ABE 51100 Drug Development Semester Project

Development of a Novel Plasminogen Inhibitor

Please note due dates and connections to September symposium

activities at end of document

NOTES:

- This is a comprehensive semester project aimed at exposing you to

drug development from the GMP viewpoint.
- The case study outlines a new drug PlasminStop but you will need to assume
PlasminStop has the same structure, properties, and marketing image as
Tranexamic Acid to answer the questions. For example, if you are asked for the
TPP for plasmin stop you will assume PlasminStop has the same structure,
solubility, dosage, synthetic method EVERYTHING as Tranexamic Acid (TXA).
- You will need to use the mockIND provided as a template to prepare your IND
- The title of your IND will be PlasminStop but the information in each section is
for Tranexamic Acid (TXA)

Background

BioPharmX is developing a novel drug "PlasminStop", a synthetic molecule designed to

inhibit plasminogen activation more effectively than tranexamic acid. This drug targets a
key step in the fibrinolysis pathway, offering potential improvements in managing
conditions associated with excessive bleeding such as surgery, trauma, and certain
bleeding disorders.

Objective

Prepare an Investigational New Drug (IND) application for PlasminStop, addressing all the
necessary regulatory, scientific, and clinical aspects to ensure successful submission and
approval to commence human clinical trials, with a strong emphasis on Good
Manufacturing Practice (GMP) compliance.
 Milestone 2: Drug Substance Characterization

Assignment:

1. Describe the chemical synthesis pathway for PlasminStop. Identify potential

challenges in scaling up production. How does the synthesis pathway illustrate
the functional role of discovery chemistry in drug development? Consider ICH
Ǫ11 for development and manufacture of drug substances. How will you ensure
GMP compliance during this process? (NOTE: As mentioned above assume
Plasmin Stop has the same structure and synthetic route as TXA to answer this
question)
2. List the analytical methods and tests required to characterize the drug
substance, including methods for determining purity, potency, impurity profiling,
and stability. How do these methods align with GMP requirements and ICH
guidelines, particularly ICH Ǫ3A/B for impurities and ICH Ǫ6A for
specifications? What is their importance in ensuring the quality and safety of the
drug substance? (NOTE: As mentioned above assume Plasmin Stop has the
same analytical methods as TXA to answer this question)
3. Evaluate potential impurities and their impact on the drug substance's quality and
safety. Use the ICH Ǫ6A decision tree to establish acceptance criteria for
impurities and degradation products. How do impurity profiling and stability
testing contribute to the overall drug development process according to ICH Ǫ3C
for residual solvents and ICH Ǫ3D for elemental impurities? (NOTE: As
mentioned above assume Plasmin Stop has the same impurities as TXA to
answer this question)

Synthesis Pathway Overview:

The synthesis of PlasminStop involves three main steps:

1. Hydrogenation of 4-Methylbenzoic Acid to 4-Methylcyclohexanecarboxylic Acid

2. Bromination of the Methyl Group to Form 4-(Bromomethyl)cyclohexanecarboxylic
Acid
3. Amination to Yield trans-4-(Aminomethyl)cyclohexanecarboxylic Acid (PlasminStop)

Step 1: Hydrogenation of 4-Methylbenzoic Acid

Chemical Reaction:
Explanation:

 Starting Material: 4-Methylbenzoic acid (p-toluic acid) contains an aromatic benzene ring
with a methyl group and a carboxylic acid group.
 Process: Catalytic hydrogenation saturates the aromatic ring by adding hydrogen atoms to
the double bonds.
 Catalyst and Conditions: Palladium on carbon (Pd/C) is used as a catalyst under high
hydrogen pressure and elevated temperatures.
 Outcome: The aromatic ring is converted into a cyclohexane ring, forming 4-
methylcyclohexanecarboxylic acid.

Visual Representation:

 The benzene ring loses its double bonds and becomes a cyclohexane ring.
 The positions of the methyl and carboxylic acid groups remain unchanged.

