiments of the invention,
from about 0.1 mg to about 10 mg daily, from about 0.1 mg 10 the antimethanogenic statin may be administered , for
to about 5 mg daily, from about 0.1 mg to about 3 mg daily, example, more than once daily, about once per day, about
from about 0.1 mg to about 1 mg daily, or from about 5 mg every other day, about every third day, about once a week ,
to about 80 mg daily. In various embodiments, the antime- about once every two weeks , about once every month , about
thanogenic statin is administered at a daily dose of about once every two months, about once every three months,
0.01 mg , about 0.02 mg , about 0.03 mg , about 0.04 mg , 15 about once every six months, or about once every year.
about 0.05 mg , about 0.06 mg , about 0.07 mg , about 0.08 In various embodiments, the antimethanogenic statin may
mg , about 0.09 mg , about 0.1 mg , about 0.2 mg , about 0.3 be administered in a patient that is fasting. In various
mg , about 0.4 mg , about 0.5 mg , about 0.6 mg , about 0.7 mg , embodiments, the antimethanogenic statin may be adminis
about 0.8 mg , about 0.9 mg , about 1 mg , about 2 mg , about tered in a patient with a meal . In various embodiments, the
3 mg , about 4 mg , about 5 mg , about 6 mg , about 7 mg , 20 antimethanogenic statin may be administered in a patient
about 8 mg , about 9 mg , about 10 mg , about 11 mg , about that is postprandial. In various embodiments, patient is on an
12 mg , about 13 mg , about 14 mg , about 15 mg , about 16 elemental diet. A comestible total enteral nutrition (TEN)
mg , about 17 mg , about 18 mg , about 19 mg , about 20 mg , formulation , which is also called an “ elemental diet ” are
about 21 mg , about 22 mg , about 23 mg , about 24 mg , about commercially available , for example, VIVONEX T.E.N.
25 mg , about 26 mg , about 27 mg , about 28 mg , about 29 25 (Nestle) and its variants, or the like. A useful total enteral
mg , about 30 mg , about 31 mg , about 32 mg , about 33 mg , nutrition formulation satisfies all the subject's nutritional
about 34 mg , about 35 mg , about 36 mg , about 37 mg , about requirements, containing free amino acids , carbohydrates,
38 mg , about 39 mg , about 40 mg , about 41 mg , about 42 lipids , and all essential vitamins and minerals, but is in a
mg , about 43 mg , about 44 mg , about 45 mg , about 46 mg , form that is readily absorbable in the upper gastrointestinal
about 47 mg , about 48 mg , about 49 mg , about 50 mg , about 30 tract, thus depriving or " starving” the methanogen syn
51 mg , about 52 mg , about 53 mg , about 54 mg , about 55 trophic microorganism of nutrients of at least some of the
mg , about 56 mg , about 57 mg , about 58 mg , about 59 mg , nutrients they use for proliferating. Thus, methanogen syn
about 60 mg , about 61 mg , about 62 mg , about 63 mg , about trophic microorganism growth is inhibited.
64 mg , about 65 mg , about 66 mg , about 67 mg , about 68 Additional Agents and Combination Therapy or Co- Formu
mg , about 69 mg , about 70 mg , about 71 mg , about 72 mg , 35 lation / Patient Selection
about 73 mg , about 74 mg , about 75 mg , about 76 mg , about Administration of the present formulations may be com
77 mg , about 78 mg , about 79 mg , about 80 mg , about 81 bined with additional therapeutic agents. Co - administration
mg , about 82 mg , about 83 mg , about 84 mg , about 85 mg , of the additional therapeutic agent and the present formu
about 86 mg , about 87 mg , about 88 mg , about 89 mg , about lations may be simultaneous or sequential. Further the
90 mg , about 91 mg , about 92 mg , about 93 mg , about 94 40 present formulations may comprise an additional therapeutic
mg , about 95 mg , about 96 mg , about 97 mg , about 98 mg , agent ( e.g. via co - formulation ).
about 99 mg , or about 100 mg , inclusive of all values and In some embodiments, the modified - release formulations
ranges therebetween. In an embodiment, the antimethano- of the present invention are administered in combination
genic statin is administered at an amount of 21 mg daily. In with an additional therapeutic agent. In an embodiment, the
another embodiment, the antimethanogenic statin is admin- 45 additional therapeutic agent and the antimethanogenic statin
istered at an amount of 42 mg daily. are combined into a single modified - release formulation . In
In some embodiments , a suitable dosage of the antime- some embodiments , the methods of treatment and / or pre
thanogenic statin (e.g. , a statin ) is in a range of about 0.01 vention comprise administering the modified - release formu
mg/kg to about 10 mg/kg of body weight of the subject, for lations of the present invention to a subject that is under
example , about 0.01 mg/kg, about 0.02 mg /kg, about 0.03 50 going treatment with an additional therapeutic agent.
mg /kg, about 0.04 mg/kg, about 0.05 mg /kg, about 0.06 In one embodiment, the additional agent and the antime
mg /kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 thanogenic statin are administered to a subject simultane
mg/kg, about 0.1 mg /kg, about 0.2 mg/kg, about 0.3 mg /kg, ously . The term “ simultaneously ” as used herein , means that
about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg /kg, about the additional agent and the antimethanogenic statin are
0.7 mg /kg, about 0.8 mg/kg, about 0.9 mg /kg, about 1 55 administered with a time separation of no more than about
mg /kg, about 1.1 mg /kg, about 1.2 mg /kg, about 1.3 mg /kg, 60 minutes, such as no more than about 30 minutes, no more
about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg /kg, about than about 20 minutes, no more than about 10 minutes, no
1.7 mg/kg, about 1.8 mg /kg, 1.9 mg /kg, about 2 mg /kg, more than about 5 minutes, or no more than about 1 minute .
about 3 mg /kg, about 4 mg/kg, about 5 mg/kg, about 6 Administration of the additional agent and the antimetha
mg/kg , about 7 mg/kg, about 8 mg/kg, about 9 mg /kg, about 60 nogenic statin can be by simultaneous administration of a
10 mg/kg body weight, inclusive of all values and ranges single formulation (e.g. , a formulation comprising the addi
therebetween . In other embodiments, a suitable dosage of tional agent and the antimethanogenic statin ) or of separate
the antimethanogenic statin is in a range of about 0.01 mg/kg formulations (e.g. , a first formulation including the addi
to about 10 mg/kg of body weight, in a range of about 0.01 tional agent and a second formulation including the antime
mg/kg to about 9 mg/kg of body weight, in a range of about 65 thanogenic statin ).
0.01 mg/kg to about 8 mg/kg of body weight, in a range of Co - administration does not require the additional thera
about 0.01 mg/kg to about 7 mg/kg of body weight, in a peutic agents to be administered simultaneously, if the
 US 11,344,501 B2
27 28
timing of their administration is such that the pharmacologi- juice constituents such as naringin and hesperidin , orange
cal activities of the additional agent and the antimethano- juice and orange juice constituents, apple juice and apple
genic statin overlap in time , thereby exerting a combined juice constituents, and green tea and green tea extracts such
therapeutic effect. For example, the additional agent and the as epicatechin gallate ( ECG ) , epigallocatechin gallate
antimethanogenic statin can be administered sequentially. 5 (EGCG) . In an embodiment, the OATP inhibitor is released
The term “ sequentially ” as used herein means that the in the intestine prior to release of the antimethanogenic
additional agent and the antimethanogenic statin are admin- statin .
istered with a time separation of more than about 60 min- In one embodiment, the additional therapeutic agent is a
utes . For example, the time between the sequential admin- prokinetic agent that facilitates movement of a mass through
istration of the additional agent and the antimethanogenic 10 the intestinal tract. Illustrative prokinetic agents include , but
statin can be more than about 60 minutes, more than about are not limited to , prucalopride (e.g. RESOLOR) or a
2 hours, more than about 5 hours, more than about 10 hours, macrolide antibiotic such as erythromycin . In another
more than about 1 day, more than about 2 days, more than embodiment, the additional therapeutic agent is a natural
about 3 days, or more than about 1 week apart. The optimal product such as peppermint oil , which alleviates abdominal
administration times will depend on the rates of metabolism , 15 pain .
excretion, and / or the pharmacodynamic activity of the addi- The present invention also contemplates the use of addi
tional agent and the antimethanogenic statin being admin- tional therapeutic agent that are useful for treating consti
istered . Either the additional agent or the antimethanogenic pation such as , for example, laxatives, guanylate cyclase C
statin may be administered first. agonist ( e.g. , linaclotide ) , a serotonin agonist (e.g. , prucalor
)

In a further embodiment, the additional therapeutic agent 20 pride, tegaserod) , a chloride channel agonist ( e.g. , lubipro
and the antimethanogenic statin are administered to a subject stone ), and combinations thereof.
simultaneously but the release of additional therapeutic In some embodiments, the additional therapeutic agent is
agent and the antimethanogenic statin from their respective an agent useful for treating IBS ( including IBS - C ) . In some
dosage forms ( or single unit dosage form if co -formulated ) embodiments, the additional therapeutic agent is a selective
in the GI tract occurs sequentially. 25 chloride channel activator, including, for example, mol
Co - administration also does not require the additional ecules derived from prostaglandins such as lubiprostone
therapeutic agents to be administered to the subject by the (e.g. AMITIZA) and those compounds described in U.S. Pat.
same route of administration . Rather, each therapeutic agent Nos . 5,284,858 , 6,414,016 and 6,583,174 , the contents of
can be administered by any appropriate route, for example, which are hereby incorporated by reference in their entire
parenterally or non -parenterally. 30 ties . In some embodiments, the additional therapeutic agent
The formulations of the present invention may comprise is an agent, including a peptide agent, that increases the
a pharmaceutically acceptable excipient. In some embodi- secretion of chloride and / or water in the intestines and /or
ments, the formulation may further include agent which soften stools and / or stimulate bowel movements , such as , for
prevents or reduces lactone ring - opening , such as an esterase example , linaclotide ( e.g. LINZESS ) and those compounds
inhibitor (e.g. grapefruit juice or components naringenin , 35 described in U.S. Pat . No. 7,304,036 , the contents of which
kaempferol) and / or a paraoxonase inhibitor (e.g. PON1 or are hereby incorporated by reference in their entirety. In
PON3 inhibitor ). In some embodiments, the esterase inhibi- some embodiments, the additional therapeutic agent is an
tor and/ or a paraoxonase inhibitor is one or more of amio- agent that relaxes the colon and / or slows the movement of
darone , anastrozole, azithromyzcin , cannabinoids, cimeti- waste through the lower bowel . In some embodiments the
dine , clarithromycin, clotrimazolem , cyclosporine , danazol, 40 additional therapeutic agent is a 5 - HT2 antagonist, includ
delavirdine , dexamethasone, diethyldithiocarbamate, dilti- ing , but not limited to , alosetron ( e.g. LOTRONEX ).
azem , dirithyromycin , disulfiram , entacapone , erythromy- In some embodiments , the additional therapeutic agent is
cin , ethinyl estradiol, fluconazole, fluoxetine , fluvoaxamine, a small molecule that acts as a peripherally selective K -opi
gestodene, grapefruit juice , indinavir, isoniazid , ketocona- oid agonist , such as , for example , EMD - 61753 ((N -methyl
zole , metronidazole, mibefradil, miconazole , nefazodone, 45 N -[ (15 )-1 -phenyl- 2- (3 S )-3 -hydroxypyrrolidin - 1 -yl)
nelfinavir , nevirapine, norfloxacin , norfluoxetine, omepra- ethyl ) -2,2 -diphenyl -acetamide hydrochloride,
zole , oxiconazole , paroxetine, propoxyphene, quinidine , ASMADOLINE) and those compounds described in U.S.
quinine, quinupristine and dalfopristin , ranitidine, ritonavir, Pat. No. 6,344,566 , the contents of which are hereby incor
saquinavir, sertindole, sertraline, troglitazone, troleandomy- porated by reference in their entirety . In some embodiments,
cin , valproic acid and /or a lactam agent selected from 50 the additional therapeutic agent is a cholecystokinin antago
oxindole , isatin , d - valerolactam , ? -caprolactam , 2 -hydroxy- nist , e.g. one selective for the CCK , subtype and /or inhibits
quinoline , and 3,4 - dihydro - 2 (1H ) -quinoline and N -bromo- gastrointestinal motility and reduces gastric secretions, such
E -caprolactam . as , for example, Dexloxiglumide ((4R ) -4 - [(3,4 -dichloroben
In various embodiments, the modified -release formula- zoyl)amino ) -5-(3 -methoxypropylpentylamino )-5 -oxopen
tion of the present invention is administered in combination 55 tanoic acid) and those compounds described in U.S. Pat . No.
with an inhibitor of the organic anion transporting polypep- 5,602,179 , the contents of which are hereby incorporated by
tide (OATP ) transporter. In an embodiment, the OATP reference in their entirety. In some embodiments, the addi
inhibitor and the antimethanogenic statin are combined into tional therapeutic agent is tapentadol ( 1R 2R )-3-( 3 -dimeth
a single modified - release formulation . Without wishing to be ylamino - 1 - ethyl -2 -methyl -propyl )-phenol ), as described in
bound by theory, it is believed that inclusion of the OATP 60 US Patent Publication No. 2013/0116334 , the contents of
inhibitor minimizes absorption of the antimethanogenic sta- which are hereby incorporated by reference in their entirety
tin from the intestine and /or reduces the enterohepatic In some embodiments, the additional therapeutic agent is
recirculation of the antimethanogenic statin , thereby maxi- a laxative, including but not limited to osmotic laxatives
mizing retention of the antimethanogenic statin in the intes- ( such as , for example, magnesium carbonate, magnesium
tine and minimizing any potential systemic side effects of 65 hydroxide ( e.g. Milk of Magnesia ), magnesium oxide , mag
the antimethanogenic statin . Illustrative OATP inhibitors nesium peroxide, magnesium sulfate, lactulose , lactitol ,
include , but are not limited to , grapefruit juice or grapefruit sodium sulfate, pentaerythritol, macrogol, mannitol, sodium
 US 11,344,501 B2
29 30
phosphate, sorbitol, magnesium citrate, sodium tartrate , ers , phenlermine, sibutramine, lorcaserin , cetilistat , rimona
laminarid , and polyethylene glycol ( e.g. , macrogol-contain- bant, taranabant, topiramate, gabapentin, valproate,
ing products, such as MOVICOL and polyethylene glycol vigabatrin , bupropion, tiagabine , sertraline, fluoxetine, tra
3350 , or SOFTLAX , MIRALAX , DULCOLAX BAL- zodone, zonisamide , methylphenidate, varenicline, naltrex
ANCE , CLEARLAX , OSMOLAX OR GLYCOLAX , 5 one, diethylpropion , phendimetrazine, repaglinide, nateglin
GOLYTELY, GAVILYTE C , NULYTELY, GLYCOLAX , ide , glimepiride, pioglitazone , rosiglilazone, and sitagliptin .
FORTRANS, TRILYTE , COLYTE , HALFLYTELY, SOFT- In an embodiment, the additional therapeutic agent is an
LAX , LAX - A -DAY, CLEARLAX AND MOVIPREP ) . In agent for treating prediabetes, diabetes , type II diabetes ,
some embodiments, the additional therapeutic agent is a insulin resistance , glucose intolerance , or hyperglycemia .
laxative, including but not limited to stimulant laxatives 10 Examples of drugs include , but are not limited to , alpha
( such as , for example, SENOKOT ) . Also provided are glucosidase inhibitors, amylin analogs, dipeptidyl pepti
contact laxatives ( e.g. oxyphenisatine , bisacodyl, dantron , dase - 4 inhibitors , GLP1 agonists , meglitinides, sulfony
phenolphthalein , castor oil , senna glycosides , cascara , lureas , biguanides , thiazolidinediones ( TZD ) , and insulin .
sodium picosulfate, and bisoxatin ) and bulk - forming laxa- Additional examples of such agents include bromocriptine
tives ( e.g. ispaghula, ethulose, sterculia, linseed, methylcel- 15 and Welchol. Examples of alpha - glucosidase inhibitors
lulose , triticum , and polycarbophil calcium) . In some include but are not limited to acarbose and miglitol . An
embodiments , the additional therapeutic agent is an enema,
? example of an amylin analog is pramlintide. Examples of
such as , for example, sodium laurilsulfate, sodium phos- dipeptidyl peptidase - 4 inhibitors include but are not limited
phate, bisacodyl, dantron , glycerol, oil , and sorbitol. Periph- to saxagliptin , sitagliptin , vildagliptin , linagliptin, and alo
eral opioid antagonists such as , for example, alvimopan and 20 gliptin . Examples of GLP1 agonist include but are not
methylnaltrexone, as well as prostaglandins such as , for limited to liraglutide, exenatide, exenatide extended release.
example , lubiprostone are also additional therapeutic agents Examples of meglitinides include but are not limited to
in some embodiments. Also , linaclotide , prucalopride, and nateglinide, and repaglinide. Examples of sulfonylureas
tegaserod may be additional therapeutics. include but are not limited to chlorpropamide, glimepiride,
In some embodiments, the additional therapeutic agent is 25 glipizide, glyburide, tolazamide, and tolbutamide. Examples
2

