Lead Drug Candidate ADMET Selection
To accelerate the rapid turn-around time from design-to-report for a number of ADMET and chemistry studies during the lead drug candidate selection process, BRI has established standard ADMET study protocols in collaboration with specialized animal facilities. These protocols can be initiated at anytime and provide rapid turn around time from design-to-report for studies involving a small population of animals.
Standard ADMET and Chemistry Screening Study Protocols:
Pharmacokinetics and Distribution Screening
- Biological half-life screening (cassette n=5 or individual dosing of compounds)
- Serum protein binding using equilibrium dialysis and / or ultra-filtration methods for CNS drugs, neuroleptics, or antiarrhythmics
- In-vitro red blood cell distribution
- Half-life modification (pegylation and formulation screening)
Oral Bioavailability Screening
- Oral formulation bioavailability screening in rodents.
- First-pass hepatic and gut metabolism screening using cannulated rats
Metabolism Screening
- In-vitro CYP450 metabolic stability screening using animal and / or human pooled liver microsomes and/or primary hepatocytes
- LC/MS and LC/MS/MS metabolite profile and characterization
Toxicology/Safety Screening
- Genotoxicity profiling
- Acute MTD study in mice or rats
Chemistry Screening
- Aqueous and solvent solubility
- Solution pH profile stability
Drug Chemistry Patents
The design of a strategic drug chemistry patent portfolio is one of the key ingredients of a successful drug development program. Drug substance patents are among the most valuable intellectual property in a NCE development program.
BRI’s chemists have been successfully involved in the review and custom synthesis of physical forms of an API resulting in the filing of drug substance patents describing an enhancement in physiochemical properties of the API. This type of drug substance patent support investigation typically involves a review of existing manufacturing processes, followed by designing proof-of-principle studies to support strategic new patent claims.
In-Vitro / In-Vivo Metabolism
During pre-clinical drug development, it is important to learn if a drug is eliminated primarily by one or more CYP450 routes of metabolism. The human liver microsomal cytochrome P450 pool has been shown to play a major role in the metabolism of many drugs, particularly those drugs exhibiting a narrow therapeutic index or a steep dose-pharmacological response profile. Therefore, if multiple drugs interact with a single CYP form, then the likelihood of a potentially hazardous drug-drug interaction is increased. Likewise, if the in-vivo clearance of a drug is largely (fm,CYP ≥ 40%) mediated by a single polymorphically expressed or allelic variant form of CYP (e.g. CYP2D6, CYP2C19, or CYP2C9), it is anticipated that “poor metabolizer” subjects will be characterized by disparate pharmacokinetics (e.g. elevated plasma AUCs and / or increased plasma T1/2).
In addition, drugs are often metabolized to pharmacologically active metabolites via CYP-mediated oxidations, which implies that the pharmacodynamics of many drugs can be modulated by induction or inhibition of certain CYPs. Some well-documented examples include the CYP3A4-catalyzed oxidation of terfenadine to carboxyterfenadine, the CYP2D6-catalyzed O-demethylation of codeine to morphine, CYP2C19-catalyzed oxidation of proguanil to cycloguanil, and the CYP2C9-catalyzed oxidation of losartan to carboxylosartan.
In-Vitro methods for determining which CYP450 forms may be involved in metabolism of a given drug candidate is available at BRI in collaboration with In-Vitro ADMET Laboratories (IVAL) under the supervision of Dr. Albert Li.
The use of in-vitro assay to determine which form(s) of CYP is involved in metabolism of a given drug, so-called “CYP reaction phenotyping”, has become well established at BRI. If in-vitro CYP reaction phenotype data is available, the information can permit a decision on which in-vivo animal drug interaction studies may be required. In the case of polymorphically-expressed enzymes, in-vitro CYP reaction phenotyping data can also alert clinicians of the need for genotyped subjects as part of a clinical protocol design. As a result, many pharmaceutical companies now regularly include such data in their submissions to various regulatory agencies.
To address these safety and efficacy concerns during preclinical development, the US Food and Drug Administration (Center for Drug Evaluation and Research CDER, April 1997) has produced a document “Drug Metabolism / Drug Interaction Studies in the Drug Development Process: Studies in-vitro” to assist industry in planning in-vitro metabolism screening studies earlier in the discovery process.
