Found in Translation: Maximizing the Clinical Relevance of Nonclinical Oncology Studies.

Purpose: The translation of nonclinical oncology studies is a subject of continuous debate. We propose that translational oncology studies need to optimize both pharmacokinetic (drug exposure) and pharmacodynamic (xenograft model) aspects. While improvements in pharmacodynamic translatability can be obtained by choosing cell lines or patient-derived xenograft models closer to the clinical indication, significant ambiguity and variability exists when optimizing the pharmacokinetic translation of small molecule and biotherapeutic agents.Experimental Design and Results: In this work, we propose a pharmacokinetic-based strategy to select nonclinical doses for approved drug molecules. We define a clinically relevant dose (CRD) as the dosing regimen in mice that most closely approximates the relevant pharmacokinetic metric in humans. Such metrics include area under the time-concentration curve and maximal or minimal concentrations within the dosing interval. The methodology is applied to six drugs, including targeted agents and chemotherapeutics, small and large molecules (erlotinib, dasatinib, vismodegib, trastuzumab, irinotecan, and capecitabine). The resulting efficacy response at the CRD is compared with clinical responses.Conclusions: We conclude that nonclinical studies designed with the appropriate CRDs of approved drug molecules will maximize the translatability of efficacy results, which is critical when testing approved and investigational agents in combination. Clin Cancer Res; 23(4); 1080-90. ©2016 AACR.

Mechanistic investigation of the preclinical pharmacokinetics and interspecies scaling of PF-05231023, a fibroblast growth factor 21-antibody protein conjugate.

PF-05231023, a long-acting fibroblast growth factor 21 (FGF21) analog, was generated by covalently conjugating two engineered [des-His1, Ala129Cys]FGF21 molecules to a nontargeting human IgG1 κ scaffold. The pharmacokinetics (PK) of PF-05231023 after i.v. and s.c. administration was evaluated in rats and monkeys using two enzyme-linked immunosorbent assays with high specificity for biologically relevant intact N termini (NT) and C termini (CT) of FGF21. Intact CT of FGF21 displayed approximately 5-fold faster systemic plasma clearance (CL), an approximately 2-fold lower steady-state volume of distribution, and at least 5-fold lower bioavailability compared with NT. In vitro serum stability studies in monkeys and humans suggested that the principal CL mechanism for PF-05231023 was degradation by serum proteases. Direct scaling of in vitro serum degradation rates for intact CT of FGF21 underestimated in vivo CL 5-fold, 1.4-fold, and 2-fold in rats, monkeys, and humans, respectively. The reduced steady-state volume of distribution and the bioavailability for intact CT relative to NT in rats and monkeys were compatible with proteolytic degradation occurring outside the plasma compartment via an unidentified mechanism. Human CL and PK profiles for intact NT and CT of FGF21 were well predicted using monkey single-species allometric and Dedrick scaling. Physiologically based pharmacokinetic models incorporating serum stability data and an extravascular extraction term based on differential bioavailability of intact NT and CT of FGF21 in monkeys improved accuracy of human PK predictions relative to Dedrick scaling. Mechanistic physiologically based pharmacokinetic models of this nature may be highly valuable for predicting human PK of fusion proteins, synthetically conjugated proteins, and other complex biologics.

Pharmacokinetics (PK), pharmacodynamics (PD) and integrated PK/PD modeling of a novel long acting FGF21 clinical candidate PF-05231023 in diet-induced obese and leptin-deficient obese mice.

Pharmacological administration of fibroblast growth factor 21 (FGF21) improves metabolic profile in preclinical species and humans. FGF21 exerts its metabolic effects through formation of beta-klotho (KLB)/FGF receptor 1c FGFR1c complex and subsequent signaling. Data from various in vitro systems demonstrate the intact C- and N-terminus of FGF21 is required for binding with KLB, and interaction with FGFR1c, respectively. However the relative roles of the termini for in vivo pharmacological effects are unclear. Here we report PF-05231023, a long-acting FGF21 analogue which is unique in that the half-life and subcutaneous (s.c.) bioavailability of the intact C-terminus are significantly different from those of the intact N-terminus (2 vs. 22 hr for half-life and 4~7 vs. ~50% SC bioavailability). Therefore, this molecule serves as a valuable tool to evaluate the relative roles of intact C-terminus vs. N-terminus in in vivo pharmacology studies in preclinical species. We determined the effects of PF-05231023 administration on body weight (BW) loss and glucose reduction during an oral glucose tolerance test (OGTT) following SC and intravenous (i.v.) administration in diet-induced obese (DIO) and leptin-deficient obese (ob/ob) mice, respectively. Our data show that the intact N-terminus of FGF21 in PF-05231023 appears to be sufficient to drive glucose lowering during OGTT and sustain BW loss in DIOs. Further, PK/PD modeling suggests that while the intact FGF21 C-terminus is not strictly required for glucose lowering during OGTT in ob/ob mice or for BW reduction in DIO mice, the higher potency conferred by intact C-terminus contributes to a rapid initiation of pharmacodynamic effects immediately following dosing. These results provide additional insight into the strategy of developing stabilized versions of FGF21 analogs to harness the full spectrum of its metabolic benefits.

