The vedotin antibody-drug conjugate payload drives platform-based nonclinical safety and pharmacokinetic profiles.

Nonclinical safety and pharmacokinetic data for MMAE and 14 vedotin ADCs were evaluated to determine patterns of toxicity, consistency of pharmacokinetic results, and species differences between rats and monkeys. Most nonclinical toxicities were antigen-independent, common across ADCs, and included hematologic, lymphoid, and reproductive toxicity related to MMAE pharmacology. Hematologic toxicity was the dose-limiting or predominant toxicity for the majority of vedotin ADCs in both species. Tissue expression of the targeted antigen of an ADC rarely correlated with dose-limiting toxicity (DLT); only two ADCs had antigen-dependent skin DLTs. For two additional ADCs, antigen-dependent delivery of MMAE in the bone marrow may have exacerbated the antigen-independent hematologic DLT. The highest tolerated doses and pharmacokinetics were similar within a given species, with rats tolerating higher doses than monkeys. Studies longer than one month in duration detected the same or fewer toxicities than one-month studies and had no additional findings that affected the human risk assessment. These data support opportunities to streamline ADC toxicity assessments without compromising human starting dose selection or target organ identification.

Pharmacokinetic drivers of toxicity for basic molecules: strategy to lower pKa results in decreased tissue exposure and toxicity for a small molecule Met inhibitor.

Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd>3l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd=1.0l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.

Preclinical stereoselective disposition and toxicokinetics of two novel MET inhibitors.

The R- and S-enantiomer of N-(4-(3-(1-ethyl-3,3-difluoropiperidin-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide are novel MET kinase inhibitors that have been investigated as potential anticancer agents. The effect of the chirality of these compounds on preclinical in vivo pharmacokinetics and toxicity was studied. The plasma clearance for the S-enantiomer was low in mice and monkeys (23.7 and 7.8 mL min(-1) kg(-1), respectively) and high in rats (79.2 mL min(-1) kg(-1)). The R/S enantiomer clearance ratio was 1.5 except in rats (0.49). After oral single-dose administration at 5 mg kg(-1) the R/S enantiomer ratio of AUC(inf) was 0.95, 1.9 and 0.41 in mice, rats and monkeys, respectively. In an oral single-dose dose-ranging study at 200 and 500 mg kg(-1) and multi-dose toxicity study in mice plasma AUC exposure was approximately 2- to 3-fold higher for the R-enantiomer compared to the S-enantiomer. Greater toxicity of the S-enantiomer was observed which appeared to be due to high plasma C(min) values and tissue concentrations approximately 24 h after the final dose. Both enantiomers showed low to moderate permeability in MDCKI cells with no significant efflux, no preferential distribution into red blood cells and similar plasma protein binding in vitro. Overall, the differences between the enantiomers with respect to low dose pharmacokinetics and in vitro properties were relatively modest. However, toxicity results warrant further development of the R-enantiomer over the S-enantiomer.

Gut-Selective Design of Orally Administered Izencitinib (TD-1473) Limits Systemic Exposure and Effects of Janus Kinase Inhibition in Nonclinical Species.

Izencitinib (TD-1473), an oral, gut-selective pan-Janus kinase (JAK) inhibitor under investigation for treatment of inflammatory bowel diseases, was designed for optimal efficacy in the gastrointestinal tract while minimizing systemic exposures and JAK-related safety findings. The nonclinical safety of izencitinib was evaluated in rat and dog repeat-dose and rat and rabbit reproductive and developmental toxicity studies. Systemic exposures were compared with JAK inhibitory potency to determine effects at or above pharmacologic plasma concentrations (≥1× plasma average plasma concentration [Cave]:JAK 50% inhibitory concentration [IC50] ratio). In rats and dogs, 1000 and 30 mg/kg/day izencitinib, respectively, produced minimal systemic findings (ie, red/white cell changes) and low systemic concentrations (approximately 1× plasma Cave:JAK IC50 ratio) with an 8× nonclinical:clinical systemic area under the curve (AUC) margin compared with exposures at the highest clinically tested dose (300 mg, quaque die, once daily, phase 1 study in healthy volunteers). In dogs, it was possible to attain sufficient systemic exposures to result in immunosuppression characteristic of systemic JAK inhibition, but at high AUC margins (43×) compared with systemic exposures observed at the highest tested dose in humans. No adverse findings were observed in the gastrointestinal tract or systemic tissues. Izencitinib did not affect male or female fertility. Izencitinib did not affect embryonic development in rats and rabbits as commonly reported with systemic JAK inhibition, consistent with low maternal systemic concentrations (2-6× plasma Cave:JAK IC50 ratio, 10-33× nonclinical:clinical AUC margin) and negligible fetal exposures. In conclusion, the izencitinib gut-selective approach resulted in minimal systemic findings in nonclinical species at pharmacologic, clinically relevant systemic exposures, highlighting the impact of organ-selectivity in reducing systemic safety findings.

Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple Myeloma.

Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.

Analysis of strain difference in sensitivity to cadmium-induced hepatotoxicity in Fischer 344 and Sprague-Dawley rats.

Acute administration of cadmium (Cd) in rats results in hepatotoxicity that appears to involve the activation of Kupffer cells and the subsequent production of proinflammatory chemokines and cytokines. However, the importance of these endogenous mediators in Cd-induced hepatotoxicity is unknown. Therefore, this study was conducted to define and utilize a rat strain difference in sensitivity to Cd-induced hepatotoxicity to elucidate the role of cytokines and chemokines in Cd-induced hepatotoxicity. Doses were selected from a dose-response study of the effect of Cd on serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities. Hepatotoxic doses of 2.0 mg Cd/kg in Fischer 344 (F344) rats and 3.0 mg Cd/kg in Sprague-Dawley (SD) rats, as well as a relatively nontoxic dose of 2.0 mg Cd/kg in SD rats, were chosen for the time-course experiment. Blood and liver from F344 (saline or 2.0 mg Cd/kg iv) and SD rats (saline or 2.0 or 3.0 mg Cd/kg iv) were collected at 0, 1, 3, 6, 10, 18, 24, and 48 h after Cd administration. Cadmium treatment caused an increase in serum ALT and SDH by 3 h and peaked between 18 and 24 h in both strains. Hepatic Cd content, metallothionein (MT) induction, and nonprotein sulfhydryl (NPSH) content were quantified and determined to be consistent with dosing rather than strain differences. Total RNA samples isolated from liver samples were analyzed for chemokine (CINC-1 and MCP-1) and cytokine (TNF-alpha, IL-1beta, IL-6, and IL-10) mRNA levels by the Quantigene branched DNA signal amplification assay. Lipopolysaccharide treatment served as a positive control for chemokine and cytokine induction. After Cd administration, F344 rat livers did not contain higher levels or earlier induction of chemokine and cytokine mRNAs than SD rats. Therefore, this study demonstrates a strain difference in sensitivity to Cd-induced hepatotoxicity that appears to be unrelated to Cd, MT, NPSH, or cytokine expression.

iNOS-null mice are not resistant to cadmium chloride-induced hepatotoxicity.

Acute administration of cadmium (Cd) to rats results in hepatotoxicity. Recent reports indicate that Kupffer cells, the resident macrophages of the liver, participate in the manifestation of Cd-induced hepatotoxicity. Nitric oxide (NO) is a reactive nitrogen radical produced by activated Kupffer cells via the induction of inducible nitric oxide synthase (iNOS). Nitric oxide can combine with superoxide to form peroxynitrite, a molecule that may participate in the toxic mechanisms of hepatotoxins, such as acetaminophen and bacterial endotoxin. It has been speculated that Cd also may exert its hepatotoxicity, in part, via the production of NO by iNOS. Therefore, this study was undertaken to determine whether iNOS contributes to Cd-induced hepatotoxicity. Wild-type (WT) mice were administered selective iNOS inhibitors (AMT and 1400W) concurrently and 3 h after administration of a hepatotoxic dose of Cd (4.0 mg Cd/mg). Additionally, WT and iNOS-null (iNOS-KO) mice were dosed iv with saline or 2.0, 2.5, 3.0, 3.5 or 4.0 mg Cd/kg. Serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities were quantified to assess liver injury. Administration of iNOS inhibitors failed to prevent Cd-induced hepatotoxicity. Also, Cd caused a dose-dependent increase in liver injury in both WT and iNOS-KO mice. The liver injury produced by Cd in the iNOS-KO mice was not different from that in WT at any dose. These data indicate that iNOS does not appear to mediate Cd-induced hepatotoxicity.