an agent used for long -term pain and cramping, including of biguanides include but are not limited to metformin ,
but not limited to anticholinergics ( antispasmodics ), such as , Riomet , Glucophage , Glucophage XR , Glumetza . Examples
for example , dicyclomine (BENTYL ) and or antidepres- of thiazolidinedione include but are not limited to rosigli
sants , including, for example , desipramine ( such as , for tazone and pioglitazone . Examples of insulin include but are
example, NORPRAMIN ), imipramine ( TOFRANIL ) or nor- 30 not limited to Aspart, Detemir, Glargine, Glulisine , and
triptyline ( PAMELOR ), which are optionally administered Lispro . Examples of combination drugs include but are not
at low doses. In low doses, they can help with pain caused limited to glipizide/metformin , glyburide/metformin, piogli
by IBS . tazone/glimepiride, pioglitazone/metformin , repaglinide/
In some embodiments , the additional therapeutic agent is metformin , rosiglitazone/ glimepiride, rosiglitazone/met
fiber supplement, such as , for example, psyllium 35 formin , saxagliptin /metformin , sitagliptin /simvastatin ,
(METAMUCIL ) or methylcellulose ( CITRUCEL ). sitagliptin /metformin , linagliptin /metformin , alogliptin /met
In some embodiments , the additional therapeutic agent is formin , and alogliptin /pioglitazone.
an agent useful for treating obesity. Illustrative agents In another embodiment, the additional therapeutic agent is
include , but are not limited to , orlistat, loracaserin , phenter- a probiotic . In some embodiments , enteric dietary formula
mine - topiramate, sibutramine, rimonabant, exenatide, pram- 40 tions containing low residual material, such as pre -digested
lintide, phentermine, benzphetamine, diethylpropion , or basic amino acid formulations and other methods and
phendimetrazine, bupropion, and metformin . In various products as described in U.S. Pat . No. 8,110,177 ( the con
embodiments, the additional agent is an agent that that tents of which are incorporated herein by reference) may be
interfere with the body's ability to absorb specific nutrients employed . In a further embodiment, such low residual
in food , such as orlistat, glucomannan , and guar gum . 45 enteric dietary formulations may be formulated in low
Agents that suppress appetite are also among the additional carbohydrate and low fat forms either with or without
agents, e.g. catecholamines and their derivatives ( such as immediate or sustained release statins or red yeast rice
phentermine and other amphetamine-based drugs ), various which may be particularly useful for weight loss and dia
anti- depressants and mood stabilizers ( e.g. bupropion and betes. In various embodiments, the probiotic may comprise
topiramate ), anorectics (e.g. dexedrine, digoxin ). Agents that 50 the following illustrative cells : E. coli Nissle 1917 , a lac
increase the body's metabolism are also among the addi- tobacillus ( e.g. acidophilus, Lactobacillus brevis, L. bulga
tional agents. In some embodiments, additional agents may ricus, L. plantarum , L. rhamnosus, Rhamnosus L. fermen
be selected from among appetite suppressants, neurotrans- tum , L. caucasicus, L. helveticus, L. lactis, L. reuteri and L.
mitter reuptake inhibitors, dopaminergic agonists , seroton- casei ) or a bifidobacteria ( Bifidobacterium bifidum , B. infan
ergic agonists , modulators of GABAergic signaling, anti- 55 tis) Streptococcus thermophiles, and Enterococcus faecium .
convulsants, antidepressants, monoamine oxidase inhibitors, Other suitable probiotics and prebiotics are disclosed for
substance P (NK1 ) receptor antagonists , melanocortin example in R. Spiller, Aliment Pharmacol Ther 28 , 385-396 ,
receptor agonists and antagonists, lipase inhibitors, inhibi- the contents of which are hereby incorporated by reference
tors of fat absorption , regulators of energy intake or metabo- in their entirety.
lism , cannabinoid receptor modulators, agents for treating 60 In some embodiments, a probiotic agent that optionally
addiction, agents for treating metabolic syndrome, peroxi- inhibits the growth of methanogens, for example, Bifido
some proliferator - activated receptor ( PPAR ) modulators ; bacterium spp . or Lactobacillus species or strains, e.g. , L.
and dipeptidyl peptidase 4 ( DPP - 4 ) antagonists . In some acidophilus, L. rhamnosus, L. plantarum , L. reuteri, L.
embodiments , additional agents may be selected from paracasei subsp . paracasei, or L. casei Shirota , or probiotic
among amphetamines, benzodiazepines, sulfonyl ureas, 65 Saccharomyces species , e.g. , S. cerevisiae, is selected and /or
meglitinides, thiazolidinediones, biguanides, beta -blockers , administered . The probiotic agent that inhibits methanogen
ACE inhibitors, diuretics, nitrates, calcium channel block- esis may be administered in a pharmaceutically acceptable
 US 11,344,501 B2
31 32
ingestible formulation , such as in a capsule , or for some Methanosaeta thermophile, Methanosarcina acetivorans,
subjects, consuming a food supplemented with the inoculum Methanosarcina barkeri, Methanosarcina mazei, Methano
is effective, for example a milk , yogurt, cheese, meat or sphaera stadtmanae, Methanospirillium hungatei , Metha
other fermentable food preparation . Probiotic agents can nothermobacter defluvii (Methanobacterium defluvii ),
inhibit the growth of methanogens, for example, by com- 5 Methanothermobacter thermautotrophicus (Methanobacte
peting against methanogens for growth and thus reduce or rium thermoautotrophicum ), Methanothermobacter thermo
inhibit the growth of methanogens . flexus (Methanobacterium thermoflexum ), Methanothermo
Methods of Treatment bacter wolfei (Methanobacterium wolfei ), and Methanothrix
In one aspect , the present invention provides methods of sochngenii.
treating or preventing a methanogen -associated disorder by 10 In one aspect , the present invention provides methods of
administering a modified - release formulation comprising at reducing or eliminating methane produced by Methanobre
least one anti-methanogenic agent, such as an antimethano vibacter smithii in the GI tract. In another aspect, the present
genic statin as described herein to the intestine (i.e. , small invention provides methods of reducing or eliminating
and / or large intestine) in a subject in need thereof. methane produced by Methanobrevibacter smithii, in the GI
In some embodiments , the methanogen -associated disor- 15 tract by administering a modified -release formulation com
der is aa disease or disorder or condition caused by, resulted prising at least one anti -methanogenic agent, such as an
from , or related to one or more of the abnormal presence or antimethanogenic statin as described herein to the intestine
absence of methanogens, abnormal levels of methanogens, (i.e. , small and / or large intestine) in a subject in need
overgrowth of methanogens, elevated levels of methanogen- thereof. In some embodiments , administration of the modi
esis , elevated enteric methane levels, excessive hydrogen 20 fied - release formulation comprising at least one anti -metha
scavenging by hydrogen - consuming methanogens or colo- nogenic agent reduces or eliminates methane produced by
nization of methanogens in an abnormal location (e.g. , in the Methanobrevibacter smithii in the small intestines (e.g. , one
small bowel rather than large bowel ) , either alone or in or more of duodenum , jejunum , ileum) . In an embodiment,
combination with non -methanogen syntrophic organisms. administration of the modified -release formulation compris
Illustrative methanogen -associated disorders include, but 25 ing at least one anti -methanogenic agent reduces or elimi
are not limited to , enteric methanogen colonization , IBS , nates methane produced by Methanobrevibacter smithii in
IBS - C , IBS - M , constipation , diabetes, type 2 diabetes , meta- the ileum . In some embodiments, administration of the
bolic syndrome, insulin resistance, metabolic syndrome, modified - release formulation comprising at least one anti
obesity, constipation , chronic constipation , chronic intesti- methanogenic agent reduces or eliminates methane pro
nal pseudo -obstruction, systemic sclerosis, systemic lupus , 30 duced by Methanobrevibacter smithii in the large intestine
erythematosus, dermatomysitis /polymyositis, periartiytis (e.g. , one or more of cecum , ascending, transverse, descend
nodosa , mixed connective tissue disorder, rheumatoid arthri- ing or sigmoid portions of the colon , and rectum ).
tis , spinal cord injury, Parkinson's disease , hypothyroidism / In one aspect , the present invention provides methods of
hypoparathyroidism , Hirschsprung's disease , Chagas' dis- reducing or eliminating the methane derived from Metha
ease , intestinal hypoganglionosis, and Ehlers - Danlos 35 nobrevibacter smithii in the GI tract. In another aspect , the
Syndrome. present invention provides methods of reducing or eliminat
In one aspect , the present invention provides methods of ing methane, for example as produced by Methanobrevi
reducing or eliminating the production and / or accumulation bacter smithii , in the GI tract by administering a modified
of methane in the GI tract by administering a modified- release formulation comprising at least one anti
release formulation comprising at least one anti -methano- 40 methanogenic agent, such as an antimethanogenic statin as
genic agent, such as an antimethanogenic statin as described described herein to the intestine (i.e. , small and /or large
herein to the intestine ( e.g. the small and / or large intestine ) intestine ) in a subject in need thereof.
of a subject in need thereof. In another aspect , the present In various embodiments, the present invention relates to
invention provides methods of reducing or eliminating the substantial reduction of methane gas in a subjects GI
methane, for example as produced by a methanogen in the 45 tract (e.g. eradication of intestinal methane ). In some
GI tract by administering a modified - release formulation embodiments the present formulations and methods prevent
comprising at least one anti -methanogenic agent, such as an the increase in levels of methane gas in a subject's GI tract.
antimethanogenic statin as described herein to the intestine In some embodiments, the patient's GI methane levels (as
( i.e. , small and / or large intestine) of a subject in need assessed by methods described herein and methods known
thereof. 50 in the art) are reduced to about 1 ppm , or about 2 ppm , or
In various embodiments, the methanogen is a microor- about 3 ppm , or about 4 ppm , or about 5 ppm , or about 10
ganism that produces methane as a metabolic byproduct. ppm , or about 15 ppm , or about 20 ppm , or about 25 ppm ,
Methanogens are classified as archaea . Examples of metha- or about 30 ppm , or about 35 ppm , or about 40 ppm , or about
nogens include but are not limited to Methanobacterium 45 ppm , or about 50 ppm , or about 55 ppm , or about 60 ppm ,
bryantii, Methanobacterium formicum , Methanobrevibacter 55 or about 65 ppm , or about 70 ppm , or about 75 ppm , or about
arboriphilicus, Methanobrevibacter gottschalkii, Methano- 80 ppm , or about 85 ppm , or about 90 ppm , or about 100
brevibacter ruminantium , Methanobrevibacter smithii, ppm . In various embodiments, the present formulations and
Methanocalculus chunghsingensis, Methanococcoides bur- methods reduce the patient's GI methane levels to less than
tonii, Methanococcus aeolicus, Methanococcus deltae, about 250 ppm , or less than about 225 ppm , or less than
Methanococcus jannaschii, Methanococcus maripaludis, 60 about 200 ppm , or less than about 175 ppm , or less than
Methanococcus vannielii, Methanocorpusculum labreanum , about 150 ppm , or less than about 125 ppm , or less than
Methanoculleus bourgensis (Methanogenium olentangyi, about 100 ppm , or less than about 50 ppm . In various
Methanogenium bourgense ), Methanoculleus marisnigri, embodiments, substantial reduction of methane gas is not
Methanofollis liminatans, Methanogenium cariaci, Metha- accompanied by a substantial reduction in hydrogen gas .
nogenium frigidum , Methanogenium organophilum , Metha- 65 In various embodiments, the present invention relates to
nogenium wolfei, Methanomicrobium mobile, Methanopy- the treatment of IBS , including IBS - C as described by
rus kandleri, Methanoregula boonei, Methanosaeta concilii, ICD - 10 ( International Statistical Classification of Diseases
 US 11,344,501 B2
33 34
and Related Health Problems, WHO edition) . In various In some embodiments, the stage or severity of the disease
embodiments, the present invention relates to the treatment in the patient to be treated is assessed by the Kruis scale
of irritable colon , as classified in ICD - 10 as [ K58 ] . IBS may (Gastroenterology 87 : 1-7 , the contents of which are hereby
include irritable bowel syndrome without diarrhea, as clas- incorporated by reference ). This scale incorporates both the
sified in ICD - 10 as [ K58.9 ] . Irritable bowel syndrome 5 “ cardinal ” symptoms ( pain , bloating , altered bowel func
without diarrhea may also include irritable bowel syndrome tion ) and “ red flag " signs of potential underlying organic
not otherwise specified (NOS ). Further, the diseases as disease that would thus exclude an IBS diagnosis . IBS is
classified in ICD - 10 as K59 are also included (e.g. consti
pation ; K59.1 Functional diarrhea; K59.2 Neurogenic diagnosed if the sum of scores >44 .
bowel , not elsewhere classified; K59.3 Megacolon , not 10 TABLE 1
elsewhere classified (including dialatation of colon , toxic
megacolon, megacolon in Chagas disease ( B57.3 ) , congeni- Kruis Scoring System . IBS is diagnosed if the sum of scores > 44 .
tal ( aganglionic ) ( Q43.1 ) , and Hirschsprung disease Parameter Score
( Q43.1) ); K59.4 Anal spasm ( including Proctalgia fugax );
K59.8 Other specified functional intestinal disorders (in- 15 Signs
cluding atony of colon ) and K59.9 Functional intestinal
disorder, unspecified ). Pain , flatulence, or bowel irregularity 34
Duration of symptoms > 2 yr 16
In various embodiments, the present invention relates to Description of abdominal pain ( Scale from burning to " not so bad ” ) 23
the treatment of spastic colon , nervous colitis , mucous Alternating diarrhea and constipation 14
colitis , functional colitis or colonic neurosis . In various 20 Red Flags
embodiments, the present invention relates to the treatment Abnormal physical findings or history pathognomonic of other -47
of diseases that have been described as sigma elongatum disease
mobile , cecum mobile , chronic colitis , splanchnoptosia and ESR > 10 mm / h -13
the like . Typological classification of the disease generally WBC > x109
Anemia
-50
-98
include convulsive large bowel , diarrhea nervosa and colica 25 History of blood in stool -98
mucosa , and the disease may also be classified in convulsive
constipation type , atonic constipation type, intestinal gas
syndrome, or chronic celiopathy. In some embodiments, the patient is evaluated with the
Furthermore, IBS may also include cholangiodyskinesia , assessment described in Francis, et al Aliment Pharmacol
gastric emptying hypofunction, hysteric globus , non - specific 30 Ther 1997 ; 11 : 395-402 , the contents of which are hereby
esophagus functional abnormalities, nervous vomiting, incorporated by reference in their entirety. For instance, a
recurrent abdominal pain, simple constipation, chronic idio- scoring system based on patient ranking of pain , distension ,
pathic constipation and the like . As diagnostic criteria of IBS bowel dysfunction and quality of life /global well - being on a
those of NIH , Manning, Cook et al . and the like are suitable scale of up to 500 is used . Mild , moderate and severe cases
( see Asakura , Clinical Digestive Internal Medicine, 8 ( 8 ) : 35 were indicated by scores of 75 to 175 , 175 to 300 and > 300 .
1373-1381 ( 1993 ) , the contents of which are hereby incor In some embodiments, the patient of the present invention
porated by reference in their entirety ). 2

