**4.3 Identification of a backup molecule with limited potential for CNS toxicity**

In order to identify a backup molecule with limited potential for the observed CNS toxicity of the lead drug candidate, screening potential backup molecules for CNS toxicity in monkey would be resource intensive. Furthermore from an animal usage and management perspective, reduction of potential primate mortality was optimal. Since toxicological screening for a potential backup was unfavorable, reduction in the distribution of a backup to the CNS was a possible solution. Marked structural alterations of the physiochemical

Fed Beagle dogs (n=18) were administered a single oral dose or repeated daily oral doses for 14 days of 10 mg/kg drug. Liver, kidney, fat, and plasma were collected at 24 hours post dose from three dogs at each time point with and without formic acid (formic acid was added to potentially increase the stability of the acyl glucuronide metabolite). Bioanalysis of liver, kidney, fat, and plasma for drug (Parent) and it metabolites (M1, M2, M3, and M4) was performed. Peak areas were integrated for both parent and metabolites in each matrix. Data

**4. Case example: Toxicokinetics and central nervous system (CNS) toxicity**  This case example (described below) will highlight an investigation into CNS toxicity where the lead drug candidate displayed CNS toxicity in the monkey and a backup molecule needed to be identified. This example highlights utilization of the efflux transporter, Pglycoprotein (Pgp), to limit the tissue distribution of the backup drug candidate to the CNS

In a Cynomolgus monkey toleration study at the 100 mg/kg/day dose (repeat daily oral dosing), test article-related clinical signs observed in the male monkey were characterized by vomiting, ptosis, decreased activity, prostration, tremors, convulsion and ataxia. A slight safety margin was identified (approximately 7-fold); however, this margin was not large enough to confidently advance this drug candidate into longer GLP safety studies in

Unfortunately, the brains of these monkeys were not sampled after the monkey toleration study. However, plasma and brain exposures in the mouse were known for this drug candidate. Mice express similar membrane proteins (e.g., Pgp and BCRP) in their blood brain barrier compared to Cynomolgus monkeys (Ito et al., 2011); therefore, we hypothesized that brain penetration of this drug candidate in mouse may approximate the

The brain to plasma ratio of this drug candidate was large (i.e., 22) in mouse; furthermore, drug was retained in the mouse brain compared to plasma (Figure 3). These results suggested that the drug candidate was preferentially distributed to the brain with a large accumulation potential. This large accumulation potential suggested that the safety margin (established in the monkey toleration study) might decrease with the increased duration of

In order to identify a backup molecule with limited potential for the observed CNS toxicity of the lead drug candidate, screening potential backup molecules for CNS toxicity in monkey would be resource intensive. Furthermore from an animal usage and management perspective, reduction of potential primate mortality was optimal. Since toxicological screening for a potential backup was unfavorable, reduction in the distribution of a backup to the CNS was a possible solution. Marked structural alterations of the physiochemical

the safety studies, further compromising the developability of this lead candidate.

**4.3 Identification of a backup molecule with limited potential for CNS toxicity** 

from kidney and fat are not shown.

in order to limit CNS toxicity potential.

respective brain penetration in monkey.

**4.2 Role of toxicokinetics in monkey CNS toxicity** 

**4.1 CNS toxicity in monkey** 

monkey.

properties for this chemical series to alter CNS distribution were not possible since these alterations markedly reduced potency for the pharmacological receptor. Interestingly, some of these molecules (in the same chemical series) were identified as substrates for Pgp. In the MDR1-MDCK cell model, the efflux ratio of the Pgp substrates was between 2 and 3. Since Pgp is known to reduce CNS distribution through efflux of drug candidate from the apical membrane of the endothelial cells in the blood brain barrier into the blood (Cordon-Cardo et al., 1989), the effect of Pgp on the CNS distribution of these potential backup molecules was determined in the mouse (as discussed previously, monkeys were not a practical model for this exploration). CNS concentrations were approximately 10-fold less for one of these backup drug candidates compared to the lead drug candidate (Figure 4). Therefore, this backup drug candidate was advanced into clinical trials and CNS toxicity was never observed in monkey and human.