DPP8 and DPP9 expression in cynomolgus monkey and Sprague Dawley rat tissues.

Dipeptidyl peptidases (DPPs) are proteolytic enzymes that regulate many physiological systems by degrading signaling peptides. DPP8 and DPP9 are distinct from DPP4 in sequence, cellular localization and expression levels, thus implying distinct functions. However, DPP8 and DPP9 expression needs further delineation. We evaluated DPP4, DPP8 and DPP9 expression using three independent methods at the mRNA, protein, and functional levels to better understand the local physiological contribution of each enzyme. Sprague Dawley rats and cynomolgus monkeys were selected for DPP4, DPP8 and DPP9 expression profiling to represent animal species commonly utilized for drug preclinical safety evaluation. A novel Xhibit assay of DPP protease activity was applied in addition to newly available antibodies for immunohistochemical localization. This combined approach can facilitate a functional evaluation of protease expression, which is important for understanding physiological relevance. Few inter-species differences were observed. Tissue mRNA and protein levels generally correlated to functional DPP4 and DPP8/9 enzymatic activity. All three proteins were seen in epithelial cells, lymphoid cells and some endothelial and vascular smooth muscle cells. Combined DPP8/DPP9 enzymatic activity was uniformly intracellular across tissues at approximately 10-fold lower levels than non-renal DPP4. Consistent levels of each DPP were detected among most non-renal tissues in rats and monkeys. DPP4 was ubiquitous, principally detected on cell membranes of epithelial and endothelial cells and was greatest in the kidney. The expression patterns suggest that DPP8 and DPP9 may act similarly across tissues, and that their actions might in part overlap with DPP4.

Liver deformation in Ahr-null mice: evidence for aberrant hepatic perfusion in early development.

Mice harboring mutations in the Ahr locus display a patent ductus venosus and smaller livers throughout life. We tested the hypothesis that these hepatic aberrations are secondary to a developmental defect in hepatovascular blood flow by performing a detailed analysis of hepatic development in wild-type and Ahr-/- mice. This study revealed necrotic lesions in the peripheries of Ahr-/- fetal livers as early as embryonic day 15.5, with an increasing incidence up to postnatal day 1 and resolution by 2 weeks post partum. To visualize perfusion of fetal livers, we injected fluorescein isothiocyanate-labeled dextran into the cranial artery and monitored hepatic fluorescence by microscopy. The peripheries of the median and left lobes displayed decreased perfusion in regions corresponding to those regions that displayed necrosis at later developmental times. An examination of adult Ahr-/- animals revealed that smaller livers are predominantly due to decreased sizes of the left and right lobes, corresponding to regions of decreased perfusion and hepatic necrosis observed in fetal livers. Histological aberrations in the portal vein also support a model in which perfusion is compromised in the Ahr-/- liver. Taken in sum, these results indicate that the Ahr locus is required for normal perfusion of the developing liver and that disruption of the AHR signaling pathway gives rise to fetal hepatic necrosis and consequent liver deformation which persists through adult-hood.

Tumor necrosis factor-alpha-null mice are not resistant to cadmium chloride-induced hepatotoxicity.

Acute administration of cadmium results in hepatotoxicity. Recent reports indicate that Kupffer cells, the resident macrophages of the liver, participate in the manifestation of chemical-induced hepatotoxicity. Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that is a major product of Kupffer cells and mediates the hepatotoxic effects of lipopolysaccharide (LPS). It has been speculated that cadmium also may exert its hepatotoxicity via the production of TNF-alpha by the Kupffer cells. Therefore, this study was undertaken to determine whether mice deficient in TNF-alpha are resistant to Cd-induced hepatotoxicity. TNF-alpha-null (TNF-KO) and wild-type (WT) mice were dosed ip with saline, LPS (0.1 mg/kg)/Gln (d-galactosamine, 700 mg/kg), or CdCl2 (2.2, 2.8, 3.4, and 3.9 mg Cd/kg). Serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities were quantified to assess liver injury. Caspase-3 activity was quantified to assess hepatocellular apoptosis. LPS/Gln treatment increased ALT (17-fold) and SDH (21-fold) in WT mice. In contrast, LPS/Gln-treatment did not significantly increase ALT or SDH in TNF-KO mice. LPS/Gln-treatment caused a 7.8-fold increase in caspase-3 activity in WT mice but did not increase caspase-3 in TNF-KO mice. Cadmium caused a dose-dependent increase in liver injury in both WT and TNF-KO mice. However, the liver injury produced by Cd in the TNF-KO mice was not different from that in WT at any dose. No significant increase in caspase-3 activity was detected in any of the Cd-treated mice. These data indicate that, in contrast to LPS/Gln-induced hepatotoxicity, TNF-alpha does not appear to mediate Cd-induced hepatotoxicity.