has a score of 75 to 175. In some embodiments, the patient

In various embodiments , the present invention relates to of the present invention has a score of 175 to 300. In some
the treatment of IBS , including IBS - C of varying stages or embodiments , the patient of the present invention has a score
severity. In one embodiment, stages or severity of the IBS 40 of >300 . In some embodiments the scales described in Wong
may be evaluated with a health - related quality of life
( HRQOL ) evaluation . In some embodiments, the stage or and Drossman ( Expert Rev. Gastroenterol. Hepatol. 4 ( 3 ) ,
severity of the disease in the patient to be treated is assessed (2010) , the contents of which are hereby incorporated by
by an evaluation of one or more of patient pain , distension , reference in their entirety ). For example, in some embodi
bowel dysfunction and quality of life / global well -being . 45 ments, the patients of the present invention are evaluated for
In some embodiments, the stage or severity of the disease the parameters of dysphoria, activity interference, body
in the patient to be treated is assessed by the Rome Scale ( for image , health worry, food avoidance, social reaction , and
the last 3 months with symptom onset at least 6 months prior sexual relationships and optionally scored on a 0-100 as
to diagnosis: recurrent abdominal pain or discomfort (e.g. described on the Patrick scale ; and / or the patients of the
uncomfortable sensation not described as pain . ) at least 3 50 present invention are evaluated for the parameters of daily
days/month in the last 3 months associated with two or more activities , emotional impact, family relations, food , sleep
of improvement with defecation , onset associated with a and fatigue, social impact, sexual relations symptoms and
change in frequency of stool , and onset associated with a optionally scored on a 0-216 as described on the Groll scale ;
change in the form ( appearance) of stool . In various embodi- the patients of the present invention are evaluated for the
ments, the present compositions and methods provide 55 parameters of activities , anxiety, diet, sleep , discomfort,
patient improve as assessed by the Rome Scale . health perception , disease coping and stress and optionally
In some embodiments, the stage or severity of the disease scored on a 0-100 as described on the Chassany scale ; the
in the patient to be treated is assessed by abdominal pain patients of the present invention are evaluated for the
intensity score of 0-10 . In various embodiments, values 23 parameters of emotional health , mental health , sleep , energy,
are considered to be suffering from pain requiring treatment. 60 physical functioning, diet, social role, physical role, and
In various embodiments, the patient has an abdominal pain sexual relations and optionally scored on a 0-100 as
intensity score of great than about 9 , or about 8 , or about 7 , described on the Hahn scale ; and / or the patients of the
or about 6 , or about 5 , or about 4 , or about 3. In various present invention are evaluated for the parameters of bowel
embodiments, the present compositions and methods reduce symptoms, fatigue , activity impairment, emotional dysfunc
the abdominal pain intensity score by about 1 , or about 2 , or 65 tion and optionally scored as domain average scores (cal
about 3 , or about 4 , or about 5 , or about 6 , or about 7 , or culated by dividing the domain sum score by the number of
about 8 , or about 9 , or about 10 . items: range 1-7 ) as described on the Wong scale .
 US 11,344,501 B2
35 36
In some embodiments, patients may be stratified based on tration for weeks , months, years and even life of the patient,
one or more of methane detection ( e.g. via breath test) and inclusive of chronic administration ). Further, in some
methanogen detection (e.g. via PCR , e.g. qPCR) . In some embodiments, non -responders are not administered a full
embodiments, the patient is considered methane breath test treatment period of a one or more of the formulations
positive if the subject presents with greater than about 3 ppm 5 described herein and instead are treated with an alternative
methane . In some embodiments, the patient of the present therapy.
invention has greater than about 104, or about 10 % , or about In various embodiments, the present invention provides
106 copies of M. smithii per grams of wet stool . In some methods of treating various methanogen -associated disor
embodiments , the patient of the present invention is defined ders, including by way of non - limiting example IBS - C , in
by a measurement of the fractional methanogen contribution 10 which a subject is evaluated as a responder or a non
to the total microbial content of the feces. In some embodi- responder and treated accordingly. For example , in some
ments , the patient has greater than about 0.5 % , or about embodiments , a subject may be evaluated for a baseline
0.6 % , or about 0.7 % , or about 0.8 % , or about 0.9 % , or about level of intestinal methane . Such a measurement may use
1.0 % , or about 1.1 % , or about 1.2 % , or about 1.3 % , or about any of the techniques described herein , including without
1.4 % , or about 1.5 % , or about 2.5 % M. smithii fraction of 15 limitation methane breath testing. Subsequently, the subject
the total microbial content of the feces . is administered one or more of the formulations described
In various embodiments, the present invention provides herein for an initial treatment period of less than about 1
methods for inhibiting or reducing methanogenesis, includ- week ( e.g. about 1 day, or about 2 days , or about 3 days , or
ing in subjects afflicted with one or more of IBS - C , obesity about 4 days, or about 5 days, or about 6 days , or about 7
and diabetes, in which a subject is evaluated as a responder 20 days ) and then re - evaluated for a post - initial treatment level
or a non - responder and treated accordingly. For example, in of intestinal methane. Such a measurement may use any of
some embodiments, a subject may be evaluated for aa base- the techniques described herein , including without limitation
line level of intestinal methane. Such a measurement may methane breath testing . This second evaluation allows of
use any of the techniques described herein, including with- classification of subjects as responders or non - responders ;
out limitation methane breath testing . Subsequently the 25 for example responders show a reduction in post - initial
subject is administered one or more of the formulations treatment level of intestinal methane while non -responders
described herein for an initial treatment period of less than do not . Accordingly, in some embodiments, responders are
about 1 week ( e.g. about 1 day, or about 2 days, or about 3 administered aa full treatment period of a one or more of the
days, or about 4 days, or about 5 days, or about 6 days, or formulations described herein ( e.g. administration for
about 7 days) and then re - evaluated for aa post - initial treat- 30 weeks, months, years and even life of the patient, inclusive
ment level of intestinal methane. Such a measurement may of chronic administration ). Further, in some embodiments ,
use any of the techniques described herein , including with- non - responders are not administered a full treatment period
out limitation methane breath testing. This second evalua- of a one or more of the formulations described herein and
tion allows classification of subjects as responders or non- instead are treated with an alternative therapy.
responders; for example responders show a reduction in 35 In various embodiments, the present invention provides
post - initial treatment level of intestinal methane while non- methods for identifying a patient that is likely to respond to
responders do not . Accordingly, in some embodiments , long term ( including chronic ) treatment one or more of the
responders are administered a full treatment period of a one formulations described herein for the treatment of one or
or more of the formulations described herein (e.g. adminis- more of inhibiting or reducing methanogenesis, including in
tration for weeks, months, years and even life of the patient, 40 subjects afflicted with one or more of IBS - C , obesity and
inclusive of chronic administration ). Further, in some diabetes ; treating constipation ; and treating various metha
embodiments , non - responders are not administered a full nogen- associated disorders, including by way of non - limit
treatment period of a one or more of the formulations ing example IBS - C . In various embodiments, the methods
described herein and instead are treated with an alternative include the steps of evaluating a subject for aa baseline level
therapy 45 of intestinal methane (e.g. using any of the techniques
In various embodiments, the present invention provides described herein , including without limitation methane
methods of treating constipation in a subject. In various breath testing ) ; administering one or more of the formula
embodiments, the subject is evaluated as a responder or a tions described herein for an initial treatment period of less
non -responder and treated accordingly. For example , in than about 1 week ( e.g. about 1 day, or about 2 days, or about
some embodiments, a subject may be evaluated for a base- 50 3 days , or about 4 days, or about 5 days, or about 6 days, or
line level of intestinal methane. Such a measurement may about 7 days ) ; and re -evaluating the subject for a post - initial
use any of the techniques described herein , including with- treatment level of intestinal methane (e.g. using any of the
out limitation methane breath testing . Subsequently, the techniques described herein , including without limitation
subject is administered one or more of the formulations methane breath testing ) . This re - evaluation allows of clas
described herein for an initial treatment period of less than 55 sification of subjects as responders or non - responders ; for
about 1 week ( e.g. about 1 day, or about 2 days, or about 3 example responders show a reduction in post -initial treat
days, or about 4 days, or about 5 days, or about 6 days, or ment level of intestinal methane while non -responders do
about 7 days) and then re - evaluated for a post - initial treat- not . Responders are those patients that are likely to respond
ment level of intestinal methane. Such a measurement may to long term ( including chronic ) treatment one or more of the
use any of the techniques described herein , including with- 60 formulations described herein for the treatment of one or
out limitation methane breath testing. This second evalua- more of inhibiting or reducing methanogenesis, including in
tion allows for classification of subjects as responders or subjects afflicted with one or more of C - IBS , obesity and
non - responders; for example responders show aa reduction in diabetes ; treating constipation ; and treating various metha
post - initial treatment level of intestinal methane while non- nogen- associated disorders, including by way of non - limit
responders do not . Accordingly, in some embodiments , 65 ing example C - IBS .
responders are administered a full treatment period of a one In some embodiments , methods of the present invention
or more of the formulations described herein (e.g. adminis- treat or prevent constipation . Constipation may be associ
 US 11,344,501 B2
37 38
ated with , for example, chemotherapy, vinca alkaloids , oxa- about 11 % , about 10% , about 9 % , about 8 % , about 7 % ,
liplatins , taxanes, thalidomide, opioids , sedatives , anticho- about 6 % , about 5 % , about 4 % , about 3 % , or about 2 % in
linergics, gastrointestinal antispasmodics, antiparkinsonism serum LDL - C levels after treatment.
agents , antidepressants, phenothiazines, calcium- and alu- In some embodiments, the patient is one who does not
minum -based antacids, diuretics, tranquilizers, sleeping 5 require statins for their cardiovascular therapeutic uses . In
medications , general anesthesia , pudendal blocks , inad- some embodiments, the patient is one who does not require
equate fluid intake, excessive use of laxatives and / or statins for their cardiovascular therapeutic uses and is meth
enemas, prolonged immobility, inadequate exercise . spinal ane -positive ( e.g. as assessed by the methods described
cord injury or compression , fractures, fatigue, weakness, herein such as the methane breath test and qPCR ).
inactivity, bedrest, cardiac problems, diverticulitis, neuro- 10 By maximizing retention of the antimethanogenic statins
logical lesions , cerebral tumors , spinal cord injury, spinal to the intestines, the methods of the invention also minimize
cord compression, paraplegia, cerebrovascular accident with the side effects associated with systemic release of the statin .
paresis, weak abdominal muscles, hypothyroidism , lead For example, the present method prevents and / or minimizes
poisoning , uremia, dehydration , hypercalcemia, hypokale- various adverse effects associated with statin usage includ
mia , hyponatremia, anorexia, immobility , antidepressants, 15 ing , muscle - associated adverse effects, such as myositis,
inability to increase intra - abdominal pressure , emphysema, myalgia, rhabdomyolysis, drug -drug - interactions, cognitive
neuromuscular impairment of the diaphragm , neuromuscu- effects, increased cancer risk , increases in liver enzymes ,
lar impairment of abdominal muscles , abdominal hernias, hemorrhagic stroke, increase in blood glucose levels , sleep
malnutrition , cachexia , anemia , carcinoma, and senility. In disorders, peripheral neuropathy, sexual dysfunction, thy
some embodiments, methods of the invention increase the 20 roid dysfunction , renal toxicity, irritability , shortness of
number of bowel movements in a subject suffering from breath , hyperkalemia, weight gain , neurodegenerative dis
constipation . For example, methods of the invention may ease , pancreatitis, liver pathology, mitochondrial syn
increase the number of bowel movements in the subject by dromes, dermatologic conditions, dry mouth , cataracts,
at least 1 , 2 , 3 , 4 , or 5 movements per week . In some olfaction , hematalogic and bone marrow adverse effects,
embodiments, methods of the invention increase the stool 25 hypotension, gastrointestinal adverse effects, including ,
wet weight in a subject suffering from constipation . For ulcerative colitis and gastric ulceration , fatigue and head
example , the methods of the invention may increase the ache . In some embodiments, the methods of the invention
stool wet weight by at least 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , also minimizes the following side effects associated with
35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70% , 75 % , 80 % , systemic release of a statin : muscle pain , tenderness, or
85 % , 90 % , 95 % , or 100 % . 30 weakness, lack of energy , weakness , fever, dark colored
In various embodiments, the constipation is associated urine, jaundice, pain in the stomach , including the upper
with IBS , but the present invention , in some embodiments , right part of the stomach , nausea , unusual bleeding or
can also relate to chronic functional constipation . bruising, loss of appetite , flu - like symptoms, rash , hives ,
In various embodiments, the present invention relates to itching , difficulty breathing or swallowing , and swelling of
the treatment of increased visceral hypersensitivity. In vari- 35 the face, throat, tongue , lips , eyes, hands, feet, ankles, or
ous embodiments, the present invention relates to the treat- lower legs , hoarseness.
ment of one or more of stomachaches, pain , nausea , strain- Accordingly, the modified - release formulation of the
ing , and bloating and / or gas . The present formulations and present invention may be used to target subjects where
methods also treat one or more of as hard stools , infrequent systemic statin levels are undesirable . In one embodiment,
stools , difficulty or straining at stools , feeling of being 40 the subject may be women and children who are otherwise
unable to completely empty during a bowel movement, and healthy and have no need for aa cardiovascular medicine (as
the sensation of wanting to go but not being able to . characterized, for example , as having low or zero myocar
In various embodiments, the present invention relates to dial event risk factors as per the ATP III Guideline ). In
the treatment for diabetes ( type 1 or type 2 ) and / or glucose another embodiment, the subject may be aa child with IBS - C
intolerance . In some embodiments, the present invention 45 who has no need for aa cholesterol -lowering agent. In such
relates to a method for treating patient at risk of diabetes , one embodiments , administration of the modified - release formu
or more of insulin resistance, prediabetes, impaired fasting lation of the present invention results in an average reduc
glucose (IFG) , impaired glucose tolerance (IGT ) , and acan- tion of less than about 20% , about 19 % , about 18 % , about
thosis nigricans. 17 % , about 16 % , about 15 % , about 14 % , about 13 % , about
In some embodiments, methods for inducing weight loss 50 12 % , about 11 % , about 10 % , about 9 % , about 8 % , about
or preventing weight gain ( or treating or preventing obesity 7 % , about 6 % , about 5 % , about 4 % , about 3 % , or about 2 %
or inducing weight loss or preventing weight gain in a in serum LDL - C levels after treatment.
patient that does not substantially change caloric intake ), The modified - release formulation of the present invention
comprising administering the present formulations are pro- may also be utilized as part of a treatment regimen wherein
vided. Patients may have undertaken or will undertake a 55 a subject is provided with an initial anti-methanogenic
surgery ofthe digestive system ; be greater than about 80-100 therapy followed by a chronic anti -methanogenic or meth
pounds overweight; have a BMI of greater than about 35 ane - reducing and / or eliminating maintenance therapy.
kg /m²; or have a health problem related to obesity The initial anti-methanogenic therapy may employ agents
In some embodiments , administration of the modified- other than statins such as , for example, antibiotics which
release formulation of the present invention does not confer 60 eradicate the methanogens. For example nitroimidazoles
cholesterol- lowering cardiovascular effects associated with such as metronidazole, metronidazole esters and /or isomers
systemic administration of statins. For example, the present or hydrophobic imidazole derivatives or rifaximin or neo
formulations and methods may avoid or reduce a subject's mycin sufficient to eradicate , substantially reduce, or reduce
systemic exposure to a statin . For example , the present the enteric methanogen colonization may be used . Such
formulations and methods may provide an average reduction 65 initial therapy may be for 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 14 , 28 ,
9 2