Step 2: Bromination of the Methyl Group

Chemical Reaction:

Explanation:

 Process: The methyl group attached to the cyclohexane ring undergoes bromination via a
free radical substitution mechanism.
 Reagents: Bromine (Br2\text{Br}_2Br2) or N-bromosuccinimide (NBS) can be used in the
presence of heat (Δ\DeltaΔ) or light (hνh\nuhν) to initiate the reaction.
 Mechanism: A hydrogen atom from the methyl group is replaced by a bromine atom.
 Outcome: Formation of 4-(bromomethyl)cyclohexanecarboxylic acid.
Visual Representation:

Step 3: Amination

Chemical Reaction:

Explanation:

 Process: Nucleophilic substitution where the bromine atom is replaced by an amino group.
 Reagents: Excess ammonia (NH3\text{NH}_3NH3) acts as a nucleophile.
 Mechanism: Ammonia attacks the carbon atom attached to bromine, displacing the
bromide ion.
 Outcome: Formation of trans-4-(aminomethyl)cyclohexanecarboxylic acid, which is
PlasminStop.

Visual Representation:

Potential Challenges in Scaling Up Production:

Safety Risks:

1. High-Pressure Reactions:
o Issue: The hydrogenation step requires high-pressure hydrogen gas, which is
flammable and poses explosion risks.
o Mitigation: Use of specialized high-pressure reactors with safety features and strict
operational protocols.
2. Handling Hazardous Chemicals:
 o Issue: Bromine and brominated compounds are toxic and corrosive.
o Mitigation: Employ closed systems, proper personal protective equipment (PPE),
and efficient ventilation.

Process Efficiency:

1. Reaction Control:
o Challenge: Achieving complete hydrogenation without over-reduction or side
reactions.
o Solution: Optimize catalyst loading, temperature, pressure, and reaction time.
2. Purity Levels:
o Challenge: Removal of impurities, especially residual bromine and by-products.
o Solution: Implement effective purification steps like recrystallization or
chromatography.

Environmental Concerns:

1. Waste Management:
o Issue: Disposal of hazardous waste, including spent catalysts and brominated by-
products.
o Mitigation: Treat waste streams according to environmental regulations and explore
recycling options.
2. Emission Control:
o Issue: Release of toxic gases like hydrogen bromide (HBr).
o Mitigation: Use scrubbers and containment systems to capture and neutralize
emissions.

Equipment and Facility Requirements:

1. Specialized Equipment:
o Need: High-pressure reactors, corrosion-resistant materials, and explosion-proof
facilities.
o Action: Invest in appropriate equipment and conduct regular maintenance and
validation.
2. Facility Upgrades:
o Consideration: Scaling up may require larger reactors and enhanced safety
systems.
o Action: Plan facility expansions and upgrades to accommodate increased
production volume.

Functional Role of Discovery Chemistry in Drug Development:

Process Optimization:

1. Efficiency Improvement:
o Goal: Develop synthetic routes with higher yields and fewer steps.
o Approach: Modify reaction conditions, catalysts, or solvents to enhance efficiency.
2. Scalability Assessment:
 o Importance: Ensure that laboratory-scale methods can be translated to industrial-
scale production.
o Method: Conduct pilot-scale studies to identify potential scale-up issues.

Innovation in Synthesis:

1. Alternative Pathways:
o Exploration: Investigate new reactions or catalysts that might simplify the
synthesis.
o Example: Using biocatalysis for hydrogenation to reduce pressure requirements.
2. Sustainable Practices:
o Objective: Reduce environmental impact by minimizing waste and using greener
reagents.
o Strategy: Apply principles of green chemistry, such as using renewable solvents.

Quality by Design (QbD):

1. Understanding Critical Parameters:

o Focus: Identify key variables that affect product quality (e.g., temperature, pH,
reagent purity).
o Tool: Use design of experiments (DoE) to systematically study parameter effects.
2. Risk Mitigation:
o Proactive Measures: Anticipate potential problems in manufacturing and develop
contingency plans.
o Documentation: Create risk assessment reports and control strategies.

Ensuring GMP Compliance During the Process:

Adherence to ICH Q11 Guidelines:

1. Process Development:
o Requirement: Thorough understanding of the process, including chemistry,
controls, and potential impurities.
o Documentation: Develop detailed process descriptions and flow diagrams.
2. Control Strategy:
o Implementation: Establish specifications for raw materials, intermediates, and
critical process parameters.
o Monitoring: Use in-process controls to ensure consistency and quality.

