**2.1 Testicular toxicity in rat**

In a 13-week rat safety study, testicular atrophy was observed in rats at all doses tested (10, 50, and 250 mg/kg/day); however, these findings were not observed in the 2-week study. At the dose of 250 mg/kg/day, testicular atrophy was observed in approximately 50% of all rats. At doses of 10 and 50 mg/kg/day, these findings were observed in only 10% of rats but responsibility of Drug A for this toxicity could not be discounted (i.e., unequivocal). Therefore, no NOAEL could be assigned in this study, which markedly complicated the further development of this drug candidate.

#### **2.2 Role of toxicokinetics in rat testicular toxicity**

From the rat quantitative whole body autoradiography (QWBA) study, preferential distribution of Drug A-derived radioactivity to the testes was observed; furthermore, this radioactivity was retained in the testes markedly longer compared to other tissues (Figure 1). Since distribution of radioactivity included both parent and its metabolites and the dose in the rat QWBA study was based on the lowest dose of the rat safety study (i.e., 10 mg/kg/day), a cold study was initiated where rats were dosed with a single oral dose of Drug A at 50 mg/kg (similar to the mid dose in the rat safety study). After this single oral dose, the plasma, testes, and epididymes of the rats were collected at different time points and analyzed for Drug A and its two known metabolites (M1 and M2). Interestingly, the predominant metabolite in plasma (i.e., M2) was not the predominant metabolite in testes. M1 preferentially distributed to the testes from plasma; whereas, M2 had limited distribution to this tissue (Table 1 and 2). Furthermore, the Tmax of M1 was 48 hours in testes suggesting a large accumulation potential of this metabolite in testes compared to plasma. Indeed after a follow-up study for six months of repeated daily oral dosing, M1 accumulated approximately five-fold in the testes; whereas, the parent did not accumulate (Figure 2). Furthermore, parent and M1 did not accumulate in the plasma during the 6 month rat safety study (data not shown).

surrogate for tissue levels of drug and its metabolites, especially for the assessment of risk

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

This case example (described below) will highlight 1) preferential distribution of parent and metabolites to tissue, 2) a predominant metabolite that is different in the tissue versus plasma, and 3) accumulation of parent and metabolite that occurs in tissue and not in plasma. Furthermore, the case example will highlight that focusing on tissue burden of the drug and its metabolites (and not plasma concentrations) may actually ensure that a backup

In a 13-week rat safety study, testicular atrophy was observed in rats at all doses tested (10, 50, and 250 mg/kg/day); however, these findings were not observed in the 2-week study. At the dose of 250 mg/kg/day, testicular atrophy was observed in approximately 50% of all rats. At doses of 10 and 50 mg/kg/day, these findings were observed in only 10% of rats but responsibility of Drug A for this toxicity could not be discounted (i.e., unequivocal). Therefore, no NOAEL could be assigned in this study, which markedly complicated the

From the rat quantitative whole body autoradiography (QWBA) study, preferential distribution of Drug A-derived radioactivity to the testes was observed; furthermore, this radioactivity was retained in the testes markedly longer compared to other tissues (Figure 1). Since distribution of radioactivity included both parent and its metabolites and the dose in the rat QWBA study was based on the lowest dose of the rat safety study (i.e., 10 mg/kg/day), a cold study was initiated where rats were dosed with a single oral dose of Drug A at 50 mg/kg (similar to the mid dose in the rat safety study). After this single oral dose, the plasma, testes, and epididymes of the rats were collected at different time points and analyzed for Drug A and its two known metabolites (M1 and M2). Interestingly, the predominant metabolite in plasma (i.e., M2) was not the predominant metabolite in testes. M1 preferentially distributed to the testes from plasma; whereas, M2 had limited distribution to this tissue (Table 1 and 2). Furthermore, the Tmax of M1 was 48 hours in testes suggesting a large accumulation potential of this metabolite in testes compared to plasma. Indeed after a follow-up study for six months of repeated daily oral dosing, M1 accumulated approximately five-fold in the testes; whereas, the parent did not accumulate (Figure 2). Furthermore, parent and M1 did not accumulate in the plasma during the 6-

generated from the author's personal experience in the pharmaceutical industry.

**2. Case example: Toxicokinetics and testicular toxicity** 

for some types of organ-specific toxicity.

does not produce the same toxicity.

further development of this drug candidate.

month rat safety study (data not shown).

**2.2 Role of toxicokinetics in rat testicular toxicity** 

**2.1 Testicular toxicity in rat** 

Fig. 1. Total Radioactivity (TR) Concentrations versus Time Profile of Drug A-derived Radioactivity in Rat Plasma, Testicle, Liver, and Lung.

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 approximately -20°C. The frozen carcasses were embedded in chilled carboxymethylcellulose and frozen into blocks. Embedded carcasses were stored at approximately -20°C in preparation for autoradiographic analysis.


Table 1. Toxicokinetic Profile of Drug A and its Metabolites in Rat Plasma, Testes, and Epididymes.

