IND-Enabling Toxicology & ADME

In order to gain ‘Investigational New Drug’ (IND) status from the FDA, the Sponsor must ‘submit data to show that that the drug is reasonably safe for use in initial, small-scale clinical studies’. This data should include ‘an evaluation of the drug's toxic and pharmacologic effects through in vitro and in vivo laboratory animal testing’. At the pre-clinical stage, the FDA recommends the following studies:

• Investigation of drug absorption;
• Assessment of the distribution of the compound and any tissue accumulation;
• An understanding of the major routes of metabolism and the identification of the major metabolites;
• Determination of the toxicity of the metabolites;
• Assessment of the propensity for CYP450 inhibitions or inductions;
• Determination of the speed with which the drug and its metabolites are excreted from the body;
• Determination of a pharmacological profile of the drug;
• Determination of the acute toxicity of the drug in at least two species of animals, including one non-rodent species;
• Conduct of short-term toxicity studies ranging from 2 weeks to 3 months, depending on the proposed duration of use of the substance in the proposed clinical studies;
• Determination of the no observed adverse effect level (NOAEL) to assign the maximum recommended starting dose (MRSD) for "first in human" clinical trials;
• Genotoxicity screening;
• Assessment of cardiac, pulmonary and neurological safety pharmacology.

Ricerca offers services to help you to gather the ADME and toxicology data to satisfy these requirements and more. We will help you to truly understand the nature of your compound and to confidently progress into the clinic and beyond.

Absorption

• Determination of absorptive and exsorptive apparent permeability in caco-2 or MDCK monolayers;
• Determination of the involvement of active transporters in the efflux or influx of the test article;
• Determination of bioavailability in the rat and dog.

Distribution

• Determination of plasma protein binding in toxicological species and man utilizing equilibrium dialysis or ultrafiltration;
• Determination of binding to specific human plasma proteins (such as albumin and a₁-acid glycoprotein);
• Potential for drug-drug interactions caused by plasma protein binding competition;
• Red blood cell binding;
• Hemolysis and flocculation;
• Tissue distribution in rats or dogs;
• Determination of volume of distribution and clearance in rats or dogs;

Metabolism

• Interspecies comparison of in vitro metabolic profile utilizing microsomes or hepatocytes from toxicological species and man. Analysis by HPLC with UV, fluorescent; MS or radiochemical detection;
• Comparison of in vivo rat or dog plasma metabolic profiles to the profiles generated in vitro.
• Isolation, purification and identification of metabolites utilizing LC/MS/MS and NMR techniques;
• Determination of specific CYP450 isozymes involved in the metabolism by chemical inhibition of specific isozymes in microsomes, use of cDNA expressed isozymes and correlation of activities utlizing characterized microsome libraries;
• Determination of Michaelis-Menten parameters associated with the metabolism of the test article;
• Determination of potential for CYP450 inhibition in microsomes or hepatocytes, utilizing specific chemical probe substrates. Confirmation with cDNA expressed CYP450 isozymes utilizing substrates producing fluorescent metabolites;
• Calculation of IC₅₀ and K_i of significant CYP450 inhibitions;
• Investigation into the effect of the test article on the metabolic profile of potentially co-administered compounds and vice versa;
• Determination of any CYP450 induction in rat liver microsomes following repeat administration;
• Determination of any CYP450 induction in human hepatocytes following repeat dosing;
• Confirmation of CYP450 inductions by Western blot analysis.

Excretion

• Excretion balance studies in rat or dog;
• Determination of biliary excretion using bile duct cannulated rats;
• Metabolic profiling of plasma and excreta following dosage to rats or dogs;

Toxicology

In Vivo

• Toxicity studies (14- or 28-day) in two or more animal species, including one non-rodent. A variety of dosing regimens is supported;
• Extended duration studies to support chronic dosing regimens;
• Toxicokinetic/pharmacokinetic studies;
• Single dose toxicity studies;
• Repeated dose toxicity studies;
• Local tolerance studies;
• Carcinogenicity studies;
• Neuro toxicity studies;
• Acute toxicity studies;
• Animal efficacy models;

In Vitro Cytotoxicity Assays

• Acute cytotoxicity in hepatocytes, HepG2, NIH3T3 cells, etc. using Alamar Blue;
• Cell Death ELISA analysis for apoptosis;
• DNA laddering for apoptosis;
• Apoptosis quantification by caspase activity;
• Proliferation assays by Alamar Blue and ³H-thymidine incorporation;
• Red blood cell lysis.