To address the need for CYP450 metabolism data, BRI, in collaboration with IVAL, has established the following in-vitro and in-vivo metabolism studies:
In-Vitro Metabolism Studies (Using Materials Microsomes and / or Hepatocytes from Rodents, Dogs and Non-Human Primates)
- Metabolic Stability – For evaluation of stability of a drug candidate upon incubation in metabolic systems, the most common systems being liver microsomes for oxidative pathways and hepatocyctes for both phase-I and phase-2 metabolic pathways. Multiple time point data on the drug candidate concentrations remaining can be used to generate pharmacokinetics data for the prediction of in-vivo metabolic clearance.
- Metabolite Profiling – For characterization of metabolites based on chemical profiling approach by LC/MS and LC/MS/MS techniques. Can be performed with or without the use of 14C-labeled drug. The use of in vitro metabolic systems (e.g. hepatocytes) from multiple animal species, including man, allows the selection of the most appropriate animal species for in vivo pharmacology, pharmacokinetics, and safety studies.
- CYP450 Inhibition – For assessment of drug-drug interaction potential for a drug candidate. The drug candidate is incubated with liver microsomes or hepatocytes in the presence of isoform-specific CYP450 substrates. If the metabolism of a specific substrate is inhibited by the drug candidate, this information would infer potential inhibitory interaction with the P450 isoform involved in the metabolism of the substrate. The CYP450 isoforms evaluated include CYP1A2, 2A6, 2B6, 2C8, 2C9 , 2C19, 2D6, 2E1 and 3A4 and assays are supported by LC/MS/MS for the quantification of the formation of metabolites from the substrates.
- CYP450 Induction - As with enzyme inhibition, enzyme induction is a major mechanism of drug-drug interactions. The induction assay evaluates the ability of a drug candidate to induce the metabolic activity of CYP450 isoforms. Hepatocytes are cultured and treated with the drug candidate for three days followed by assessment of CYP450 isoform activities. The isoforms being evaluated include CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4.
- CYP450 Reaction Phenotyping – Characterization of specific CYP450 isoforms involved in the metabolism of a drug candidate is a critical part of the definition of its drug-drug interaction potential. In this assay, a drug candidate is incubated with liver microsomes in the presence and absence of specific P450 isoform-specific inhibitors. If metabolic activity of the drug candidate is inhibited by a specific inhibitor, inference is made that the drug candidate is metabolized by the isoform sensitive to the inhibitor. The definition of P450 isoforms involved in the metabolism of a drug candidate allows the evaluation of potential drug interactions with existing drugs known to be P450 inhibitors (e.g. Ketoconazole) and inducers (e.g. Rifampin).
In-Vivo Metabolism Studies
- Rat, mice, dog and monkey models can be performed
- 14C-labeled trace dose study for metabolic mass balance and metabolite characterization
A key element of success in BRI’s effective management of accelerated preclinical development programs has been its central role in managing all analytical and bioanalytical studies throughout all phases of preclinical development.
BRI’s central role in vertically integrating analytical and bioanalytical chemistry requirements from CMC to PK and TK studies has brought to bear the complexity of managing the logistics and accelerated timeline targets that are often encountered during a development program.
The following is a brief review of the process:
Preclinical Program Planning
At the concept stage, a preclinical development program is designed based on pre-existing knowledge of each lead candidate obtained from lead selection studies (chemistry, ADME, and toxicology screening data).
API utilization requirements are planned based on a proposed therapeutic use for phase I/II working with a proposed dosage form and its associated manufacturing requirements.
CMC studies relating to both API and clinical finished products are planned following a systematic review of all relevant study components. Considerations may include API synthetic / isolation scale-up, technology transfer to CMOs, impurities, degradation products, physiochemical characterization, formulation design, manufacturing outsourcing, as well as stability parameters relating to both API and clinical products in its final package unit. All assays supporting the development of the API and drug product are planned and conducted by BRI.
Safety and toxicology study requirements are planned with a review of pre-existing concerns relating to the chemical class of the drug candidate. Relevant studies required for a synthetic NCE drug candidate may include consideration of all aspects of ADME with a dose ranging component, various toxicology parameters, acute and chronic MTD, and various mandatory safety studies including pharmacology, acute and sub-acute studies. These studies are often required to be conduct in more than one species of animals in addition to rodents.
A listing of the studies by specialization delivered by BRI working within its consortium of specialized CMOs, animal facilities and regulatory companies is given under the heading Drug Development Services to the left of the page.
It is important to define API and clinical product utilization requirements early in preclinical development. The procurement of commercially available API’s, outsourcing of custom synthesis or scale-up synthesis of API are projects that have been well managed by BRI.