To aid the identification of this backup, the rat QWBA study of a prior drug candidate for this target (referred to as Drug B) was assessed where Drug B did not induce testes toxicity in rat during long-term safety studies. Interestingly, Drug B-derived radioactivity was approximately equivalent in blood and testes (Table 3), suggesting that the reduced burden of this tissue may have markedly lowered the susceptibility for this toxicity compared to the structurally similar molecule, Drug A. This markedly lowered distribution to the testes was also mirrored in the volume of distribution calculated after an single intravenous administration of Drug A and B, where the volume of distribution was markedly lower for Drug B compared to Drug A in every animal species tested (e.g., rat, dog, and monkey). Therefore to select future backups of Drug A into development, the volume of distribution was calculated from similar studies with administration of potential backup drug candidates via the intravenous route. These studies led to the identification of a potential drug candidate with similar distribution properties of Drug B (i.e, lower volume of distribution in every animal species tested after a single intravenous dose compared to Drug A). This potential backup to Drug A (referred to as Drug C) was then assessed in a rat QWBA study. In this study, Drug C-derived radioactivity was approximately equivalent in blood and testes (Table 4). From these encouraging results, Drug C was advanced into further development where no testicular toxicity has been observed in long-term rat safety studies. These results support the hypothesis that reduced tissue burden of the drug and its

metabolites may actually predict that a backup does not produce the same toxicity.

0.5 2 4 8 12 24 48 72 120

Blood 21.0 17.0 15.5 8.22 3.09 0.153 ND ND ND Testis 2.84 7.54 15.4 9.98 5.26 0.346 0.135 BLQ BLQ

Table 3. Tissue Concentrations (µg equivalents/g) of Drug B-derived Radioactivity in Rat

Long Evans rats were dosed with single oral dose of 30 mg/kg [14C]-labeled Drug B. See

Time (hr) 1 4 8 24 72 168 336

Blood 4530 1280 310 58.3 BLQ ND ND Testis 2240 989 456 105 BLQ BLQ BLQ

Table 4. Tissue Concentrations (µg equivalents/g) of Drug C-derived Radioactivity in Rat

Long Evans rats were dosed with a single oral dose of 10 mg/kg [14C]-labeled Drug C. See

**2.3 Identification of a backup molecule with limited potential for testicular toxicity**  In order to identify a backup to this molecule (e.g., Drug A), screening potential backups in terms of their toxicity potential to rat testicular atrophy was not practical because of the time required for the toxicity to be observed (i.e., more than 2 weeks). Therefore, another method

of screening potential backups needed to be initiated.

Time (hr)

Plasma and Testis.

Plasma and Testis.

description of Figure 1 for experimental details.

description of Figure 1 for experimental details.

Fed Sprague Dawley rats (n=27) were administered a single oral dose of 50 mg/kg Drug A. Testes, epididymes, and plasma were collected at 1, 4, 8, 24, 48, 72, 96, 168, and 336 hours post dose from three rats at each time point. Bioanalysis of plasma, testes, and epididymes for Drug A (Parent) and its metabolites M1 and M2 was performed. Toxicokinetic parameters were determined on plasma, testes, and epididymes.


Table 2. Tissue to Plasma Ratios of Drug A and its metabolites in Rat Plasma, Testes, and Epididymes.

See description of Table 1 for experimental details. After toxicokinetic parameters were determined on testes, epididymes, and plasma, tissue to plasma ratios were calculated.

Fig. 2. Distribution of Drug A and its Metabolite, M1, in the Rat Testes after 6 Months of Repeated Daily Dosing (50 mg/kg/day) Compared to a Single Oral Dose (50 mg/kg)

Fed Sprague Dawley rats (n=4) were administered a single oral dose or repeated daily oral doses of 50 mg/kg/day Drug A for 6 months. Testes were collected at 24 hours post dose. Bioanalysis of testes for Drug A (Parent) and M1 was performed. Concentrations of Drug A and M1 after 6 months of repeated daily oral dosing (50 mg/kg/day) were compared to a single oral dose (50 mg/kg) at 24 hours post dose (see description of Table 1 for experimental details of the single oral dose study).

Fed Sprague Dawley rats (n=27) were administered a single oral dose of 50 mg/kg Drug A. Testes, epididymes, and plasma were collected at 1, 4, 8, 24, 48, 72, 96, 168, and 336 hours post dose from three rats at each time point. Bioanalysis of plasma, testes, and epididymes for Drug A (Parent) and its metabolites M1 and M2 was performed. Toxicokinetic

 Cmax AUClast AUCinf Testes M1 40 391 386 Testes M2 0.07 0.03 0.08 Testes Parent 2 16 16 Epididymes M1 49 65 64 Epididymes M2 0.22 0.10 0.24 Epididymes Parent 2 3 3 Table 2. Tissue to Plasma Ratios of Drug A and its metabolites in Rat Plasma, Testes, and

See description of Table 1 for experimental details. After toxicokinetic parameters were determined on testes, epididymes, and plasma, tissue to plasma ratios were calculated.

