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DMPK

Aragen provides comprehensive and cost-effective drug metabolism and pharmacokinetics (DMPK) studies to evaluate and optimize the drug-like properties of new chemical entities (NCEs). We incorporate customized approaches and advanced technologies such as high-throughput screening to provide decision enabling, high quality data with rapid turnaround time. The extensive experience and problem solving capabilities of our team, along with our large capacity for bioanalysis, ensures flexible solutions and continuous support. Locally present breeding partners, Taconic Biosciences and Charles River Laboratories, enable us to provide highest quality PK data from our AAALAC accredited vivarium. Our project management team ensures the journey from compound procurement to sharing results, is seamless for clients.

DMPK studies at Aragen span all stages of drug discovery, including exploratory, hit-to-lead, lead optimization and candidate selection, as well as preclinical IND enabling studies.

In Vitro ADME

Through our range of ADME assays, we help identify and mitigate limitations against absorption, distribution, metabolism and excretion related issues very early in drug discovery projects. In addition to small molecules, we conduct comprehensive druggability studies with other modalities such as proteolysis targeting chimeras (PROTACs), molecular glues, nucleotides, nucleosides, peptides and proteins. With a focus on quality and efficiency, our ADME scientists working closely with our synthetic organic chemists, accelerate the design and testing cycles of drug discovery programs.

Physicochemical Studies
  • Solubility (pION, kinetic, thermodynamic, FaSSIF, FeSSIF and FaSSGF)
  • Stability (buffer, pH, plasma etc.)
  • Lipophilicity (LogP, LogD and pKa by Spectramax®)
Metabolism
  • Metabolic stability: hepatic and extrahepatic (microsomes, S9, hepatocytes): Phase 1 and 2; CYP-mediated and non-CYP mediated
  • Reactive metabolites (glutathione and potassium cyanide adduct)
  • CYP inhibition (8 isoforms: cocktail and individual approaches)
  • CYP induction (PXR, CAR and AhR) in induction qualified hepatocytes
  • Time dependent inhibition/mechanism dependent inhibition/drug-drug interactions
Distribution
  • Plasma protein binding across species, rapid equilibrium dialysis (RED), high-throughput dialysis and ultra-centrifugal methods
  • Matrix protein binding
  • Blood cell partitioning
Permeability/ Transporters
  • PAMPA-GIT, BBB, skin
  • Caco-2/MDCKII, MDCKII-MDR1 permeability
  • ABC/SLC transporters
  • Transdermal diffusion (porcine and human skin)
  • Buccal permeability

The compound requirement for  in vitro studies is typically 5 mg (to conduct all assays) with a turnaround time of 5-7 working days.

Pharmacokinetics (PK)

Rodent pharmacokinetics (in life) vivarium studies are conducted in-house on different strains of mice and rats, in accordance with the following certifications, compliances and quality systems:

Certification
& Compliance
  • AAALAC accredited rodent vivarium facility
  • In-house facility for rodent equipped IVC/Conventional cages
  • OHSAS (Occupational Health and Safety Assessment Series) compliant
  • Procedures compliant to Institutional Animal Ethics Committee (IAEC) comprised of external members and a Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) nominee
  • IAEC approved and committee reviewed protocols of study experimentation
  • Institutional Bio Safety Committee (IBSC) registered under the Department of Biotechnology, India
  • Bio-waste Disposal Management registered under the State Pollution Board
  • EMS (Environment Management System) as per ISO 14001
Quality
Systems
  • Procurement of animals from certified vendors (local subsidiaries of Taconic Biosciences, Charles River Laboratories and The Jackson Laboratory)
  • Documentation of animal health status at receipt
  • Subjecting all newly arrived animals to quarantine
  • Physical examination of animals by a veterinarian
  • Clinical tests:
    – Parasitological examination
    – Haematological
    – Biochemical
    – Microbiological examination of feces for pathogenic organisms on blood agar
    – Feed, water and bedding material analysis
  • Ethical efforts to avoid pain and distress to experimental animals
  • Anaesthesia and euthanasia as per CPCSEA guidelines

Ninety SOPs have been established for the regular functioning of the vivarium and for the various studies being conducted.

Typical Compound Requirements for PK Studies
Study TypeTAT (Days)Compound Requirement (mg)
Rat PK525
Mouse PK: serial sampling510
Mouse PK: parallel sampling520
Rat Blood Brain Barrier515
Mouse Blood Brain Barrier515
Cassette dosing525 in rats

Metabolite Identification

Metabolic profiling or metabolite identification is very important in early drug discovery to help understand the fate of a compound towards a pharmacologically active or toxic metabolite. The FDA recommends the metabolic profiling of any compound whose preclinical toxicity needs to be evaluated. Metabolite profiling helps identify disproportionate metabolites that are solely present in human or at higher concentrations in human than in any preclinical animal species. Such metabolites are considered for further safety assessment. Metabolite profiling also helps in optimizing fast metabolising compounds by proposing soft spots and then blocking the labile soft spots, thereby increasing the metabolic stability of a compound. At Aragen, we use different analytical (XB500-TOF, tandem mass spectrophotometry) and software (Lightsight, MetaboLynx) tools to evaluate the metabolite profile of a compound.

Soft Spot Elucidation

Based on stability red flags or compound clearance, we conduct soft spot elucidations by understanding the fragmentation pattern of compounds and metabolites. Our medicinal chemists use this information in the synthesis/optimization of a series of metabolically stable compounds.