of less than about 20 % , about 19 % , about 18 % , about 17 % , 42 , 56 , 60 , 90 , 120 or 180 days or more . Examples of
about 16 % , about 15 % , about 14 % , about 13 % , about 12 % , antibiotics include but are not limited to aminoglycosides
 US 11,344,501 B2
39 40
( e.g. , amikacin, gentamicin, kanamycin , neomycin , netilmi- In certain embodiments, the human has an age in a range
cin , streptomycin , tobramycin , paromomycin ), ansamycins of from about 1 to about 18 months old, from about 18 to
( e.g. , geldanamycin , herbimycin ), carbacephems ( e.g. , lora- about 36 months old, from about 1 to about 5 years old , from
carbef), carbapenems ( e.g. , ertapenem , doripenem , imipe- about 5 to about 10 years old , from about 10 to about 15
nem , cilastatin, meropenem ), cephalosporins (e.g. , first gen- 5 years old, from about 15 to about 20 years old , from about
eration: cefadroxil, cefazolin , cefalotin or cefalothin , 20 to about 25 years old , from about 25 to about 30 years
cefalexin ; second generation : cefaclor, cefamandole , cefoxi- old , from about 30 to about 35 years old , from about 35 to
tin , cefprozil, cefuroxime; third generation : cefixime, cef- about 40 years old , from about 40 to about 45 years old, from
dinir, cefditoren , cefoperazone , cefotaxime, cefpodoxime, about 45 to about 50 years old, from about 50 to about 55
ceftazidime, ceftibuten , ceftizoxime, ceftriaxone; fourth 10 years old, from about 55 to about 60 years old , from about
generation: cefepime; fifth generation: ceftobiprole ), glyco- 60 to about 65 years old , from about 65 to about 70 years
peptides ( e.g. , teicoplanin , vancomycin ) , macrolides ( e.g. , old , from about 70 to about 75 years old , from about 75 to
azithromycin , clarithromycin , dirithromycin , erythromycin , about 80 years old , from about 80 to about 85 years old, from
roxithromycin , troleandomycin , telithromycin , spectinomy- about 85 to about 90 years old, from about 90 to about 95
cin) , monobactams (e.g. , aztreonam ), penicillins (e.g. , 15 years old or from about 95ato about 100 years old . In one
amoxicillin , ampicillin, azlocillin , carbenicillin , cloxacillin , embodiment, the human is a child . In one embodiment, the
dicloxacillin , flucloxacillin , mezlocillin , meticillin, nafcillin , human is a female .
oxacillin , penicillin , piperacillin , ticarcillin ) , antibiotic poly- Methods to Determine Methanogen Levels/ Diagnostic and
peptides (e.g. , bacitracin , colistin , polymyxin b ) , quinolones Patient Selections
( e.g. , ciprofloxacin , enoxacin , gatifloxacin, levofloxacin , 20 Intestinal methanogen and / or methane levels can be deter
lomefloxacin , moxifloxacin , norfloxacin , ofloxacin , trova- mined by breath tests that measure breath methane levels .
floxacin ), rifamycins ( e.g. , rifampicin or rifampin , rifabutin , Breath testing may be utilized to identify subjects who are
rifapentine, rifaximin ), sulfonamides (e.g. , mafenide, pron- “ methane-positive” and who can potentially benefit from
tosil , sulfacetamide, sulfamethizole, sulfanilamide, sulfasa- methods of the present invention . Further, breath testing can
lazine , sulfisoxazole , trimethoprim , trimethoprim - sulfame- 25 also be used to monitor the efficacy of treatment. Breath
thoxazole ( co - trimoxazole , " tmp- smx " ), and tetracyclines testing analysis methods and equipment are known in the art
( e.g. , demeclocycline, doxycycline , minocycline , oxytetra- ( see , for example, PCT/US14 /27697 , the entire contents of
cycline , tetracycline ) as well as arsphenamine, chloram- which are incorporated by reference herein ). Examples of
phenicol, clindamycin , lincomycin , ethambutol, fosfomycin , such equipment include , for example, the QuinTron Breath
fusidic acid , furazolidone, isoniazid , linezolid, metronida- 30 Tracker gas chromatographic ( GC ) analyzer or the Quin Tron
zole , mupirocin , nitrofurantoin , platensimycin , pyrazina- Breath Tracker device (QuinTron Instrument Company, Inc. ,
mide, quinupristin /dalfopristin combination , and tinidazole . Milwaukee, Wis .).
Following the initial therapy, a subject may be placed on Further, abnormal lactulose breath test results are com
maintenance therapy in order to maintain reduced methano- mon in subjects with IBS and therefore the present invention
gen and / or methane levels. In some embodiments, the main- 35 provides for the use of lactulose breath tests in evaluating
tenance therapy utilizes a modified -release formulation of patients. In some embodiments, a patient is evaluated with
the present invention . In an embodiment, the initial therapy a lactulose breath test before and /or after administration with
includes an antibiotic followed by a chronic maintenance the present formulations.
regimen of low dose statin formulations . In various embodi- In general, individuals having a breath methane level of at
ments, the maintenance regiment may be administered for at 40 least about 3 ppm are generally associated with methanogen
least 1 week , at least 2 weeks , at least 3 weeks, at least 4 associated disorders and are likely to benefit from methods
weeks, at least one month , at least two months, at least three of the present invention . Alternatively, methods of the
months, at least four months, at least five months, at least six invention may be practiced on subjects having a breath
months, at least seven months, at least eight months, at least methane level of at least 1 ppm , at least 1.5 ppm , at least 2
nine months, at least ten months, at least eleven months, at 45 ppm , at least 2.5 ppm , at least 3 ppm , at least 3.5 ppm , at
least 1 year, at least 2 years , at least 3 years , at least 4 years , least 4 ppm , at least 5 ppm , at least 6 ppm , at least 7 ppm ,
at least 5 years , at least 10 years , or indefinitely. at least 8 ppm , at least 9 ppm , at least 10 ppm .
The modified - release formulation of the present invention One method for measuring methanogen levels involves
may be utilized solely for chronic maintenance therapy. In calculation of a subject's breath methane area under the
various embodiments, the present invention provides a 50 curve ( BM - AUC ). This method involves obtaining multiple
method of treating previously methane positive patients who breath samples averaging about 15 minutes apart for a
do not have one or more of cardiovascular disease , an LDL period of about 90 minutes, or about 120 minutes , or for up
level of 190 mg/dL or higher, Type 2 diabetes who are to 4 hours or more at potentially less frequent intervals. The
between 40 and 75 years of age , an estimated 10 -year risk time period results are used to calculate a person's BM
of cardiovascular disease of 7.5 percent or higher who are 55 AUC . For example, a subject may undergo a such as
between 40 and 75 years of age with a modified -release lactulose , xylose , lactose , or glucose breath test after a 12
formulation herein in order to maintain their methane nega- hour fast. The breath test may comprise a baseline breath
tive status. Accordingly, in some embodiments, the modi- measurement after which the subject ingests about 10 g of
fied - release formulation of the present invention finds use as such as lactulose , xylose , lactose , or glucose . Following
a prevention measure in a high risk patient. 60 lactulose ingestion , the subject is then asked to provide a
In various embodiments, methods of the invention are breath sample about every 15 minutes for about 90 to about
useful in treatment a human subject. In some embodiments , 120 minutes to determine methane production. BM -AUC
the human is a pediatric human . In other embodiments, the may be utilized for more precisely determining and moni
human is an adult human . In other embodiments, the human toring, for example, the efficacy of the anti -methanogenic
is a geriatric human . In other embodiments, the human may 65 therapy. BM -AUC measurements could also be utilized to
be referred to as a patient. In some embodiments, the human segregate “ methane positive” from “ methane negative” sub
is a female . In some embodiments, the human is a male . jects for improved clinical decision making. BM -AUC may
 US 11,344,501 B2
41 42
be compared to or utilized with measurement of methanogen small intestine bacterial overgrowth , Gastroenterol. 91 ( 6 ) :
levels in stool samples via PCR , e.g. qPCR . Alternatively, 1447-51 [ 1986 ] ; A. Schneider et al . , Value of the 14C - D
measurement of methanogen levels in stool samples via xylose breath test in patients with intestinal bacterial over
PCR , e.g. qPCR may supplant the use of a breath test . More growth , Digestion 32 ( 2 ) : 86-91 [ 1985 ] ) .
precise techniques may also involve measurement of breath 5 In various embodiments, treatments using the modified
methane taking into account and subtracting ambient meth- release formulation of the invention result in a reduction of
ane levels. breath methane level of at least about 1 ppm , at least about
Spot breath methane analysis via commercially available 2 ppm , at least about 3 ppm , at least about 4 ppm , at least
equipment such as Breath Tracker may be used in discrimi- about 5 ppm , at least about 66 ppm , at least about 7 ppm , at
nating “ methane- positive ” from “ methane -negative” indi- 10 least about 8 ppm , at least about 9 ppm , at least about 10
viduals, and monitoring the success , failure , dose titration , ppm , at least about 20 ppm , at least about 30 ppm , at least
dosing schedule (daily or non -daily, for example) of the about 40 ppm , at least about 50 ppm , at least about 60 ppm ,
modified - release formulations, such as various antimetha- at least about 70 ppm , at least about 80 ppm , at least about
nogenic statins . For example, the lowest minimum effective 90 ppm , at least about 100 ppm , at least about 110 ppm , at
dose may be identified as such . Additional instruments and 15 least about 120 ppm , at least about 130 ppm , at least about
techniques for measuring methane levels include , but are not 140 ppm , at least about 150 ppm , at least about 160 ppm , at
limited to , cavity enhanced absorption techniques such as a least about 170 ppm , at least about 180 ppm , at least about
LGR - FMR methane measurement instrument having a 190 ppm , at least about 200 ppm , at least about 210 ppm , at
range as low as 0.01 ppm ( Los Gatos Research, Inc. , least about 220 ppm , at least about 230 ppm , at least about
Mountain View , Calif .), wavelength - scanned cavity down- 20 240 ppm , and at least about 250 ppm .
ring spectroscopy, carbon isotope analysis ( G2132-113C , The samples used for the present invention include a
Picarro , Inc, Santa Clara, Calif .), gas chromatography, mass patient's breath . In various embodiments, measurement of
