P.D. Ward

*Johnson & Johnson, Pharmaceutical Research and Development, L.L.C., USA* 

#### **1. Introduction**

456 Toxicity and Drug Testing

Williams, A. (2008). Principles of the EURACHEM/CITAC Guide 'Use of Uncertainty

Toxicokinetics (TK) refers to the kinetics of absorption, distribution, metabolism, and elimination (ADME) processes where both first and zero order kinetics are expected and these processes can vary over a wide range of doses. The goal of TK and pharmacokinetic studies are similar, which is to define the ADME properties of a drug candidate (Dixit & Ward, 2007). Therefore, the wide range of studies to define these ADME properties (e.g., in vitro and in vivo metabolism, animal mass balance, and distribution studies) performed in the pharmacokinetic evaluation of the drug candidate can also serve to help guide the toxicokinetic evaluation of the same drug candidate with the knowledge that first and zero order kinetics might be expected in the ADME processes at the higher doses of this drug candidate in the safety studies.

Now it is widely accepted that toxic effects can be better extrapolated from animals to humans when these comparisons are based on TK instead of dose alone. For example, the safety margin that is based on the ratio of the animal exposure at no observed adverse effect level (NOAEL) to human exposure at the efficacious dose is a key predictor of human safety risk. To calculate this safety margin, the animal and human exposure is determined by analyzing drug and metabolites concentrations in plasma, which is the most practical and widely accepted way of assessing this risk (Dixit & Ward, 2007). However, most safety issues are not observed in the plasma but in the organs and/or tissues. Therefore, is sampling plasma a good measure of the safety margin for the risk assessment of safety?

Sampling plasma and extrapolating this exposure to organs or tissues assumes that 1) concentration of drug in plasma is in equilibrium with concentrations in tissues, 2) changes in plasma drug concentrations reflect changes in tissue drug concentrations over time, and 3) distribution of drug and its metabolites is not affected by polarized cells (e.g., drug transporters and enzymes) that protect a lot of these tissues. Drug transport into tissues may not be a passive process and may depend on drug transporters (Ward, 2008), thus these assumptions may result in an inaccurate assessment of target organ exposure to drug and metabolites. Even without a drug candidate being a substrate for a drug transporter, lysosomal trapping of weak bases (e.g., liver and lung) or accumulation in membranes (e.g., muscle) can occur that can give rise to preferential distribution of the drug and its metabolites (MacIntyre & Cutler, 1988). Therefore, plasma is sometimes not a good

Fig. 1. Total Radioactivity (TR) Concentrations versus Time Profile of Drug A-derived

0 100 200 300 400

**Time (hr)**

Long Evans rats were dosed with a single oral dose of 10 mg/kg [14C]-labeled Drug A. At different times after this dose, rats were sacrificed via exsanguination (cardiac puncture) under isoflurane anesthesia and blood (approximately 2 to 10 mL) was collected into tubes containing K2EDTA immediately prior to collection of carcasses for QWBA. Samples were maintained on wet ice and refrigerated until aliquoted and centrifuged to obtain plasma. Immediately after blood collection the animals were prepared for QWBA. The carcasses were immediately frozen in a hexane/dry ice bath for approximately 8 minutes. Each carcass was drained, blotted dry, placed into an appropriately labelled bag, and placed on dry ice or stored at approximately -70°C for at least 2 hours. Each carcass was then stored at

Radioactivity in Rat Plasma, Testicle, Liver, and Lung.

0.001

0.01

0.1

**TR Concentrations (ug eq./g)**

1

10

approximately -20°C. The frozen carcasses were embedded in chilled

Tmax (hr)

approximately -20°C in preparation for autoradiographic analysis.

Half Life (hr)

Epididymes.

carboxymethylcellulose and frozen into blocks. Embedded carcasses were stored at

Cmax (ng/mL or g)

Plasma M1 4 4 29 401 410 Plasma M2 6 4 1033 11983 12025 Plasma Parent 5 4 3712 43454 43491 Testes M1 46 48 1182 156763 158157 Testes M2 7 4 76 412 947 Testes Parent 54 8 9061 684074 692652 Epididymes M1 9 8 1441 25949 26064 Epididymes M2 7 4 231 1215 2908 Epididymes Parent 51 8 6676 115682 116647 Table 1. Toxicokinetic Profile of Drug A and its Metabolites in Rat Plasma, Testes, and

AUClast (ng\*hr/mL or g)

AUCinf (ng\*hr/mL or g)

Plasma Testicle

Liver Lung

surrogate for tissue levels of drug and its metabolites, especially for the assessment of risk for some types of organ-specific toxicity.

The following case examples will illustrate how focusing on drug and metabolites in these tissues (where toxicity is observed) instead of plasma increases understanding of the nature of the toxicity and in some cases allows the efficient identification of a backup drug that has markedly less potential to cause that specific organ toxicity under investigation. These case examples are categorized by the different organs where toxicity was investigated and are generated from the author's personal experience in the pharmaceutical industry.