Quality Management Systems:

1. Standard Operating Procedures (SOPs):

o Purpose: Provide clear instructions for each manufacturing step to ensure
consistency.
o Maintenance: Regularly review and update SOPs as processes evolve.
2. Documentation:
o Importance: Maintain records for traceability, including batch records, deviation
reports, and change controls.
 o Compliance: Ensure documentation meets regulatory standards for audits and
inspections.

Personnel Training:

1. GMP Training Programs:

o Necessity: All staff must understand GMP principles and their role in compliance.
o Content: Training on SOPs, safety protocols, and regulatory requirements.
2. Continuous Education:
o Updates: Keep personnel informed about new regulations, technologies, and best
practices.
o Methods: Regular training sessions, workshops, and certifications.

Equipment and Facility Validation:

1. Qualification of Equipment:
o Steps: Perform Installation Qualification (IQ), Operational Qualification (OQ), and
Performance Qualification (PQ).
o Outcome: Ensure equipment operates correctly and consistently.
2. Facility Compliance:
o Standards: Maintain cleanrooms, controlled environments, and proper storage
conditions.
o Monitoring: Regular environmental monitoring for parameters like temperature,
humidity, and microbial contamination.

Quality Control and Assurance:

1. In-Process Testing:
o Purpose: Detect deviations early to prevent batch failures.
o Examples: Monitoring reaction completeness, pH levels, and impurity profiles.
2. Final Product Testing:
o Rigorous Testing: Analyze the final product for purity, potency, and compliance
with specifications.
o Release Criteria: Only batches that meet all quality standards are approved for use.

By thoroughly understanding and controlling each step of the synthesis and ensuring
compliance with GMP and ICH guidelines, the development of PlasminStop can proceed
efficiently while maintaining the highest standards of quality and safety.

2. Analytical Methods and Tests Required to Characterize the Drug Substance:

To ensure the quality, safety, and efficacy of PlasminStop, a comprehensive set of analytical
methods is employed:
Identification Tests:

1. Infrared Spectroscopy (IR):

o Purpose: Confirms the presence of specific functional groups (e.g., amino,
carboxylic acid).
o Process: Measures absorption of infrared light at characteristic wavelengths.
o Interpretation: Compare the IR spectrum of the sample with that of a reference
standard.
2. Nuclear Magnetic Resonance (NMR) Spectroscopy:
o Purpose: Provides detailed structural information.
o Process: Analyzes the magnetic properties of atomic nuclei (usually 1H^1\
text{H}1H and 13C^{13}\text{C}13C).
o Interpretation: Confirms the chemical structure by comparing chemical shifts and
coupling patterns.
3. Mass Spectrometry (MS):
o Purpose: Determines the molecular weight and fragmentation pattern.
o Process: Ionizes the compound and measures the mass-to-charge ratio of ions.
o Interpretation: Confirms molecular mass and identifies potential impurities.

Purity Assessment:

1. High-Performance Liquid Chromatography (HPLC):

o Purpose: Quantifies PlasminStop and detects impurities.
o Process: Separates compounds based on their interactions with the stationary and
mobile phases.
o Detection: Uses UV, DAD, or MS detectors for sensitive analysis.
2. Gas Chromatography (GC):
o Purpose: Used if volatile impurities or residual solvents are present.
o Process: Separates volatile compounds through a heated column.
o Detection: Often coupled with flame ionization detection (FID) or MS.

Impurity Profiling:

1. HPLC with Diode-Array Detection (HPLC-DAD):

o Purpose: Detects and quantifies related substances.
o Advantage: Simultaneous monitoring at multiple wavelengths increases sensitivity.
2. Liquid Chromatography-Mass Spectrometry (LC-MS):
o Purpose: Identifies unknown impurities and degradation products.
o Process: Combines separation power of LC with the identification capabilities of
MS.

Potency Determination:

 Assay by HPLC:
 o Purpose: Measures the concentration of PlasminStop in the sample.
o Requirement: Must be accurate, precise, and validated according to ICH
guidelines.

Residual Solvents Analysis:

 Headspace Gas Chromatography (Headspace GC):

o Purpose: Quantifies residual organic solvents as per ICH Q3C guidelines.
o Process: Volatilizes solvents from the sample matrix into the headspace for
analysis.

