Comparative Analyses of Poly(ADP-Ribose) Polymerase Inhibitors.

Poly(ADP-ribose) polymerase inhibitors (PARPi) are approved as monotherapies in BRCA1/2-mutated (mBRCA1/2) metastatic breast and ovarian cancers, and in advanced pancreatic and metastatic castration-resistant prostate cancers. Differential safety profiles across PARPi necessitate improved mechanistic understanding of inhibitor differences, especially with expansion of PARPi indications and drug combinations. Here, we report in vitro evaluations of PARPi (-/+ PARP trapper temozolomide, TMZ) with reference to total clinical mean concentration average or maximum (tCavg, tCmax), to elucidate contributions of primary pharmacology and structural differences to clinical efficacy and safety. In biochemical assays, rucaparib and niraparib demonstrated off-target secondary pharmacology activities, and in selectivity assays, talazoparib, olaparib, and rucaparib inhibited a broader panel of PARP enzymes. In donor-derived human bone marrow mononuclear cells, only olaparib both increased early apoptosis and decreased the cell viability half inhibitory concentration (IC50) at ≤ tCavg, whereas other PARPi only did so in the presence of TMZ. In cancer cell lines with DNA damage repair mutations, all PARPi decreased cell viability in H1048 but not TK6 cells, and only talazoparib decreased cell growth in DU145 cells at ≤ tCavg concentrations. When combined with low dose TMZ, only talazoparib left-shifted the functional consequences of PARP trapping (S-phase arrest, apoptosis, S-phase double-stranded breaks) and reduced cell viability/growth in TK6 and DU145 cell lines at ≤ tCavg, whereas the other inhibitors required high-dose TMZ. Our study suggests structural differences across PARPi may contribute to differences in PARP selectivity and off-target activities, which along with distinct pharmacokinetic properties, may influence inhibitor-specific toxicities in patients.

Enzalutamide and Apalutamide: In Vitro Chemical Reactivity Studies and Activity in a Mouse Drug Allergy Model.

Enzalutamide and apalutamide are two androgen receptor inhibitors approved for the treatment of castration-resistant prostate cancer (CRPC) and nonmetastatic castration-resistant prostate cancer (nmCRPC), respectively. Apalutamide is associated with an increased incidence of skin rash above the placebo groups in the SPARTAN trial in nmCRPC and in the TITAN trial in metastatic castration-sensitive prostate cancer patients. On the contrary, the rate of skin rash across all clinical trials (including PROSPER [nmCRPC]) for enzalutamide is similar to the placebo. We hypothesized that the apalutamide-associated increased skin rash in patients could be linked to a structural difference. The 2-cyanophenyl and dimethyl moieties in enzalutamide are substituted in apalutamide with 2-cyanopyridine and cyclobutyl, respectively. In our evaluations, the 2-cyanopyridine moiety of apalutamide was chemically reactive with the thiol nucleophile glutathione, resulting in rearranged thiazoline products. Radiolabeled apalutamide, but not radiolabeled enzalutamide, was shown to react with mouse and human plasma proteins. Thiol nucleophiles decreased the extent of covalent binding to the model protein bovine serum albumin, whereas amine and alcohol nucleophiles had no effect, suggesting reactivity with cysteine of proteins. Subcutaneous administration of apalutamide dose dependently increased lymphocyte cellularity in draining lymph nodes in a mouse drug allergy model (MDAM). Enzalutamide, and its known analogue RD162 in which the cyanophenyl was retained but the dimethyl was replaced by cyclobutyl, demonstrated substantially less covalent binding activity and negative results in the MDAM assay. Collectively, these data support the hypothesis that the 2-cyanopyridine moiety in apalutamide may react with cysteine in proteins forming haptens, which may trigger an immune response, as indicated by the activity of apalutamide in the MDAM assay, which in turn may be leading to increased potential for skin rash versus placebo in patients in the SPARTAN and TITAN clinical trials.

Industry perspective on the nonclinical safety assessment of heterobifunctional degraders.

Targeted protein degraders (TPDs), which act through the ubiquitin proteasome system (UPS), are one of the newest small-molecule drug modalities. Since the initiation of the first clinical trial in 2019, investigating the use of ARV-110 in patients with cancer, the field has rapidly expanded. Recently, some theoretical absorption, distribution, metabolism, and excretion (ADME) and safety challenges have been posed for the modality. Using these theoretical concerns as a framework, the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) Protein Degrader Working Group (WG) conducted two surveys to benchmark current preclinical practices for TPDs. Conceptually, the safety assessment of TPDs is the same as for standard small molecules; however, the techniques used, assay conditions/study endpoints, and timing of assessments might need to be modified to address differences in mode of action of the class.

L-type calcium channel antagonism - Translation from in vitro to in vivo.