Study of CRTC2 pharmacology using antisense oligonuceotides.

The cAMP response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is a key component of the transcription complex regulating glucagon driven hepatic glucose production and previous evidence suggests that "inhibition" of CRTC2 improves glucose homeostasis in multiple rodent models of type 2 diabetes. Here we describe a process of identifying potential therapeutic antisense oligonucleotides (ASOs) directed against CRTC2. These ASOs were designed as locked nucleic acid (LNA) gapmers and a panel of approximately 400 sequences were first screened in vitro within both human and mouse liver cell lines. A group of active and selective compounds were then profiled in acute studies in mice to determine the level of CRTC2 mRNA reduction in liver as well as to obtain a preliminary indication of safety and tolerability. The compounds with the best activity and safety profiles were then evaluated in subchronic efficacy studies using the diet induced obese (DIO) mouse model of type 2 diabetes and primary human hepatocytes. Efficacy findings broadly confirmed the beneficial effect of reducing CRTC2 mRNA levels towards improving glucose control and other markers of metabolic function. Additionally, for the first time, translation to human cells has been established with demonstration of a reduction in glucagon-mediated glucose production in primary human hepatocytes and a potential clinical biomarker source identified to assess modulation of CRTC2 mRNA following ASO treatment. While the compounds identified herein did not demonstrate a therapeutic index sufficient for further development, this study should facilitate more efficient prosecution of compounds within an in vivo setting.

Design and synthesis of orally-active and selective azaindane 5HT2c agonist for the treatment of obesity.

Based on our original pyrazine hit, CP-0809101, novel conformationally-restricted 5HT2c receptor agonists with 2-piperazin-azaindane scaffold were designed. Synthesis and structure-activity relationship (SAR) studies are described with emphasis on optimization of the selectivity against 5HT2a and 5HT2b receptors with excellent 2c potency. Orally-active and selective compounds were identified with dose-responsive in vivo efficacy in our pre-clinical food intake model.

Genotoxicity of 2-(3-chlorobenzyloxy)-6-(piperazinyl)pyrazine, a novel 5-hydroxytryptamine2c receptor agonist for the treatment of obesity: role of metabolic activation.

2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) is a potent and selective 5-HT(2C) agonist that exhibits dose-dependent inhibition of food intake and reduction in body weight in rats, making it an attractive candidate for treatment of obesity. However, examination of the genotoxicity potential of 3 in the Salmonella Ames assay using tester strains TA98, TA100, TA1535, and TA1537 revealed a metabolism (rat S9/NADPH)- and dose-dependent increase of reverse mutations in strains TA100 and TA1537. The increase in reverse mutations was attenuated upon coincubation with methoxylamine and glutathione. The irreversible and concentration-dependent incorporation of radioactivity in calf thymus DNA after incubations with [14C]3 in the presence of rat S9/NADPH suggested that 3 was bioactivated to a reactive intermediate that covalently bound DNA. In vitro metabolism studies on 3 with rat S9/NADPH in the presence of methoxylamine and cyanide led to the detection of amine and cyano conjugates of 3. The mass spectrum of the amine conjugate was consistent with condensation of amine with an aldehyde metabolite derived from hydroxylation of the secondary piperazine nitrogen-alpha-carbon bond. The mass spectrum of the cyano conjugate suggested a bioactivation pathway involving N-hydroxylation of the secondary piperazine nitrogen followed by two-electron oxidation to generate an electrophilic nitrone, which reacted with cyanide. The 3-chlorobenzyl motif in 3 was also bioactivated via initial aromatic ring hydroxylation followed by elimination to a quinone-methide species that reacted with glutathione or with the secondary piperazine ring nitrogen in 3 and its monohydroxylated metabolite(s). The metabolism studies described herein provide a mechanistic basis for the mutagenicity of 3.