Elemental Impurities Testing:

 Inductively Coupled Plasma Mass Spectrometry (ICP-MS):

o Purpose: Detects trace levels of elemental impurities (e.g., Pd, Br) according to
ICH Q3D.
o Sensitivity: Capable of detecting parts per billion (ppb) levels.

Stability Testing:

1. Stress Testing:
o Purpose: Exposes the drug to stress conditions (heat, light, humidity) to identify
degradation products.
o Outcome: Helps in understanding degradation pathways and shelf-life.
2. Stability-Indicating Methods:
o Requirement: Analytical methods (usually HPLC) that can separate and quantify
the drug substance and its degradation products.

Physical Characterization:

1. Melting Point Determination:

o Purpose: Assesses purity and identifies polymorphic forms.
o Process: Uses melting point apparatus or differential scanning calorimetry (DSC).
2. Particle Size Analysis:
o Importance: Affects dissolution rate and bioavailability, especially for oral
formulations.
o Methods: Laser diffraction, microscopy, or sieve analysis.

Water Content:
  Karl Fischer Titration:
o Purpose: Measures moisture content, critical for stability.
o Process: Volumetric or coulometric titration methods.

pH Measurement:

 pH Meter:
o Purpose: Determines the pH of aqueous solutions, important for stability and
compatibility.

Alignment with GMP Requirements and ICH Guidelines:

ICH Q3A/B (Impurities):

1. Identification and Qualification:

o Thresholds: Impurities above 0.05% (for doses ≤2g/day) must be identified.
o Qualification: Toxicological evaluation for impurities above the qualification
threshold.
2. Analytical Method Validation:
o Parameters: Accuracy, precision, specificity, detection limit, quantitation limit,
linearity, and range.
o Documentation: Validation reports must be available for regulatory review.

ICH Q6A (Specifications):

1. Setting Specifications:
o Criteria: Establish acceptance criteria for identity, purity, potency, and impurities.
o Justification: Based on analytical data, manufacturing capability, and clinical
relevance.
2. Test Procedures:
o Suitability: Methods must be appropriate for their intended use and validated
accordingly.

Importance in Ensuring Quality and Safety:

Ensuring Efficacy:

 Potency Tests:
o Verification: Confirms that the drug substance contains the correct amount of
active ingredient.
o Impact: Directly relates to the therapeutic effect.

Patient Safety:
  Impurity Profiling:
o Detection: Identifies potentially harmful impurities.
o Control: Ensures impurities are within safe limits.
 Residual Solvents and Elemental Impurities Testing:
o Compliance: Ensures toxic substances are within acceptable daily exposure levels.

Regulatory Compliance:

 Meeting Standards:
o Requirement: Compliance with GMP and ICH guidelines is essential for regulatory
approval.
o Audit Readiness: Proper documentation and validation facilitate inspections.
 Global Acceptance:
o Benefit: Adherence to international standards enables market access in multiple
regions.

Product Consistency:

 Quality Control:
o Routine Testing: Ensures each batch meets the established quality criteria.
o Batch Release: Only products meeting specifications are released.
 Stability Assurance:
o Shelf Life: Stability testing confirms the drug remains effective and safe over its
shelf life.
o Storage Conditions: Determines optimal conditions to maintain quality.

By employing rigorous analytical methods aligned with GMP and ICH guidelines, the
characterization of PlasminStop ensures that the drug substance is of high quality, safe for
patient use, and effective in its therapeutic action.

3. Evaluation of Potential Impurities and Their Impact on Quality and Safety:

Potential Impurities:

Process-Related Impurities:

1. Unreacted Starting Materials:

o Examples: Residual 4-methylbenzoic acid or intermediates like 4-
methylcyclohexanecarboxylic acid.
o Control Measures: Purification steps such as recrystallization or chromatography.
2. By-Products:
o Formation: Side reactions during bromination or amination may produce
structurally related compounds.
o Identification: Use of LC-MS and NMR for structural elucidation.
Degradation Products:

1. Chemical Degradation:
o Causes: Exposure to heat, light, or moisture.
o Examples: Oxidation products, hydrolysis of the amine group.
2. Physical Degradation:
o Polymorphic Changes: Different crystal forms can affect solubility and
bioavailability.
o Detection: DSC and X-ray diffraction analyses.