A number of API CMC relevant studies may include:
- Chemistry specifications (purity and chemical structural data)
- Physicochemical characterization (log P, solvent and pH profile solubility, hygroscopicity)
- Impurity chemical profile by mass spectrometry
- Residual volatile solvents and impurities in API and excipients
- Structural elucidation of impurities and degradation products
- API storage stability studies (long-term and accelerated conditions)
- Stress stability evaluation
- Certification of primary / secondary reference standards
- Development and validation of integrated purity and stability-indicating assays in support of purity QC
A number of clinical finished product relevant studies may include:
- Formulation design for oral, topical and parenteral administration (please refer to Formulation Studies page)
- Formulation bioavailability assessment (oral, skin penetration and terminal half-life studies)
- Physiochemical characterization of bulk drug in support of manufacturing (bulk-tap density, excipient-active compatibility, anti-caking excipient evaluations)
- Clinical product manufacturing release testing against product specifications
- Clinical product storage stability studies (long-term and accelerated conditions)
- Development and validation of integrated purity and stability-indicating assays in support of label claim QC
CMC Documentation Review and Preparation:
- BRI’s management team is well versed with relevant current CMC requirements in reviewing and editing Manufacturing Batch Records for API and clinical finished product
- Review, preparation, filing and annual maintenance of DMF
- BRI has adopted a multi-disciplinary approach in designing Manufacturing Release and Shelf-Life Specifications for APIs and clinical finished products
BRI’s diversified role in vertically integrating all analytical chemistry studies including development and validation of assays for API and clinical product reduces the complexity of managing the logistics of operating under an accelerated timeline and within limited budgets.
BRI’s formulation scientists have been successfully involved in the design of oral capsules (hard gelatin and soft gelatin), oral tablets, topical creams / gel formulations and parenteral products. The team has also cost effectively managed the outsourcing of manufacturing and packaging of clinical products.
BRI offers the following list of pre-formulation and formulation development studies including the management of outsourcing finished product manufacturing with CMOs:
Pre-formulation Physicochemical Characterization
- API / Formulation compatibility and stability
- Intrinsic aqueous solubility (Co) and pH-solubility profile studies
- Solubility in co-solvents, fixed oils and other formulation excipients
- Pka, Partition coefficient (Log P, n-octanol / water)
- Water content (Karl Fischer determination)
- Loss on drying (TGA or USP Method)
- Polymorphism, crystalline / amorphous state (DSC & XRD)
- Hydrates, solvates and meting behavior (DSC & TGA)
- Crystal habit (optical microscopy, image analysis & SEM)
- Particle size distribution (laser diffraction, sieve analysis)
- Specific surface are (BET method)
- Pore size distribution (Mercury intrusion porosimetry)
- Hygroscopicity
- Bulk and tap density
Formulation Development (Oral, Topical and Parenteral)
- Initial dosage form development in solids, semi-solids, suspensions, solutions, emulsions and parenterals
- Oral bioavailability enhancement excipients evaluation for sparingly water soluble drugs in conjunction with In-vivo oral bioavailability assessment in rat/mouse models
- Co-solvent solubility and stability study for parenteral formulations
- Topical formulation study with skin penetration enhancement excipients in conjunction with in-vivo skin penetration study
- In-vivo double cannulated rat model for mechanistic studies on gut and hepatic 1st-pass bioavailability
In compliance with current ICH guidelines on storage stability parameters for API and pharmaceutical products, BRI performs storage stability under long-term, intermediate and accelerated temperature and relative humidity conditions (25°C/60%RH, 30°C/60%RH and 40°C/60%RH, respectively).
All environmental chambers are temperature mapped and calibrated in accordance with a prescribed schedule according to BRI’s operating SOP. All chambers are temperature recorded, remote alarm monitored and equipped with emergency power backup generators.
A storage stability study may involve a number of relevant shelf-life assay parameters performed at BRI (listed below). A Certificate of Analysis is issued to the study sponsor for review after each pre-determined time point with a complete study report after completion of the final time point.
API Storage Stability Parameters
- Assay content LC/MS or HPLC stability indicating assay
- Related substance content
- Karl Fischer water content analysis
- Loss on drying
- Residue on ignition
- Organic volatile impurities
- Trace metals analysis
- Physical appearance
Clinical Product Stability Parameters
- Label claim LC/MS or HPLC stability indicating assay
- Related substances content
- Unit weight
- Hardness (tablet), Viscosity (semi-solids)
- Weight uniformity (tablets and capsules)
- Dimension
- Dissolution
- Disintegration
- Content uniformity
- Microbial testing (release)
- Physical appearance
During the course of an accelerated preclinical program, BRI plays a central role in vertically integrating all bioanalytical assay requirements including the development and validation of assays in support of measurement of drug substance in plasma, tissues and other biological matrices.