Fig. 3. Concentration-Time Profile of Lead Drug Candidate in Mouse Brain and Plasma after a Single Oral Dose (20 mg/kg)

Fasted CD1 mice (n=27) were administered a single oral dose (20 mg/kg) of the lead drug candidate. Brains and plasma were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48 hours post dose from three mice at each time point. Bioanalysis of brain and plasma of the lead drug candidate was performed.

The concept of sampling excreta to estimate drug and metabolites in human tissues is still evolving. The importance of understanding absolute abundance of metabolites from sampling excreta was highlighted by the need to understand the importance of metabolites in safety testing or MIST (Baillie et al., 2002; Smith & Obach, 2005). Smith and Obach concluded that the risk assessment of metabolites would seem more prudent if it was based on absolute mass and not proportion of drug-related material (Smith & Obach, 2005); therefore, sampling excreta and analyzing total amount of metabolite excreted would be more useful than sampling plasma (especially at higher dose of the drug). The recommendation for sampling excreta was based on determining the entire body burden of the metabolites for this MIST guidance and less about sampling excreta to estimate drug and

In animals, the concept of sampling excreta to estimate drug and metabolites in tissues has been applied in a limited fashion. For example in beef steers treated with gentamicin, a small residue remains bound to the kidney cortex tissue for many months (this residue is unacceptable at the time of slaughter). Interestingly, plasma levels of gentamicin declined rapidly to no detectable levels within 3 days after intramuscular administration of gentamicin, while measurable amounts in urine persisted for 75 days before the concentration of gentamicin declined to levels too low to quantitate by the available liquid chromatography tandem mass spectrometry (LC/MS/MS) technique (Chiesa et al., 2006). An estimated correlation between an extrapolation of urine gentamicin concentration to the corresponding kidney tissue sample suggested a urine to kidney tissue relationship of 1:100. A test system sufficiently sensitive to a urine gentamicin concentration of 1 ng/mL correlated with the estimated 100 ng/g gentamicin limit applied to the fresh kidney of the recently slaughtered bovine (Chiesa et al., 2006). This example highlights the utility of measuring excreta (e.g., urine) to better estimate concentrations of drug in tissue (e.g.,

The challenge of excreta being a surrogate model to assess concentrations of drug and metabolites in human tissues is the limited understanding of how concentrations of drug and metabolites in the excreta will relate to the concentrations in the respective tissue. This challenge can be minimized by establishing a relationship between the concentration of drug and metabolites for excreta and tissues in animals (as illustrated by the above example with gentamicin in beef steers). In addition, translating that relationship from animal to human with in silico tools (e.g., PBPK modelling) and in vitro and in vivo human models (e.g., primary in vitro human cell models and humanized mice) will increase the confidence in including safety margins from exposure of drug and its metabolites in the tissues (in addition to plasma) where organ specific toxicity is observed. Below is a case example where the utility of semen as a surrogate model to assess the concentrations of drug and

**5.1.1 Case example: Utility of semen as a potential matrix to estimate drug and** 

In this case example, the potential of semen was evaluated as a matrix to determine the concentration of Drug A (same drug candidate described in the section for Toxicokinetics and Testicular Toxicity) and its metabolite (M1) in dog testes (for potential extrapolation to human). For this study, dogs were given a single oral dose of Drug A and then at different

**5.1 Sampling excreta to estimate drug and metabolites in tissues** 

metabolites in tissues.

kidney).

metabolites in dog testes was investigated.

**metabolites in testes** 

Fig. 4. Dose Normalized CNS Concentration-Time Profile of Drug Candidate in Mouse Brain and Plasma after a Single Oral Dose (20 mg/kg for lead and 5 mg/kg for backup) Fasted CD1 mice (n=54) were administered a single oral dose of 20 mg/kg of the lead or 5 mg/kg of the backup drug candidate. For the lead drug candidate, brains were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48 hours post dose from three mice at each time point. For the backup drug candidate, brains were collected at 0.5, 1, 4, 6, 24, and 48 hours post dose from three mice at each time point. Bioanalysis of brains for the lead and backup drug candidate was performed.

#### **4.4 Conclusion**

Development of a backup drug candidate that is a substrate for efflux transporters which limit its distribution to the CNS (e.g., Pgp) can reduce the potential for this backup to cause CNS toxicity where the prior lead drug candidate demonstrated this toxicity in animal safety studies.