spectroscopy, membrane extracted carbon isotope analysis methanogen levels in stool samples via PCR , e.g. qPCR or
( Pollock , 2012 GSA Annual Meeting, “ Membrane Extracted other molecular biology approaches, for example, is also
Carbon Isotope Analysis of Dissolved Methane ” ), head- 25 provided . Further, aspirates of the fluid in the GI tract may
space gas chromatography with FID detector and GC com- be analyzed for methanogen and / or methane levels. Also
bustion with IRMS instruments , for example. Other instru- mucosal biopsies from aa site in the gastrointestinal tract may
ments having the ability to measure low concentration be analyzed for methanogen and /or methane levels .
breath methane levels at higher precision than the clinical Methods of “ quantitative ” amplification are well known
validated instrument marketed as the QuinTron Breath- 30 to those of skill in the art. For example, quantitative PCR
Tracker include high precision breath methane analysis involves simultaneously co -amplifying a known quantity of
( HPBMA) . Use of HPBMA may be used to test spot breath a control sequence using the same primers . This provides an
methane levels or in BM - AUC form . internal standard that may be used to calibrate the PCR
In some embodiments , detection of hydrogen quantity and reaction . Detailed protocols for quantitative PCR are pro
methane quantity is by gas chromatography with mass 35 vided in , for example, Innis, et al . ( 1990 ) PCR Protocols, A
spectrometry and / or radiation detection to measure breath Guide to Methods and Applications, Academic Press , Inc.
emissions of isotope - labeled carbon dioxide , methane, or N.Y. ). Measurement of DNA copy number at microsatellite
hydrogen , after administering an isotope - labeled substrate loci using quantitative PCR analysis is described in, for
that is metabolizable by gastrointestinal bacteria but poorly example, Ginzonger, et al . (2000 ) Cancer Research
digestible by the human host , such as lactulose , xylose , 40 60 : 5405-5409 . The known nucleic acid sequence for the
mannitol, or urea (e.g. , G. R. Swart and J. W. van den Berg , genes is sufficient to enable one of skill in the art to routinely
13C breath test in gastrointestinal practice , Scand . J. Gas- select primers to amplify any portion of the gene . Fluoro
troenterol. [ Suppl . ] 225 : 13-18 [ 1998 ] ; S. F. Dellert et al . , genic quantitative PCR may also be used in the methods of
The 13C -xylose breath test for the diagnosis of small bowel the invention . In fluorogenic quantitative PCR, quantitation
bacterial overgrowth in children, J. Pediatr. Gastroenterol. 45 is based on amount of fluorescence signals, e.g. , TaqMan
Nutr. 25 (2 ) : 153-58 [ 1997 ] ; C. E. King and P. P. Toskes, and Sybr green .
Breath tests in the diagnosis of small intestinal bacterial Other suitable amplification methods include , but are not
overgrowth , Crit. Rev. Lab. Sci. 21 (3 ) : 269-81 [ 1984 ] ) . A limited to , ligase chain reaction (LCR) ( see , for example,
poorly digestible substrate is one for which there is aa relative Wu and Wallace ( 1989 ) Genomics 4 : 560 , Landegren, et al .
or absolute lack of capacity in a human for absorption 50 ( 1988 ) Science 241 : 1077 , and Barringer et al . ( 1990 ) Gene
thereof or for enzymatic degradation or catabolism thereof. 89 : 117 ) , transcription amplification (Kwoh , et al . ( 1989 )
Suitable isotopic labels include 13C or 14C . For measuring Proc. Natl. Acad . Sci. USA 86 : 1173 ) , self - sustained
methane suitable isotopic labels can also include H and PH sequence replication (Guatelli, et al . ( 1990) Proc. Nat. Acad.
or 170 and 180 , as long as the substrate is synthesized with Sci . USA 87 : 1874 ) , dot PCR , and linker adapter PCR , etc.
the isotopic label placed in aa metabolically suitable location 55 In still other embodiments of the methods provided
in the structure of the substrate , i.e. , a location where herein , sequencing of individual nucleic molecules (or their
enzymatic biodegradation by intestinal microflora results in amplification products) is performed . In one embodiment, a
the isotopic label being sequestered in the gaseous product. high throughput parallel sequencing technique that isolates
If the isotopic label selected is aa radioisotope, such as 14C , single nucleic acid molecules of a population of nucleic acid
3H , or 150 , breath samples can be analyzed by gas chroma- 60 molecules prior to sequencing may be used . Such strategies
tography with suitable radiation detection means (e.g. , C. S. may use so - called “ next generation sequencing systems”
Chang et al . , Increased accuracy of the carbon - 14 D - xylose including, without limitation , sequencing machines and /or
breath test in detecting small -intestinal bacterial overgrowth strategies well known in the art, such as those developed by
by correction with the gastric emptying rate, Eur. J. Nucl. Illumina/ Solexa ( the Genome Analyzer; Bennett et al .
Med . 22 ( 10 ) : 1118-22 [ 1995 ] ; C. E. King and P. P. Toskes, 65 (2005 ) Pharmacogenomics, 6 : 373-20 382 ) , by Applied Bio
Comparison of the 1 -gram [ 14C ]xylose, 10 -gram lactulose- systems, Inc. ( the SOLID Sequencer; solid.appliedbiosys
H2 , and 80 - gram glucose - H , breath tests in patients with tems.com) , by Roche (e.g. , the 454 GS FLX sequencer;
 US 11,344,501 B2
43 44
Margulies et al . (2005 ) Nature , 437 : 376-380 ; U.S. Pat. Nos . Without wishing to be bound by theory, an immediate
6,274,320 ; 6,258,568 ; 6,210,891 ) and others. Other release product substantially releases higher in the GI tract
sequencing strategies such as stochastic sequencing ( e.g. , as than an extended release product, which releases low in the
developed by Oxford Nanopore) may also be used , e.g. , as GI tract. Accordingly, a polymer coated bead released from
an enteric - coated capsule as described in FIGS . 1-3 and
described in International Patent Publication No. WO/2010/ 5 various
004273 . other dual pulse formulations are made .
In still other embodiments of the methods provided Example 2 : Development of Dual Pulse
herein , deep sequencing can be used to identify and quantify Formulations
the methanogen or methanogen syntrophic microorganism . 10
These techniques are known in the art. A SYN -010 drug product was produced which was a
Kits HPMC capsule filled with enteric - coated mini -tablets from
The present invention is also directed to a kit for the which lovastatin was released at different intestinal pH
treatment of a methanogen -associated disorder. The kit is an values . The mini -tablets were designed to pass through the
assemblage of materials or components, including at least stomach unchanged then release a small amount of lovas
one of the modified - release formulations described herein . 15 tatin into the duodenum and the majority of the lovastatin
The kit may further include materials and components for dose into the ileocecal junction and colon ( FIG . 5 ). The
relative amounts of lovastatin released into the small and
the quantification of methanogens. The exact nature of the
components configured in the kit depends on its intended large intestine reflected the levels of methane - producing
purpose . In one embodiment, the kit is configured for the archaea in each location .
purpose of treating human subjects. 20 Each mini - tablet in the SYN - 010 dosage form contains
Instructions for use may be included in the kit. Instruc lovastatin combined with USP excipients and coated with a
tions for use typically include a tangible expression describ EUDRAGIT® enteric polymer that dissolves at either pH
ing the technique to be employed in using the components 5.5 (duodenal release ; DR) or pH 7.0 (ileocecal release ;
of the kit to affect a desired outcome , such as to treat a ICR) . Specifically, the SYN -010 ( 21 mg ) formulation com
disorder associated with methanogens. Optionally, the kit 25 prises an opaque, white , size 1 HPMC capsule containing
1xpH 5.5 - coated mini - tablet (DR) and 2xpH 7.0 - coated
also contains other useful components, such as , diluents, mini -tablets (ICR) . The SYN -010 (42 mg ) formulation com
buffers, pharmaceutically acceptable carriers, syringes, cath prises
eters , applicators , pipetting or measuring tools , bandaging 1xpH an5.5 opaque
- coated
, white , size 1 HPMC capsule containing
mini- tablet (DR) and 5xpH 7.0 - coated
materials or other useful paraphernalia as will be readily 30 (ICR ).
recognized by those of skill in the art.
The materials and components assembled in the kit can be withThe200SYNmL- 010 capsules are ingested orally, once daily,
water . The SYN - 010 capsules are swallowed
provided to the practitioner store in any convenience and whole and not chewed . The SYN - 010 capsules do not
suitable ways that preserve their operability and utility. For require dilution .
example, the components can be provided at room , refrig Lovastatin was produced, analyzed and released using
erated or frozen temperatures. The components are typically 35 methodology
contained in suitable packaging materials. In various is summarizedknown in the art. The properties of lovastatin
below in Table 2 :
embodiments, the packaging material is constructed by
well- known methods, preferably to provide a sterile, con TABLE 2
taminant- free environment. The packaging material may 40
have an external label which indicates the contents and / or Property Description
purpose of the kit and / or its components. Name Lovastatin
In various embodiments, a kit comprises a pill bottle CAS 75330-75-5
containing a desiccant to maintain formulation stability . Formula C24H3605
The invention is further described by reference to the 45 Appearance
MW 404.54 g /mol
following non - limiting examples. White to off -white crystalline powder
Melting Point 174.5 ° C. ( under N2); 170.6-170.8 ° C.
EXAMPLES ( crude product)
Density 1.12 g / 100 cm
Solubility Water 0.0004 mg /mL ; ethanol 16 mg/mL ;
Example 1 : Dual Pulse Formulation (room temp) methanol 28 mg /mL
50 Specific Rotation ( + ) 328.9 °
A clinical study was undertaken with a human patient. UV max 238 nm
The patient was administered ALTOPREV (i.e. extended
release lovastatin ) and the breath methane reading was about Various excipients were utilized in the SYN - 010 drug
70 ppm . When switched to MEVACOR (i.e. immediate product and their functions are listed in Table 3 below. The
release lovastatin ), the breath methane increased to 168 55 excipients and coatings were chosen to enable formulation
ppm . Surprisingly, when administering the combination of of lovastatin in appropriate enteric - coated mini- tablets and
ALTOPREV and MEVACOR , the breath methane was provide the desired lovastatin dual-pulse release profile
reduced to Oppm. detailed herein .
TABLE 3
Name Common Name Function