LC/MS/MS or LC/MS assays are designed based on a lead plasma matrix. Supplemental validation studies are subsequently performed to qualify and support the use of the assays for plasma and tissue matrices. At each supplemental validation studies, experience is gathered and accumulated in maintaining a consistent level of performance in implementation of an assay. This approach has permitted reliably consistent performance of bioanalytical results and has streamlined the logistics of meeting validation requirements under an accelerated timeline and within a limited budget.
Bioanalytical Assays Capabilities:
- Multiple LC/MS/MS and LC/MS instrumentation supporting plasma assays with LLOQ performing at 1-5 pg/ml for specific analytes
- GC/MS and HPLC assay options with a variety of detectors
- Development and validation studies in conformance with ICH, FDA and OECD requirements.
- Multiple -80°C ultra-low temperature freezers for clinical plasma sample storage with continuous temperature recording, remote alarm monitoring and equipped with emergency power backup generators
- Analytical instrument data backup and data archive audit trail management by a dedicated data server operating on CRF21 (58) part 11 compliance software system
- Pre-validated LC/MS/MS, GC/MS, HPLC and GC methods in support of routine high-throughput analysis of clinical human plasma samples
- Pre-validated HPLC-UV, HPLC-FLD and LC/MS/MS method analytical support of bioavailability and bioequivalence studies
- Chiral LC/MS/MS and GC/MS assay method development and validation
In support of phase-I and phase-II clinical investigations, BRI manages the design of plasma sample collection and handling protocol and provide plasma sample collection and shipping kits worldwide for multi-centre studies.
BRI also provides integration of clinical product manufacturing release specification testing, followed by inventory controlled and distribution to each clinical site.
Ms. Fuller’s team has a broad scope of knowledge and experience in coordinating the collection and shipment of all types of biological samples from worldwide international locations for importation to Canada. This includes working with clinical sites and specialized couriers to provide sample collection and shipping kits and providing documentation support for shipments across international borders
Therapeutic Drug Monitoring (TDM) Program:
It has become increasingly recognized clinically that therapeutic drug monitoring can play a significant role in enhancing therapeutic outcomes and patient well being in specific area therapeutic areas. The monitoring of plasma drug levels provides clinicians with the ability to evaluate individualized therapeutic plasma level changes due to unanticipated interactions.
TDM programming offered by BRI includes the following components:
- Preparation and distribution of plasma sample collection and shipping supplies to multiple clinic or hospital sites included return courier waybill for return shipping of plasma samples
- Establish single or multiple drug LC/MS/MS assays
- Rapid turn around and reporting of assay results via secured internet-based website with monograms of normal therapeutic values for use in therapeutic decision-making
The field of natural health product development has changed considerably over the past few years. As consumer demand for natural health products (NHPs) was increasing, various regulatory agencies recognized the need for more rigorous standards in the development and production industries.
Health Canada, through its Natural Health Products Directorate, regulates NHPs in Canada, and the US FDA regulates this type of products under the Dietary Supplement Health and Education Act of 1994 (DSHEA).
BRI has had a wide range of experience in the design of numerous NHP and dietary supplement products from early development to data package filing seeking regulatory approvals as clinical trial products or as market applications.
Analytical chemistry and product development studies include the following:
Raw Material Bulk Chemical Specification Testing:
- LC/MS and GC/MS analysis of chemical components profile
- Chemical component structural characterization
- Development and validation of quantitation assays (LC/MS, GC/MS, HPLC and GC)
- Organic solvent residues analysis
- Moisture content analysis
- Stability and degradation product profile studies
- Chemical adulterant identification
- Lot-by-Lot release of bulk extract label claim
- Certification of Analysis
Finished Product Testing:
- Stability testing of raw materials
- Label claim chemical analysis
- Expiry date and shelf-life determination for finished products
- Bioavailability, formulation design and dissolution testing
- Product manufacturing release specification testing
Laboratory studies are performed under the supervision of the Quality Assurance Unit in accordance with the study protocol and with the following Good Laboratory Practice Regulations / Standards / Guidelines:
- United States Food and Drug Administration, Title 21 Code of Federal Regulations Part 58, Revised April 1, 2004.
- Organization for Economic Cooperation and Development (OECD), the OECD Principles of Good Laboratory Practice, Series on Principles of Good Laboratory Practice and Compliance Monitoring, Monograph No.1 to 14, current.
Regulatory certification for BRI, including Health Canada Establishment License, can be found under Publications & Presentations.
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