Gadolinium chloride pretreatment prevents cadmium chloride-induced liver damage in both wild-type and MT-null mice.

The heavy metal cadmium (Cd) causes hepatotoxicity upon acute administration. Kupffer cells, the resident macrophages of the liver, have been suggested to play a role in Cd-induced hepatotoxicity. Gadolinium chloride (GdCl3) may prevent Cd-induced hepatotoxicity by suppressing Kupffer cells. However, GdCl3 also induces the Cd-binding protein, metallothionein (MT). Therefore, this study was conducted to determine whether GdCl3 prevents Cd-induced hepatotoxicity via the induction of MT. Hepatic MT and Kupffer cell counts were analyzed 24 h after wild-type (WT) mice were administered saline or 10, 30, or 60 mg GdCl3/kg. GdCl3 induced MT in a dose-dependent manner without affecting nonprotein sulfhydryl content. All examined doses of GdCl3 were effective at eliminating Kupffer cells from the liver. To examine the hepatoprotective effects of GdCl3, WT and MT-null mice were pretreated with saline or 10, 30, or 60 mg GdCl3 24 h prior to a hepatotoxic dose of Cd (2.5 mg Cd/kg). Blood and livers were removed 16 h later and analyzed for hepatotoxicity as well as MT, Cd, and Kupffer cell content. Hepatotoxicity was alleviated in both WT and MT-null mice that were pretreated with 30 or 60 mg GdCl3/kg, indicating that MT induction is not required for the hepatoprotective effects of GdCl3. Hepatic Cd content was not decreased by GdCl3, demonstrating that GdCl3 does not negatively affect Cd distribution to the liver. Kupffer cells were depleted at all three doses of GdCl3, whereas hepatoprotection was only observed at doses of 30 and 60 mg GdCl3/kg. This does not rule out Kupffer cells in the mechanism of Cd-induced hepatotoxicity, but it does suggest that GdCl3 exerts hepatoprotective effects on the liver aside from depleting Kupffer cells. In summary, these data substantially rule out MT induction and decrease the importance of Kupffer cells as mechanisms of GdCl3-induced protection from Cd-induced hepatotoxicity.

Patent ductus venosus and dioxin resistance in mice harboring a hypomorphic Arnt allele.

The Ah receptor nuclear translocator (ARNT) is the dimeric partner of hypoxia-inducible factors and thus plays a pivotal role in cellular adaptation to low oxygen environments. ARNT is also a dimeric partner for the Ah receptor (AHR), and this complex is essential in regulating the adaptive metabolic response to polycyclic aromatic hydrocarbons. Because of the essential role of ARNT in hypoxia-driven developmental events, it has been difficult to study the physiological significance of AHR.ARNT heterodimers in vivo. To address this issue, we developed a hypomorphic Arnt allele that displayed normal development and allowed the examination of the role of ARNT in AHR biology. In this regard, the AHR is also known to mediate two additional biological processes: the toxicological response to compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and the developmental closure of a fetal vascular structure known as the ductus venosus. Although the mechanism of the adaptive pathway has been well described, the mechanism of AHR-mediated signal transduction in the toxic and developmental pathways is not well understood. Liver perfusion studies demonstrated that ARNT hypomorphs have a patent ductus venosus, identical to that observed in the Ahr null mice. Parallel dioxin toxicity studies demonstrated that the ARNT hypomorphs exhibited resistance to the end points of dioxin exposure. Moreover, we observed that toxicity could be segregated from the classical adaptive responses such as P4501A induction. Taken in sum, these experiments demonstrate that ARNT is an essential component of AHR developmental signaling and shed light on the mechanism of dioxin toxicity.