Lovastatin Lovastatin lactone Active pharmaceutical ingredient;

reduces methane production by
intestinal archaea
 US 11,344,501 B2
45 46
TABLE 3 - continued
Name Common Name Function
Avicel PH102 Cellulose , microcrystalline Tablet diluent
Kollidon VA64 Fine Copovidone Tablet binder
Aerosil 200 Silicon dioxide ( silica) Viscosity and dispersion agent
Magnesium stearate Magnesium stearate Lubricant used to facilitate
tableting
Kollidon CL Crospovidone Tablet disintegrant
EUDRAGIT L 30 D - 55 + Enteric polymer, pH 5.5 Enteric coating that dissolves at pH
PlasACRYL HTP20 Poly (methacrylic acid -co - ethyl 5.5 , enabling the mini -tablets to
acrylate) 1 : 1 pass through the stomach
unchanged and release drug into the
duodenum ( DR ). PlasAcryl is an
anti -tacking agent coating additive
that results in shorter preparation
and spraying times
EUDRAGIT FS 30 D + Poly (methyl acrylate - co -methyl Enteric coating that dissolves at pH
PlasACRYL T20 methacrylate -co -methacrylic 7.0 , enabling, the mini - tablets to
acid ) 7 :3 : 1 pass through the stomach and upper
small intestine unchanged and
release drug into the ileocecal
junction and colon ( ICR ).
PlasAcryl is an anti - tacking agent
coating additive that results in
shorter preparation and spraying
times
FD & C Blue No.2 FD & C Blue No.2 Pigment used to differentiate the
two enteric - coated mini - tablets to
facilitate encapsulation and ensure
quality control
Vcaps HPMC , size 1 , opaque white Capsule shell
capsule

30
The compatibility of lovastatin drug substance with for- Subsequent stress testing of enteric - coated lovastatin mini
mulation excipients was evaluated in binary stress testing tablets affirmed lovastatin moisture sensitivity and demon
studies where 1 : 1 mixtures of lovastatin and each excipient strated that the small amount of lovastatin degradation
were stored for 7 days under different conditions of tem- observed in the dosage form may be prevented by storage in
perature and relative humidity (RH ) . Samples were analyzed 35 a sealed container or by storage with aa desiccant ( see Table
by HPLC (based on USP methods) at day 0 and day 7. Data 5 below ) . Moisture barrier sub -coats, including SEPI
from binary stress testing studies with the present excipients FILMTM LP014 and LP030 ( SEPPIC ) , Opadry® amb II
are presented in Table 4 below : (Colorcon ), and Aquarius® MG (Ashland Aqualon Func
TABLE 4

Lovastatin degradant peak (% of lovastatin ) after

storage for 7 days at the indicated conditiona, b
Lovastatin 1 : 1 mixture 25 ° C./60% 40 ° C./75%
with indicated excipient Day 0 5 ° C. RH RH 50 ° C.

Alone (no excipient) 0.03 0.00 0.00 0.00 0.02

Kollidon VA64 Fine 0.04 0.05 0.04 0.05 0.04
Aerosil 200 0.07 0.09 0.09 0.23 0.10
Kollidon CL- F 0.05 0.05 0.05 0.06 0.05
Citric acid 0.38 0.63 0.69 0.23 0.64
EUDRAGIT L 30 D - 55 + 0.11 0.19 0.19 0.27 0.23
PlasACRYL HTP200
EUDRAGIT FS 30 D + 0.11 0.18 0.19 0.33 0.26
Plas ACRYL T20 °

HPLC relative retention time 0.46 min = lovastatin ß -hydroxyacid.

BUSP monograph requires individual impurities to be no more than 0.2 % .
High moisture content.

Binary stress testing identified that lovastatin lactone tional Ingredients) were evaluated during formulation devel
alone was stable over a range of conditions ; however, opment. The Acryl -EZE® (Colorcon ) pH 5.5 enteric coating
formulated lovastatin underwent a small amount of hydro- 65 was also evaluated in initial coat integrity testing in 0.1 M
lytic degradation to the B -hydroxyacid. This was exacer- HC1 . The EUDRAGIT polymers were chosen for use in the
bated in the presence of acidic materials such as citric acid . SYN -010 formulations .
 US 11,344,501 B2
47 48
TABLE 5
Composition of coated lovastatin -containing mini-tablets ( % )“
FORMULATION ANH - 056 ANH - 069 ANH - 073 ANH - 069 ANH - 069

Lovastatin lactone 14.0 % 12.3 % 12.9 % 12.3 % 12.3 %

Avicel PH102 70.0 % 61.7 % 64.4% 61.7 % 61.7 %
Kollidon VA64 Fine 7.0 % 6.2 % 6.4 % 6.2% 6.2 %
Aerosil 200 2.0 % 1.8 % 1.8 % 1.8 % 1.8 %
Magnesium stearate 1.0 % 0.9 % 0.9 % 0.9 % 0.9 %
Kollidon CL- F 6.0 % 5.3 % 5.5 % 5.3 % 5.3 %
Aquarius MG 4.3 % 4.3 % 4.3 %
EUDRAGIT FS 30 D + 7.6% 7.9 % 7.6 % 7.6 %
Plas ACRYL T20
TOTAL 100.0 % 100.0 % 100 / .0 % 100.0 % 100.0 %

Lovastatin degradant peak ( % of lovastatin ) after storage at

STRESS TESTING 40 ° C./75% RH in different containers in ==
2)
Container Open Dish Open Closed Closed Closed
Bottled Bottled Bottled
Bottled
Desiccant Silica gel
Day 0 not tested 0.12 , 0.13 0.15 , 0.14 0.12 , 0.13 0.12 , 0.13
Day 7 0.64 0.46 , 0.47 0.32 , 0.34 0.23 , 0.23 0.09 , 0.09
" All mini - tablets contained the same core (ANH - 056 ) prior to coating .
Moisture barrier sub - coat.
HPLC RRT 0.46 min = lovastatin B -hydroxyacid ; USP monograph limit no more than 0.2 % .
dHigh-density polyethylene (HDPE ) bottle.

The compositions of the lovastatin -containing, enteric

coated mini -tablets and placebo enteric - coated mini -tablets
are detailed in Table 6. The mini - tablets are round ( 5.5 mm
diameterx2.5 mm high ) with normal concavity.
TABLE 6
DR mini ICR mini
tablets tablets Placebo

Component Common Name Compendia mg % mg % mg %

Lovastatin Lovastatin lactone USP /NF 7.0 12.2 7.0 12.2 0.0
Avicel ® PH102 Cellulose , USP /NF 35.0 60.9 35.0 60.9 42.0 73.0
microcrystalline
Kollidon ® Copovidone USP /NF 3.5 6.1 3.5 6.1 3.5 6.1
VA64 Fine
Aerosil ® 200 Silicon dioxide USP /NF 1.0 1.7 1.0 1.7 1.0 1.7
( silica)
Magnesium Magnesium stearate USP /NF 0.5 0.9 0.5 0.9 0.5 0.9
stearate
Kollidon ® CL - F Crospovidone USP /NF 3.0 5.2 3.0 5.2 3.0 5.2
EUDRAGIT ® L Enteric polymer, pH USP /NF 7.5 13.0
30 D - 55 + 5.5 Poly (methacrylic USP /NF
Plas ACRYL TM acid -co - ethyl
HTP200 acrylate) 1 : 1
EUDRAGIT ® Poly (methyl Non 7.5 13.0 7.5 13.0
FS 30 D + acrylate - co -methyl compendial
Plas ACRYL TM methacrylate -co
T20 methacrylic acid)
7:3:1

Coated Mini 57.5 100.0 57.5 100.0 57.5 100.0

tablet Total

* FD & C Blue No. 2 Aluminum Lake 12-14% (0.0065 % of the EUDRAGIT L30 D - 55 coated mini -tablet weight) included
to allow visual differentiation of the DR mini - tablets
 US 11,344,501 B2
49 50
The compositions of SYN -010 21 mg and 42 mg capsule ing but was not otherwise processed to reduce particle size
dosage forms and placebos are further detailed in Table 7 or convert to an amorphous state . In the present indication
below : (IBS - C ) , systemic lovastatin bioavailability may not be
required and solubility may not a primary determinant of
TABLE 7 5 potential efficacy. Rather, lovastatin needs to disperse in the
PARAMETER
intestinal lumen, and dissolution studies have demonstrated
appropriate lovastatin release from the SYN -010 dosage
21 mg 42 mg Placebo form
MINI- TABLETS per CAPSULE Development of a product with the appropriate lovastatin
No. No. No.
10 release profile required detailed dissolution testing in media
of varying pH values that represented different regions of the
DR (pH 5.5 coated ) 1 1 intestinal tract. The dissolution strategy employed during
ICR (pH 7.0 coated ) 2 5 6 SYN -010 development is represented in FIG . 7. Dissolution
Total 3 6 6
studies utilized a Type 2 apparatus (as proscribed in the
15 lovastatin USP monograph ; Lovastatin USP 37 ) and evalu
COMPONENTS per CAPSULE ated a number of variables, including paddle speed and the
concentration of sodium dodecyl sulfate ( SDS ) included in
mg % mg % mg %
the dissolution medium . During development, it was deter
Lovastatin lactone 21.0 8.5 42.0 10.0 mined that an elevated paddle speed ( 100 rpm ) was unsuit
Avicel PH102 105.0 42.4 210.0 50.0 252.0 60.0 20 able for the integrity of the enteric coating while a lower
Kollidon VA64 Fine 10.5 4.2 21.0 5.0 21.0 5.0 paddle speed ( 50 rpm ) did not provide sufficient agitation of
Aerosil 200
Magnesium stearate
3.0
1.5
1.2
0.6
6.0
3.0
1.4
0.7
6.0
3.0
1.4
0.7
the dosage forms to ensure lovastatin dissolution . The SDS
Kollidon CL- F 9.0 3.6 18.0 4.3 18.0 4.3 concentrations in the dissolution medium at pH 5.9 ( 20 g / L )
EUDRAGIT L 30 D 7.5 3.0 7.5 1.8 and pH 7.0 ( 10.75 g/ L) were sufficient to enable appropriate
55 + PlasACRYL 25 dissolution of lovastatin ; however SDS concentrations of
HTP20 "
EUDRAGIT FS 30 D + 15.0 6.1 37.5 8.9 45.0 10.7
5-20 g / L in the acid medium (0.1 M HCl ) adversely
PlasACRYL T20 impacted the pH 5.5 enteric coating. This issue was resolved
Vcaps ® HPMC 75.0 30.3 75.0 17.9 75.0 17.9 by application of aa thicker coating of enteric polymer ( 15 %
capsule; white , opaque weight increase over the mini - tablet core ) that was used in
size 1 ° 30 the SYN -010 clinical formulation . A lower concentration of
SYN -010 Total 247.5 100.0 420.0 100.0 420.0 100.0 SDS (0.625 g/ L) was employed in the 0.1 M HCl dissolution
medium without adversely impacting lovastatin dissolution .
FD & C Blue No. 2 Aluminum Lake 12-14 % ( 0.0065 % of the EUDRAGIT L30 D - 55 Data from the dissolution studies of N- 010, 42 mg
coated mini - tablet weight) included to allow visual differentiation of the DR mini -tablets capsules are presented in FIG . 8. Each mini- tablet contained
35 the ANH -056 core . The 1xDR mini - tablet was coated with
The SYN -010 formulation takes advantage of ( i ) intesti- EUDRAGIT L 30 D -55 + Plas ACRYL HTP20 ( 15.55 %
nal regional differences in lovastatin hydrolysis and absorp- weight increase over the mini - tablet core ). The 5xICR
tion, and (ii ) intrinsic absorption differences between lov- mini -tablets was coated with EUDRAGIT FS 30 D + Pla
astatin lactone and B -hydroxyacid to increase the amount of SACRYL T20 ( 15.87 % weight increase ) . The HPMC cap
lovastatin lactone in the intestinal lumen and minimize the 40 sule shell dissolved within 10 minutes in 0.1 M HCl (rep
absorption of lovastatin species into the systemic circula- resenting the stomach ) to expose the lovastatin mini - tablets.
tion . Specifically enteric protection avoids gastric absorption All mini - tablets were stable in 0.1 M HC1 for 2 hours , and
and prevents conversion of the more poorly absorbed lov- no lovastatin or lovastatin degradation products were
astatin lactone ( the active antimethanogenic agent) to the observed in the acid medium . After 2 hours in 0.1 M HCI ,
more readily absorbed B -hydroxyacid (not antimethano- 45 the mini - tablets were transferred to a new well containing
genic ). In addition, the bulk of lovastatin released from pH 5.9 phosphate buffer ( representing the duodenum ) and
SYN -010, 21 mg and 42 mg occurs after the primary the 1xDR mini - tablet disintegrated and lovastatin dissolved
absorption lovastatin windows in the small intestine , thereby completely within 10 minutes. After 60 minutes at pH 5.9 ,
increasing delivery of lovastatin lactone to the colon . the pH was raised to pH 7.2 ( representing the ileum ) by
The primary absorption window for both lovastatin lac- 50 addition of NaOH , After a 30 min lag period , complete
tone and B -hydroxyacid is the small intestine; however, disintegration of the 5xICR tablets and dissolution of lov
there appears to be a meaningful gastric component to astatin was observed at pH 7.2 .
lovastatin oral absorption . For example, -30 % of an intra- The dissolution studies on SYN -010 , 42 mg capsules
gastric dose of either lovastatin lactone or B -hydroxyacid demonstrate that a dosage form comprising HPMC capsules
exited the gastric juice of pylorus- ligated rats within 30 min . 55 containing a combination of enteric- coated lovastatin mini
There appears to be relatively little pre -portal hydrolysis of tablets has the appropriate release profile to deliver lovas
lovastatin lactone in vivo after oral administration (~ 10 % ), tatin to the duodenum and the ileocecal junction /colon.
with the bulk of the lactone to B -hydroxyacid conversion Dissolution studies have also determined that the thick
occurring in the liver and the plasma . Studies also suggest ness of the mini - tablet enteric coating particularly the
that colonic bacteria may contribute to intestinal lovastatin 60 EUDRAGIT L 30 D - 55 — was important for ensuring mini
hydrolysis, and incubation of lovastatin lactone with human tablet integrity in stomach acid and thus the appropriate
and rat fecal bacterial enzyme fractions resulted in 8-19 % lovastatin release profile. As illustrated in Table 8 , when
loss of lovastatin lactone over a 12 h period. FIG . 6 shows combinations of mini - tablets with different coating thick
the estimated lovastatin lactone levels in the gastrointestinal nesses were stirred in 0.1 M HC1 , EUDRAGIT L 30 D - 55
tract after oral dosing. 65 coating thicknesses of less than 15 % failed . Specifically,
Lovastatin is a white to off-white crystalline powder that Table 8 shows the effect of different enteric coating thick
was co - milled and blended with excipients during process- nesses and on coat integrity of mini - tablets stirred in 0.1 M
 US 11,344,501 B2
51 52
HC1 (pH 1.2 ) for 120 min in a USP type 2 dissolution Generally , the majority of the best responses were seen in
apparatus at 75 rpm . SDS added to the dissolution medium patients receiving ALTOPREV alone or in combination with
to help solubilize lovastatin also adversely impacted the pH immediate - release lovastatin (e.g. MEVACOR ).
5.5 enteric coating , and reduced levels of SDS were used in Further, evaluation of the absolute change in breath
dissolution studies of the final SYN -010, 21 mg and 42 mg 5 methane levels from baseline showed a trend towards a
clinical dosage forms. greater breath methane- lowering effect at higher ALTO
TABLE 8
Tablet integrity over 120 min period at indicated SDS conc . ( g / L ) "
Coating Thickness (B = blister, R rupture , S = swell .)
=