Metabolite & Structure/Pathway Identification

Metabolite identification is conducted on in vivo and in vitro  samples.  In vitro  matrices for metabolite ID include microsomes, S9 fractions, hepatocytes and expressed CYP enzymes across multiple species such as mice, rats, dogs, monkeys and humans. This enables the selection of the best toxicological species to predict human metabolism. The metabolic pathways involved are also elucidated to understand drug-drug interactions.

Targeted Metabolomics 

We conduct comparison studies in diseased and control preclinical species to measure differences in sugars, nucleosides, organic acids, ketones, aldehydes, amines, amino acids, small peptides, lipids, steroids, terpenes, alkaloids, drugs (xenobiotics) etc.

IND Filing Package

Investigational new drug (IND) studies are an essential prerequisite to conducting clinical trials that administer an investigational drug to humans. Aragen experts can provide customized IND enabling study packages to help sponsors in initiating their IND enabling program. The reports of our study packages complement the IND pharm-tox packages our customers submit to regulatory agencies for approval to initiate Phase I clinical trials. Our studies are aligned with clinical routes of administration, dose schedule and duration of treatment, all designed to identify PK/PD responses, target organs, dose responses, exposure multiples and safety margins.

Typical IND enabling ADME packages contain data from the following studies:

  • Bioanalytical method development and validation in one rodent and one or more non-rodent species
  • Single and multiple-dose pharmacokinetics, dose proportionality and absolute bioavailability in one rodent and one or more non-rodent species
  • Plasma protein binding tests in one rodent, one or more non-rodent species and in human
  • In vitro metabolism in animal and human hepatic preparations
  • In vitro CYP inhibition in human liver microsomes

Additional Studies

  • Mass balance and routes of excretion in rodents, including biliary excretion in rats
  • Metabolite profiling and identification in rodents and non-rodents
  • Transporters (T7) assays: inhibitors and substrate studies

Bioanalysis

All assay samples must undergo quantitative (PK) and qualitative (ADME) analyses to measure compound presence. As an integral part of drug discovery and development, bioanalysis plays an important role in the progression of any drug discovery project. Additionally, it provides the measurement of exogeneous and endogenous metabolites and biomarkers to guide drug design. Aragen’s state-of-the-art infrastructure supports all hit to IND activities, and we continually expand and update our bioanalysis capacities to maintain high data quality and aggressive turnaround time.

Biowaiver

BCS Based In Vitro Biowaiver

BCS based  in vitro  biowaiver studies replace clinical BA/BE studies to classify the equivalence of two pharmaceutical products for regulatory approval. Aragen offers the rapid and cost effective establishment of bioequivalence using USFDA/EMEA approved methods. Initially applied for scale-up and post approval changes (SUPAC) of generic products, the studies were later extended to the approval of orally administered BCS class I (high solubility and high permeability) and class III (high solubility and low permeability) IR formulations. These studies involve characterizing the comparative dissolution of test and innovator formulations, solubility, gastrointestinal (GI) stability, and Caco-2 permeability of the active pharmaceutical ingredient (API). Our client studies have been successfully reviewed and audited by USFDA.

Steps in the Design and Conduction of BCS based  In Vitro  Biowaiver Studies

  • Method development and validation of test compounds using HPLC and/or LC-MS/MS as per USFDA published analytical/bioanalytical guidelines
  • Conducting dissolution, gastrointestinal stability and solubility of IR formulations/APIs in biorelevant media as per US pharmacopeia and analyzing the samples using validated HPLC/LC-MS/MS methods
  • Assessing test compound in vitro Caco-2 permeability along with appropriate controls (high permeability, low permeability and efflux marker compounds) and analyzing the samples using validated LC-MS/MS methods
  • Validation of a Caco-2 permeability model with at least 20 model drugs binned in four different BCS classes
  • Characterization of Caco-2 cell lines with respect to the expression of various drug transporters
In Vitro Binding Studies

Conventional clinical BA/BE studies do not allow the characterization of bioequivalence for locally acting drugs. Aragen offers USFDA approved  in vitro  binding studies for the rapid and cost effective establishment of bioequivalence of complex generics for bile acid and phosphate binding drugs, and sucralfate. Bioequivalence is established by comparing the rate and extent of binding between test and reference formulations.

Steps in the Design and Conduction of In Vitro Binding Studies

  • Method development and validation of bile acids/phosphate/human serum albumin (HSA) or bovine serum albumin (BSA) using HPLC/IC/ICP-MS as per USFDA published bioanalytical guidelines
  • Conduction of kinetic binding study for test and reference formulations with bile acids/phosphate/HSA or BSA concentrations and pH variations resembling gastrointestinal pH. Free bile acids/phosphate/HSA or BSA concentration is determined and bound concentration is computed. Saturation binding with respect to time and binding similarity between the test and reference formulations is characterized
  • Conduction of equilibrium binding study for test and reference formulations with appropriate bile acids/phosphate/HSA or BSA ranging across 8 different concentrations and pH variations resembling gastrointestinal pH. Free bile acids/phosphate/HSA or BSA concentration is determined and bound concentration is computed to calculate Langmuir adsorption constants (K1 and K2)

Bioequivalence between the test and reference formulations is determined based on the capacity constant (K2)

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