T&PB Master Toxicology Protocols: The Toxicology & Pharmacology Branch maintains a set of master protocols for the performance of various toxicity studies on file with the FDA in the NCI Drug Master File. Copies of these protocols are available upon request to Dr. Elizaberth Glaze, Toxicology & Pharmacology Branch, Developmental Therapeutics Program, NCI, 6130 Executive Blvd. Room 8040, Bethesda, MD 20892.

The following toxicology and pharmacology resources are made available only via one of our Pathways to Development Resources (e.g., RAID, DDG).

Drug Plasma Method Development: Develop and validate an analytical method for quantitation of the compound in question in mouse, rat, dog, and human plasma. Characterize the stability in plasma at 37°C. If significant decomposition is observed, similar studies should be conducted at 4°C. Assess the stability in frozen plasma and/or plasma extracts to determine appropriate sample handling and storage procedures.

Determine the extent of protein binding in mouse, rat, dog, and human plasma. Additional binding studies with constituent plasma proteins (albumin, ₁-AGP) may also be performed.

Pharmacokinetics: Plasma elimination kinetics will be determined in one or more of the following species: mice, rats, dogs, and non-human primates after single intravenous doses of drug. Of primary importance will be the determination of plasma levels or AUC associated with efficacy in tumored animal models. Other routes of administration such as oral, intraperitoneal, subcutaneous, and intramuscular may be necessary to evaluate as well. Bioavailability of non-parenteral routes and plasma clearance rates will be determined in order to establish the dose required to produce effective drug concentrations in plasma for future toxicity study evaluation. The ability of a drug to cross the blood-brain barrier will be assessed in dogs or non-human primates, as needed.

A standard mathematical work-up of the plasma concentration-time data to yield half-life values, plasma clearance, AUC, etc. will be performed. Urine may be collected in selected experiments for determination of cumulative drug excretion, urinary clearance, and possible identification of drug metabolites. Major organs and tissues may be obtained at necropsy and frozen for possible characterization of the tissue distribution of the compound.

Metabolism: For those drugs that undergo metabolism, the in vitro and in vivo metabolism will be characterized and the potential effects of this compound on drug metabolizing enzymes will also be evaluated. Initially, the metabolism by mouse, rat, and human liver microsomes will be characterized. These studies shall include, as necessary, determinations of cofactor dependency and effects of CO and N₂ on metabolism.

If P450 involvement is evident, studies using cDNA-expressed P450 isoforms and/or specific substrate/inhibitors of P450 isoforms shall be performed to further characterize the enzymes responsible for metabolism.

In vivo studies in mice and/or rats may be performed to confirm and extend the preliminary investigations described above and to obtain quantities of metabolites for structure identification and comparisons with in vitro results.

If results of the in vitro and in vivo studies outlined above suggest that the compound could induce its own metabolism and/or be involved in clinically significant metabolism-based drug interactions, in vivo studies to test this hypothesis will be considered.

Preliminary (Range-Finding) Toxicity: For each drug, it will be necessary to establish a maximum tolerated dose (MTD) and dose limiting toxicities (DLT) in both rodent and non-rodent species. The following types of studies may be required in this phase:

Up/Down toxicity study in rodents.

Single or multiple dose range-finding studies in rodents, beagle dogs, or non-human primates.

IND-Directed Toxicity: For each drug, it will be necessary to establish toxicity and safety in relation to drug plasma concentrations or area-under-the-curve in both rodents and non-rodents. The following types of studies may be required in this phase:

Single or multiple daily dose schedules such as Dx1, q3hr x 3, q8hr x 15, q4D x 3, etc.

Continuous administration to mice via Alzet osmotic pumps and to beagle dogs, rats and non-human primates via infusion pumps for periods of from one hour up to 30 days.

Up to twenty-eight days or more of repeated administration of drug to rodents and non-rodents.

Multiple cycle studies may be required if delayed or cumulative toxicity is anticipated.

Special studies such as cardiotoxicity, neurotoxicity evaluations, and immunotoxicity studies may be required as part of an existing study or in a separate study.

Bone Marrow Toxicity: For each drug that produces myelosuppression in preliminary toxicity studies or is an analog of a drug known to produce bone marrow toxicity, human, canine, and murine bone marrow toxicity assessments will be performed in vitro using CFU_GM and correlated with toxicity that occurs in vivo. Assessment of toxicity in other bone marrow progenitors is performed as necessary.