pH wt . gain 0 0.625 1.25 2.5 5 20

5.56 9.56 % B, S , R B, S, R B, R
7.00 9.03 % No || 11 1

No No
change change change
7.00 11.4 % sd Se
5.55 15.55 % No No B, S, R B, S , R B, S, R
change change
7.00 15.89 % No No No No No
change change change change change
Identical ANH - 056 tablet cores
DEUDRAGIT ® L 30 D - 55 + PlasACRYL TM HTP20 .
EUDRAGIT ® FS 30 D + PlasACRYL TM T20 .
done of 6 tablets .
e4 of 6 tablets .

25
Stress - testing of enteric - coated lovastatin mini -tablets has PREV doses; however, there were a number of apparent
illustrated that SYN -010, 21 mg and 42 mg can be effec- non - responders (FIG . 9A) . This is perhaps seen more clearly
tively stored in closed HDPE containers containing a des- when comparing percentage change from baseline, where
iccant. SYN -010 ( 21 mg ) and SYN -010 ( 42 mg) clinical trial there was a division between ALTOPREV responders and
materials were packaged in separate 60 mL high - density 30 apparent non -responders with no obvious dose response
polyethylene (HDPE ) wide -mouth round bottles with aa 33 amongst the responders (FIG . 9B )
mm polypropylene child -resistant closure and an induction When reviewing the absolute change in breath methane
foil inner seal . Each bottle contained 33 SYN -010 capsules levels , there appears to be an almost linear trend, with the
with aa CAN SORB - IT® desiccant canister containing 1.0 g patients having highest baseline breath methane levels
of silica gel desiccant. The capsules are stored at 20-25 ° C. 35 showing the greatest absolute reductions in breath methane
regardless of the ALTOPREV dose (FIG . 9C ) . In this analy
Example 3 : Clinical Evaluation of Different sis , there was a group of apparent non -responders with
Release Profiles varying baseline methane levels. Comparison of the per
centage change in breath methane vs. baseline breath meth
Duodenal and ileocecal release profiles are compared 40 ane ( FIG . 9D ) showed a separation between ALTOPREV
separately and in combination to evaluate any benefit of one responders and apparent non - responders, again , with no
over the other or synergy in the combination . Further an obvious dose response amongst the responders.
evaluation of the pharmacokinetics and breath methane
effects of different doses and dosing profiles in methane 45 Example 4 : In Vivo Effects of Lovastatin on M.
positive subjects may be undertaken . smithii Colonized Rats with Constipation
Example 3 : Clinical Selection of Responder 30 adult, male Sprague - Dawley rats were placed on a
Patients high - fat diet ( 60.3 % kcal from fat, Teklad high -fat diet
TD.06414 , Harlan Laboratories Inc, Madison, Wis .) for 7
In this study, a retrospective chart review from the last 18 50 weeks. The rats were assessed for increased M. smithii by
months of clinical practice was undertaken for the use of qPCR before and after the diet , and then divided into 3
statins in treating patients with methane -positive bacterial groups. Group 1 was given lovastatin in its lactone form ,
overgrowth and the constipation -predominant form of IBS Group 2 was given lovastatin hydroxy acid (each 1.5
( C - IBS ) . While constipation and bloating severity were in mg / rat ), and Group 3 was gavaged with a placebo . Each
general proportional to the reduction in methane, this was 55 group was gavaged daily for 10 days. Three day stool
not a prospective study, and symptoms were subjective. The collections were performed to assess average stool wet
chart review therefore focused on the reduction of methane weight and daily variability prior to commencing the high
production . As data for methane were not normally distrib- fat diet , after 7 weeks of high - fat diet , and the final days of
uted , data were represented as medians and a non -parametric the lovastatin gavage ( still on high - fat diet ). On day 10 of the
test Mann -Whitney test was used to compare groups. Most 60 gavage, rats were euthanized and DNA was extracted from
of the methane positive IBS patients with constipation contents of ligated bowel segments ( duodenum , jejunum ,
evaluated were first treated with a course of rifaximin and ileum , cecum and left colon) . qPCR was performed using
neomycin. Subjects placed on statin therapy were those that primers for total luminal bacteria and M. smithii .
were resistant or refractory to this conventional antibiotic Results indicate that high - fat diet augmented stool M.
approach . This could also imply, without wishing to be 65 smithii colonization in Sprague - Dawley rats (7.58 %
bound by theory , that they are more refractory to treatment 10 16.62x104 cfu /mL at baseline to 2.60x10 + 1.95x105
in general. after 7 weeks of high - fat) ( P <0.01 ) ( FIG . 10A) . This was
 US 11,344,501 B2
53 54
coupled with a reduction in the stool wet-weights ( 62.4 % at
a and B -hydroxyacid concentrations (ITlag 1.0-2.0h) and a later
baseline to 48.6 % after 7 weeks) ( P <0.01 ) ( FIG . 10B ) . At first peak plasma concentration ( Tpeak
, ,1 2.0-6.0 h) for Dose
this point rats were divided into 3 groups. With respect to the A compared to the MEVACOR immediate release formula
total bacteria by qPCR , levels were not different between tion (Tlag 0.5-1.0 h and Tpeak ,1 1.0-2.0 h) . As observed for
placebo and either lovastatin group . For M. smithii, the ratio 5 MEVACOR, Dose A also demonstrated a large mean Cpeak,2
of M. smithii to total bacteria was reduced in the ileum of that was predominantly due to two dogs . This second peak
rats given the lovastatin lactone but not hydroxy acid . M. may reflect delayed release of one or more mini -tablets from
smithii levels in the colon were unaffected (FIG . 11 ). the stomach of these animals. Published reports have iden
tified that the dog pylorus is more restrictive than the human
Example 5 : Pharmacokinetics of SYN - 010 in Dogs 10 pylorus, and particles 25 mm in diameter ( such as the
SYN - 010 mini - tablets ) tend to be retained in the stomach
The SYN -010 formulation comprises capsules containing until expelled with the next GI housekeeper wave (Phase III
a combination of different enteric - coated mini - tablets of the migrating motor complex ), regardless of prandial
designed to pass through the stomach unchanged and release state . The time between housekeeper waves in fed dogs
lovastatin in different areas of the intestinal tract. The 15 ( 5-13 h ) is highly variable and significantly longer than
present study evaluated the plasma pharmacokinetics of observed in fed humans ( 2-5 h) . In the present study, food
lovastatin lactone and B -hydroxyacid after administration of was restored to dogs 2.0-2.5 h post - dose . If SYN - 010
the different SYN - 010 lovastatin enteric - coated mini-tab- mini -tablets were administered to fasted dogs immediately
lets alone and in combination to beagle dogs, Animals after a housekeeping wave , and one or more mini- tablets did
were also administered commercially available immediate 20 not exit the stomach, these mini- tablets could be retained in
release and extended release formulations of lovastatin . the stomach for a significant period of time prior to release
Dogs have previously been shown to be appropriate for with the next housekeeper wave .
studying lovastatin disposition and have a gastrointestinal The results obtained with the ICR mini - tablets ( Dose B )
tract with many similarities to humans. Five dogs ( 6.4-8.0 kg and the 1xDR + 5xICR combination (Dose C ) were compel
body weight) were randomized to receive each of the 25 ling with respect to the potential utility of these formulations
following doses using a Latin square dose design, i.e. , each in IBS - C . The very low to undetectable levels of lovastatin
dog received each dose during the study, separated by a one lactone and B -hydroxyacid after administration of Dose B
week washout period: Dose A 6xpH 5.5 - coated lovastatin ( 7 suggest negligible lovastatin absorption from the GI tract
mg ) mini - tablets ( duodenal release ; DR) ; total dose 42 mg ; and retention of lovastatin lactone in the intestinal lumen .
Dose B : 6xpH 7.0 - coated lovastatin ( 7 mg ) mini - tablets 30 No undisintegrated mini - tablets or tablet fragments were
( ileocecal release ; ICR) ; total dose 42 mg ; Dose C : 1xDR + reported in dog feces during routine cage - side observations .
5xICR lovastatin ( 7 mg ) mini -tablets; total dose 42 mg ; Dose C delivered low systemic lovastatin levels (i.e. the
Dose D : 1xMEVACOR immediate release lovastatin tablet ; mean dose -normalized lovastatin lactone AUC was 56 % of
total dose 40 mg ; Dose E : 1xALTOPREV extended release the mean dose - normalized MEVACOR AUC ) and exhibited
lovastatin tablet ; total dose 40 mg . 35 a dual pulse release profile, with two peak concentrations for
All doses were administered in a single Torpac size 000 each analyte separated by ~ 14 h . As for Dose A , the second
gelatin capsule. Dogs were fasted overnight prior to dosing peak was largely due to two dogs that had very large Cpeak ,2.
and food was restored 2.0-2.5 h post - dose . Blood samples Considering the negligible plasma levels of lovastatin lac
were taken from each dog over a 36 h time - period and tone and B -hydroxyacid observed with the ICR component
plasma was analyzed for lovastatin lactone and lovastatin 40 alone (Dose B ) , the plasma concentration vs. time profiles
B -hydroxyacid using a qualified LC - MS /MS method . Phar- for these analytes in Dose C appears to be predominantly
macokinetic parameters were calculated using non -compart- due to the DR component of the formulation .
mental methods. SYN -010 mini- tablets were among the drug products used
Mean concentration versus time profiles for the different in this study. Each enteric - coated mini -tablet contains 7 mg
doses are presented in FIG . 12. Plasma levels of lovastatin 45 of lovastatin combined with USP excipients and coated with
B -hydroxyacid tracked almost identically with lovastatin a EUDRAGIT® enteric polymer that dissolves at either pH
lactone , consistent with published reports that conversion of 5.5 ( DR) or pH 7.0 (ICR) . Each mini- tablet is circular in
lactone to B -hydroxyacid occurs predominantly after shape, with diameter ~ 5 mm , height ~ 3 mm , and weight ~ 54
absorption from the GI tract . The AUCacid / AUClactone mg . DR mini - tablets have a pale blue color while ICR
ratio ( 1.5-1.7 ) was not different for Doses A , C , D and E ; but 50 mini -tablets are white . MEVACOR 40 mg IR lovastatin
was only 0.8 for Dose B , due to very low lovastatin tablets ; ALTOPREV 40 mg XR lovastatin tablets and vet
absorption from the Dose B formulation . erinary size 000 porcine gelatin capsules ( Torpac, Fairfield
The comparative pharmacokinetic behaviors of MEVA- N.J. ) were also used . All materials were ready to use and
COR and ALTOPREV in this dog study were consistent with maintained at room temperature; ALTOPREV and MEVA
published clinical studies and pharmacokinetic parameters 55 COR were stored desiccated in the dark .
for these formulations were similar to those reported in
published dog studies . A key difference in the current work Example 6 : Phase 2 Clinical Trial of SYN -010 for
was the presence of a large second peak concentration IBS - C
( Cpeak ,2) of both lactone and B -hydroxyacid in some dogs , 60
which has not previously been reported. A Phase 2 , randomized , double -blind , parallel -group, pla
The DR mini- tablets ( Dose A) provided similar overall cebo - controlled, multi - dose study is being conducted . The
lovastatin exposure ( AUC ) to the MEVACOR and ALTO- primary objective of this study is to evaluate the change
PREV formulations; however, unlike MEVACOR , the phar- from baseline in breath methane, as determined by a lactu
macokinetic profile for Dose A indicated that the pH 5.5 lose breath test , in methane -positive patients with IBS - C
enteric coating delayed lovastatin release until the mini- 65 after seven days of treatment with one of two formulations
tablets reached the upper small intestine . This was reflected of SYN -010 compared with placebo . Approximately 60
in longer times before the first measurable lovastatin lactone patients are being enrolled and randomly assigned in a 1 : 1 : 1
 US 11,344,501 B2
55 56
ratio to one of three groups , including two different SYN- at least about 10 -fold , at least about 50 - fold , at least about
010 dose groups, 21 mg and 42 mg , and a placebo group . 100 - fold , in the presence of an agent or stimulus, relative to
Patients are scheduled to receive single oral doses of SYN- the absence of such agent or stimulus.
010 each day for 28 days. Sixty subjects with who are As referred to herein , all compositional percentages are by
between the ages of 18 and 65 , inclusive, are being enrolled. 5 weight of the total composition, unless otherwise specified.
Inclusion criteria are: subjects must have IBS - C and have As used herein , the word " include, " and its variants , is
a positive breath methane test result (> 10 ppm) at screening, intended to be non - limiting, such that recitation of items in
subject must meet the modified Rome III criteria for IBS - C , a list is not to the exclusion of other like items that may also
subject must have an average abdominal pain intensity score be useful in the compositions and methods of this technol
of 23 ( scale 0-10) reported at screening and baseline, subject 10 ogy. Similarly, the terms “ can ” and “ may ” and their variants