Residual Solvents:

 Examples: Methanol, ethanol, or other solvents used in synthesis or purification.

 Control: Limit residual solvents according to ICH Q3C guidelines.

Elemental Impurities:

1. Catalyst Residues:
o Sources: Palladium from hydrogenation catalysts.
o Control: Implementing purification steps and using metal scavengers.
2. Reagent Impurities:
o Examples: Trace elements from reagents or contaminated water.
o Control: Use high-purity reagents and deionized water.

Impact on Quality and Safety:

Toxicity:

1. Adverse Effects:
o Risk: Impurities may be toxic, causing side effects or allergic reactions.
o Evaluation: Conduct toxicological assessments for significant impurities.
2. Mutagenicity:
o Concern: Some impurities could be genotoxic or carcinogenic.
o Testing: Ames test and other genotoxicity assays.

Efficacy:

1. Drug Performance:
o Interference: Impurities may inhibit or alter the drug's mechanism of action.
o Assurance: Maintain high purity to ensure consistent therapeutic effects.
2. Stability:
o Degradation: Breakdown products can reduce potency.
o Monitoring: Stability studies under various conditions.

Establishing Acceptance Criteria Using ICH Q6A Decision Tree:

Identification Thresholds:

 Based on Maximum Daily Dose:

o Thresholds: For drugs with doses ≤2g/day, impurities above 0.05% need
identification.
o Action: Identify and characterize impurities exceeding this level.

Qualification Thresholds:

 Safety Assessment:
o Requirement: Impurities above the qualification threshold require toxicological
evaluation.
o Thresholds: Varies depending on maximum daily dose.

Setting Specifications:

 Analytical Data:
o Use: Set acceptable limits based on analytical results and process capability.
o Justification: Provide rationale for specifications in regulatory submissions.
 Regulatory Guidelines:
o Alignment: Ensure specifications meet ICH recommendations and local regulatory
requirements.

Contribution of Impurity Profiling and Stability Testing:

According to ICH Q3C (Residual Solvents):

1. Classification of Solvents:
o Class 1 Solvents: Should be avoided (e.g., benzene).
o Class 2 Solvents: Limited due to inherent toxicity (e.g., methanol).
o Class 3 Solvents: Low toxic potential; less stringent limits.
2. Control Measures:
o Analytical Testing: Regular testing to ensure levels are below permissible limits.
o Process Optimization: Use safer solvents or solvent-free methods when possible.

According to ICH Q3D (Elemental Impurities):

1. Risk Assessment:
o Source Identification: Determine potential sources in raw materials, water,
equipment.
o Exposure Evaluation: Calculate total daily intake from all sources.
2. Control Strategy:
o Specifications: Set limits based on permitted daily exposures (PDEs).
o Analytical Methods: Use validated methods sensitive enough to detect impurities
at required levels.
Overall Contribution to Drug Development:

Safety Assurance:

1. Patient Protection:
o Objective: Minimize the risk of adverse effects from impurities.
o Outcome: Increased patient safety and trust in the medication.
2. Regulatory Compliance:
o Necessity: Meeting stringent health authority requirements to market the drug.

Quality Control:

1. Consistency:
o Goal: Ensure each batch meets quality standards.
o Method: Implement robust manufacturing and quality control processes.
2. Shelf Life Determination:
o Importance: Stability testing informs expiration dates and storage conditions.

Process Improvement:

1. Feedback Loop:
o Use of Data: Impurity profiles can indicate where process improvements are
needed.
o Continuous Improvement: Adopt new technologies or methods to enhance purity.
2. Innovation:
o Encouragement: Development of cleaner, more efficient synthesis routes.

Regulatory Approval:

1. Comprehensive Dossier:
o Requirement: Detailed impurity and stability data support the IND application.
o Benefit: Facilitates a smoother review process.
2. Global Standards:
o Advantage: Compliance with ICH guidelines eases approvals in multiple countries.

In summary, evaluating potential impurities and conducting thorough impurity profiling and
stability testing are crucial steps in the drug development process. These activities ensure that
PlasminStop is safe, effective, and of high quality, ultimately leading to successful regulatory
approval and patient well-being.