INTRODUCTION: Although therapeutically beneficial in the treatment of certain diseases, L-type calcium channel antagonism can result in unwanted off-target pharmacology leading to adverse drug reactions and to the termination of the development of otherwise promising compounds. In the present study three marketed calcium channel inhibitors, nifedipine, verapamil and diltiazem were profiled in a series of in vitro and ex-vivo assays in an effort to determine the ability of these assays to discriminate, between dihydropyridine versus non-dihydropyridine-like compounds, and how well they can predict the cardiovascular effects observed in a conscious telemetered rat model. METHODS: Standard calcium channel antagonists were profiled in radioligand binding, patch clamp and calcium flux assays. In addition, cardiovascular endpoints related to calcium channel activity were also examined in ex vivo tissue bath preparations, including relaxation of pre-constricted rat aorta and the guinea pig Langendorff isolated heart model. The data generated were correlated with in vivo blood pressure and heart rate data from conscious telemetered rats. RESULTS: Our results show that the binding, FLIPR and aorta assays allow differentiation of the compounds in two distinct classes of L-type calcium channel antagonists, and are good predictors of in vivo outcomes. DISCUSSION: These results suggest that in vitro and ex vivo profiling remains a valuable tool in predicting potential in vivo cardiovascular safety issues, and can aid in the selection of novel development compounds that show inherent inhibitory activity against L-type calcium channels.

Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Statistical power analysis of cardiovascular safety pharmacology studies in conscious rats.

UNLABELLED: Cardiovascular (CV) toxicity and related attrition are a major challenge for novel therapeutic entities and identifying CV liability early is critical for effective derisking. CV safety pharmacology studies in rats are a valuable tool for early investigation of CV risk. Thorough understanding of data analysis techniques and statistical power of these studies is currently lacking and is imperative for enabling sound decision-making. METHODS: Data from 24 crossover and 12 parallel design CV telemetry rat studies were used for statistical power calculations. Average values of telemetry parameters (heart rate, blood pressure, body temperature, and activity) were logged every 60s (from 1h predose to 24h post-dose) and reduced to 15min mean values. These data were subsequently binned into super intervals for statistical analysis. A repeated measure analysis of variance was used for statistical analysis of crossover studies and a repeated measure analysis of covariance was used for parallel studies. Statistical power analysis was performed to generate power curves and establish relationships between detectable CV (blood pressure and heart rate) changes and statistical power. Additionally, data from a crossover CV study with phentolamine at 4, 20 and 100mg/kg are reported as a representative example of data analysis methods. RESULTS: Phentolamine produced a CV profile characteristic of alpha adrenergic receptor antagonism, evidenced by a dose-dependent decrease in blood pressure and reflex tachycardia. Detectable blood pressure changes at 80% statistical power for crossover studies (n=8) were 4-5mmHg. For parallel studies (n=8), detectable changes at 80% power were 6-7mmHg. Detectable heart rate changes for both study designs were 20-22bpm. DISCUSSION: Based on our results, the conscious rat CV model is a sensitive tool to detect and mitigate CV risk in early safety studies. Furthermore, these results will enable informed selection of appropriate models and study design for early stage CV studies.

Discovery of 1-{(3R,4R)-3-[({5-Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (PF-06459988), a Potent, WT Sparing, Irreversible Inhibitor of T790M-Containing EGFR Mutants.

First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR. A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (i) high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR.

Effects of chronic PPAR-agonist treatment on cardiac structure and function, blood pressure, and kidney in healthy sprague-dawley rats.

PPAR-γ agonists have been associated with heart failure (HF) in diabetic patients. These incidences have been reported mostly in patient populations who were at high risk for HF or had pre-existing impaired cardiovascular function. However, whether there are similar effects of these agents in subjects with no or reduced cardiovascular pathophysiology is not clear. In this study, the effects of chronic treatment with PD168, a potent peroxisome proliferator activated receptor (PPAR) subtype-γ agonist with weak activity at PPAR-α, and rosiglitazone (RGZ), a less potent PPAR-γ agonist with no PPAR-α activity, were evaluated on the cardiovascular-renal system in healthy male Sprague-Dawley (SD) rats by serial echocardiography and radiotelemetry. Rats were treated with vehicle (VEH), PD168, @ 10 or 50 mg/kg.bw/day (PD-10 or PD-50, resp.) or RGZ @ 180 mg/kg.bw/day for 28 days (n = 10/group). Relative to VEH, RGZ, and both doses of PD168 resulted in a significant fall in blood pressure. Furthermore, RGZ and PD168 increased plasma volume (% increase from baseline) 18%, 22%, and 48% for RGZ, PD-10, and PD-50, respectively. PD168 and RGZ significantly increased urinary aldosterone excretion and heart-to-body weight ratio relative to VEH. In addition, PD168 significantly decreased (10-16%) cardiac ejection fraction (EF) and increased left ventricular area (LVA) in systole (s) and diastole (d) in PD-10 and -50 rats. RGZ significantly increased LVAd; however, it did not affect EF relative to VEH. In conclusion, chronic PPAR-γ therapy may predispose the cardiorenal system to a potential sequela of structural and/or functional changes that may be deleterious with regard to morbidity and mortality.