must have an average of fewer than 3 complete spontaneous are intended to be non - limiting, such that recitation that an
bowel movement (CSBMs ) per week and subject must agree embodiment can or may comprise certain elements or fea
to refrain from making any lifestyle changes that may affect tures does not exclude other embodiments of the present
IBS - C symptoms from the time of screening to the end of the technology that do not contain those elements or features .
study. 15 Although the open - ended term “ comprising , ” as a syn
Exclusion Criteria are : subject has taken IBS treatments onym of terms such as including, containing, or having , is
( prescription or over -the-counter ), proton pump inhibitors , used herein to describe and claim the invention , the present
laxatives, antibiotics, subject currently has any structural invention , or embodiments thereof, may alternatively be
abnormality of the gastrointestinal ( GI ) tract or a disease or described using alternative terms such as “ consisting of ” or
condition that can affect GI motility, or any unexplained and 20 “ consisting essentially of . ”
clinically significant symptoms such as lower GI bleeding, As used herein , the words “ preferred ” and “ preferably ”
rectal bleeding , heme -positive stool , iron -deficiency anemia , refer to embodiments of the technology that afford certain
weight loss , or systemic signs of infection, subject has been benefits, under certain circumstances. However, other
diagnosed with or has a family history of familial adeno- embodiments may also be preferred, under the same or other
matous polyposis , hereditary nonpolyposis colorectal can- 25 circumstances. Furthermore, the recitation of one or more
cer, or any other form of familial colorectal cancer, and preferred embodiments does not imply that other embodi
subject reports loose (mushy) or watery stools (Bristol Stool ments are not useful, and is not intended to exclude other
Form Scale [ BSFS ] score of 6 or 7 ) . embodiments from the scope of the technology.
A decrease from baseline in breath methane, as deter- The amount of compositions described herein needed for
mined by a lactulose breath test , in methane- positive 30 achieving a therapeutic effect may be determined empiri
patients with IBS - C is expected . cally in accordance with conventional procedures for the
particular purpose. Generally, for administering therapeutic
Definitions agents (e.g. , antimethanogenic statins and /or additional
therapeutic agents described herein ) for therapeutic pur
As used herein , “ a , ” “ an ,” or “ the ” can mean one or more 35 poses , the therapeutic agents are given at a pharmacologi
than one . cally effective dose . A “ pharmacologically effective
Further, the term “ about” when used in connection with a amount , ” “ pharmacologically effective dose,” “ therapeuti
referenced numeric indication means the referenced numeric cally effective amount," or " effective amount” refers to an
indication plus or minus up to 10 % of that referenced amount sufficient to produce the desired physiological effect
numeric indication . For example , the language “ about 50 % ” 40 or amount capable of achieving the desired result, particu
covers the range of 45 % to 55 % . larly for treating the disorder or disease . An effective amount
An “ effective amount, ” when used in connection with as used herein would include an amount sufficient to , for
medical uses is an amount that is effective for providing a example , delay the development of a symptom of the
measurable treatment, prevention, or reduction in the rate of disorder or disease , alter the course of a symptom of the
pathogenesis of a disorder of interest. 45 disorder or disease ( e.g. , slow the progression of a symptom
As used herein, something is “ decreased ” if a read - out of of the disease) , reduce or eliminate one or more symptoms
activity and /or effect is reduced by a significant amount , or manifestations of the disorder or disease , and reverse a
such as by at least about 10% , at least about 20 % , at least symptom of aa disorder or disease . Therapeutic benefit also
about 30% , at least about 40 % , at least about 50 % , at least includes halting or slowing the progression of the underlying
about 60% , at least about 70 % , at least about 80 % , at least 50 disease or disorder, regardless of whether improvement is
about 90% , at least about 95 % , at least about 97 % , at least realized .
about 98 % , or more , up to and including at least about 100 % , Effective amounts, toxicity, and therapeutic efficacy can
in the presence of an agent or stimulus relative to the be determined by standard pharmaceutical procedures in cell
absence of such modulation . As will be understood by one cultures, tissue samples, tissue homogenates or experimental
of ordinary skill in the art, in some embodiments, activity is 55 about
decreased and some downstream read - outs will decrease but
animals50, e.g., for determining the LD50 (the dose lethal to
% of the population) and the ED50 ( the dose
others can increase. therapeutically effective in about 50 % of the population ).
Conversely, activity is “ increased ” if a read - out of activity The dosage can vary depending upon the dosage form
and / or effect is increased by a significant amount, for employed and the route of administration utilized . The dose
example by at least about 10% , at least about 20% , at least 60 ratio between toxic and therapeutic effects is the therapeutic
about 30% , at least about 40 % , at least about 50 % , at least index and can be expressed as the ratio LD50/ED50 . In some
about 60% , at least about 70 % , at least about 80 % , at least embodiments , compositions and methods that exhibit large
about 90% , at least about 95 % , at least about 97 % , at least therapeutic indices are preferred. A therapeutically effective
about 98 % , or more , up to and including at least about 100 % dose can be estimated initially from in vitro assays , includ
or more, at least about 2 - fold , at least about 3 - fold , at least 65 ing , for example, cell culture assays or measurements or
about 4 -fold , at least about 5 - fold , at least about 6 - fold , at methane production in stool samples. Also , a dose can be
least about 7 - fold , at least about 8 - fold , at least about 9 - fold , formulated in animal models to achieve a circulating plasma
 US 11,344,501 B2
57 58
concentration range that includes the IC50 as determined in about 0.1-3.0 % by weight magnesium stearate ;
cell culture, or in an appropriate animal model. Levels of the about 1-10 % by weight crospovidone; and
described compositions in plasma can be measured, for about 10-20% by weight enteric polymer;
example , by high performance liquid chromatography. The wherein aa first type of modified -release particle comprises
effects of any particular dosage can be monitored by a 5 a first enteric polymer that dissolves at a pH of about
suitable bioassay. The dosage can be determined by a 5.5 and a second type of modified - release particle
physician and adjusted , as necessary , to suit observed effects comprises a second enteric polymer that dissolves at a
of the treatment.
pH of about 7.0 ,
wherein the first type of modified - release particle and the
In certain embodiments, the effect will result in a quan 10 second type of modified - release particle are present in
tifiable change of at least about 10 % , at least about 20 % , at the pharmaceutical formulation at a ratio of from about
least about 30 % , at least about 50 % , at least about 70% , or 1 : 2 to about 1 : 5 , and
at least about 90 % . In some embodiments , the effect will wherein the formulation has a unit dosage of about 21 to
result in a quantifiable change of about 10 % , about 20 % , 42 mg of the antimethanogenic statin .
about 30 % , about 50 % , about 70 % , or even about 90 % or 2. The method of claim 1 , wherein each modified -release
more . Therapeutic benefit also includes halting or slowing 15 particle contains:
about 12 % by weight antimethanogenic statin ;
the progression of the underlying disease or disorder, regard about 61 % by weight microcrystalline cellulose ;
less of whether improvement is realized . about 6 % by weight copovidone;
As used herein , “ methods of treatment” are equally appli- about 2 % by weight silicon dioxide ;
cable to use of a composition for treating the diseases or 20 about 1 % by weight magnesium stearate;
disorders described herein and / or compositions for use and / about 5 % by weight crospovidone; and
or uses in the manufacture of a medicaments for treating the about 15 % by weight of the first or the second enteric
diseases or disorders described herein . polymer, and
about 7 mg of the antimethogenic statin .
EQUIVALENTS 25 3. The method of claim 1 , wherein the antimethanogenic
statin is lovastatin in the lactone form .
While the invention has been described in connection 4. The method of claim 2 , wherein the antimethanogenic
with specific embodiments thereof, it will be understood that statin is lovastatin in the lactone form .
it is capable of further modifications and this application is 5. The method of claim 3 , wherein the systemic absorp
intended to cover any variations, uses , or adaptations of the 30 tion of the lovastatin is insufficient to provide a clinically
invention following, in general, the principles of the inven effective
6. The
reduction in cholesterol.
method of claim 1 , wherein the first type of
tion and including such departures from the present disclo modified - release
sure as come within known or customa practice within the release particle areparticle and the second type of modified
art to which the invention pertains and as may be applied to tion at a ratio of about 1 : 2 . in the pharmaceutical formula
present
the essential features hereinbefore set forth and as follows in 35 7. The method of claim 1 , wherein the first type of
the scope of the appended claims . modified -release particle and the second type of modified
Those skilled in the art will recognize, or be able to release particle are present in the pharmaceutical formula
ascertain , using no more than routine experimentation, tion at a ratio of about 1 : 4 .
numerous equivalents to the specific embodiments described 8. The method of claim 1 , wherein the first type of
specifically herein . Such equivalents are intended to be 40 modified - release particle and the second type of modified
encompassed in the scope of the following claims . release particle are present in the pharmaceutical formula
tion at a ratio of about 1 : 5 .
INCORPORATION BY REFERENCE 9. The method of claim 1 , wherein each modified -release
particle is aa microbead or mini - tablet.
All patents and publications referenced herein are hereby 45 10. The method of claim 3 , wherein the first enteric
incorporated by reference in their entireties . polymer releases the lovastatin into the duodenum .
The publications discussed herein are provided solely for 11. The method of claim 3 , wherein the second enteric
their disclosure prior to the filing date of the present appli- polymer releases the lovastatin into the ileocecal junction .
cation. Nothing herein is to be construed as an admission 12. The method of claim 1 , wherein the pharmaceutical
that the present invention is not entitled to antedate such 50 formulation is a capsule or tablet .
publication by virtue of prior invention . 13. The method of claim 1 , wherein the pharmaceutical
As used herein , all headings are simply for organization formulation is suitable for oral administration .
and are not intended to limit the disclosure in any manner. 14. The method of claim 1 , wherein the first enteric
The content of any individual section may be equally polymer that dissolves at a pH of about 5.5 is poly (meth
applicable to all sections . 55 acrylic acid -co-ethylacrylate) 1 : 1.
15. The method of claim 1 , wherein the second enteric
What is claimed is : polymer that dissolves at a pH of about 7.0 is poly (methyl
1. A method of treating constipation - associated IBS ( IBS- acrylate - co -methyl methacrylate -co -methacrylic acid ) 7 : 3 : 1 .
C ) , comprising administering an effective amount of a 16. The method of claim 1 , comprising administering to
pharmaceutical formulation to a patient in need thereof, the 60 the patient an additional therapeutic agent .
pharmaceutical formulation comprising at least two types of 17. The method of claim 1 , wherein the additional thera
modified - release particles, each modified - release particle peutic agent comprises a laxative, guanylate cyclase C
comprising : agonist , a serotonin agonist , a chloride channel agonist, or a
about 5-20 % by weight antimethanogenic statin ; combination thereof.
about 50-70% by weight microcrystalline cellulose ; 65 18. The method of claim 16 , wherein the additional
about 1-10 % by weight copovidone ; therapeutic agent comprises a selective chloride channel
about 0.1-3.0 % by weight silicon dioxide ; activator.
 US 11,344,501 B2
59 60
19. The method of claim 18 , wherein the selective chlo
ride channel activator is derived from prostaglandins.
20. The method of claim 16 , wherein the additional
therapeutic agent comprises an anticholinergic or antide
pressant. 5
21. The method of claim 16 , wherein the additional
therapeutic agent comprises a fiber supplement.
22. The method of claim 16 , wherein the additional
therapeutic agent comprises a probiotic that inhibits growth
of methanogens. 10