Single dose

6 month daily dosing

Fig. 2. Distribution of Drug A and its Metabolite, M1, in the Rat Testes after 6 Months of Repeated Daily Dosing (50 mg/kg/day) Compared to a Single Oral Dose (50 mg/kg)

Parent M1

experimental details of the single oral dose study).

0

**Testes concentration (ng/g)**

Fed Sprague Dawley rats (n=4) were administered a single oral dose or repeated daily oral doses of 50 mg/kg/day Drug A for 6 months. Testes were collected at 24 hours post dose. Bioanalysis of testes for Drug A (Parent) and M1 was performed. Concentrations of Drug A and M1 after 6 months of repeated daily oral dosing (50 mg/kg/day) were compared to a single oral dose (50 mg/kg) at 24 hours post dose (see description of Table 1 for

parameters were determined on plasma, testes, and epididymes.

Epididymes.

#### **2.3 Identification of a backup molecule with limited potential for testicular toxicity**

In order to identify a backup to this molecule (e.g., Drug A), screening potential backups in terms of their toxicity potential to rat testicular atrophy was not practical because of the time required for the toxicity to be observed (i.e., more than 2 weeks). Therefore, another method of screening potential backups needed to be initiated.

To aid the identification of this backup, the rat QWBA study of a prior drug candidate for this target (referred to as Drug B) was assessed where Drug B did not induce testes toxicity in rat during long-term safety studies. Interestingly, Drug B-derived radioactivity was approximately equivalent in blood and testes (Table 3), suggesting that the reduced burden of this tissue may have markedly lowered the susceptibility for this toxicity compared to the structurally similar molecule, Drug A. This markedly lowered distribution to the testes was also mirrored in the volume of distribution calculated after an single intravenous administration of Drug A and B, where the volume of distribution was markedly lower for Drug B compared to Drug A in every animal species tested (e.g., rat, dog, and monkey). Therefore to select future backups of Drug A into development, the volume of distribution was calculated from similar studies with administration of potential backup drug candidates via the intravenous route. These studies led to the identification of a potential drug candidate with similar distribution properties of Drug B (i.e, lower volume of distribution in every animal species tested after a single intravenous dose compared to Drug A). This potential backup to Drug A (referred to as Drug C) was then assessed in a rat QWBA study. In this study, Drug C-derived radioactivity was approximately equivalent in blood and testes (Table 4). From these encouraging results, Drug C was advanced into further development where no testicular toxicity has been observed in long-term rat safety studies. These results support the hypothesis that reduced tissue burden of the drug and its metabolites may actually predict that a backup does not produce the same toxicity.



Long Evans rats were dosed with single oral dose of 30 mg/kg [14C]-labeled Drug B. See description of Figure 1 for experimental details.


Table 4. Tissue Concentrations (µg equivalents/g) of Drug C-derived Radioactivity in Rat Plasma and Testis.

Long Evans rats were dosed with a single oral dose of 10 mg/kg [14C]-labeled Drug C. See description of Figure 1 for experimental details.

**Liver Liver +** 

Day 1 2 292 373 130 168 209 180 59 Mean

6 286 284 131 96 181 123 65 Mean

24 54 48 46 28 68 34 61 Mean

Day 14 2 295 381 226 140 245 90 73 Mean

6 293 275 284 128 187 94 71 Mean

24 39 37 41 29 52 36 72 Mean

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 2, 6, and 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

Metabolite Type Day 1 Day 14 Day 1 Day 14 Day 1 Day 14 Day 1 Day 14 Parent - 116,860,326 123,174,663122,389,879122,716,07227,431,25236,110,99528,993,390 29,924,921

M2 Glucuronidation 5,299,597 3,790,520 3,552,836 2,817,400 ND ND ND ND M3 Oxidation ND ND ND ND ND ND ND ND M4 Oxidation 453,680 1,035,337 553,582 1,045,264 ND ND ND ND

Table 6. Peak Area Counts Versus Time Profile of Parent and its Metabolites in Dog Plasma,

Sulfation ND ND ND ND ND ND ND ND

Table 5. Concentration-Time Profile of Parent in Dog Plasma, Liver, Kidney, and Fat.

liver, kidney, fat, and plasma for drug candidate was performed.

**Acid** 

35 114 17 24 46 68 11 SD

112 92 35 12 102 57 4 SD

44 37 24 16 37 25 5 SD

141 208 100 27 160 38 12 SD

89 110 72 29 27 35 5 SD

54 53 40 24 61 43 3 SD

Plasma Plasma + Acid Liver Liver + Acid

**Kidney Kidney +** 

**Acid** 

**Fat** 

 **Concentration (**µ**g/mL or g)** 

**Acid** 

**Plasma Plasma +** 

**Day Time (hr)** 

M1 Oxidation +

ND = not detected

Liver, Kidney, and Fat.
