Christopher Houle

Discovery and preclinical profiling of 3-[4-(morpholin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]benzonitrile (PF-06447475), a highly potent, selective, brain penetrant, and in vivo active LRRK2 kinase inhibitor.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinsons disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.

Evaluation of Serum Bile Acid Profiles as Biomarkers of Liver Injury in Rodents

Bile acids (BAs) have been studied as potential biomarkers of drug-induced liver injury. However, the relationship between levels of individual BAs and specific forms of liver injury remains to be fully understood. Thus, we set out to evaluate cholic acid (CA), glycocholic acid (GCA), and taurocholic acid (TCA) as potential biomarkers of liver injury in rodent toxicity studies. We have developed a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay applicable to rat and mouse serum and evaluated levels of the individual BAs in comparison with the classical biomarkers of hepatotoxicity (alanine aminotransferase [ALT], aspartate aminotransferase [AST], glutamate dehydrogenase (GLDH), alkaline phosphatase, total bilirubin, gamma-glutamyl transferase, and total BAs) and histopathology findings in animals treated with model toxicants. The pattern of changes in the individual BAs varied with different forms of liver injury. Animals with histopathologic signs of hepatocellular necrosis showed increases in all 3 BAs tested, as well as increases in ALT, AST, GLDH, and total BAs. Animals with histopathologic signs of bile duct hyperplasia (BDH) displayed increases in only conjugated BAs (GCA and TCA), a pattern not observed with the other toxicants. Because BDH is detectable only via histopathology, our results indicate the potential diagnostic value of examining individual BAs levels in serum as biomarkers capable of differentiating specific forms of liver injury in rodent toxicity studies.

Kinase domain inhibition of leucine rich repeat kinase 2 (LRRK2) using a [1,2,4]triazolo[4,3-b]pyridazine scaffold.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinsons disease (PD). The most common mutant, G2019S, increases kinase activity, thus LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the structure, potential ligand-protein binding interactions, and pharmacological profiling of potent and highly selective kinase inhibitors based on a triazolopyridazine chemical scaffold.

Drug-induced Skin Lesions in Cynomolgus Macaques Treated with Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulators.

Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

Perspectives on Drug-induced Vascular Injury

Drug-induced vascular injury in nonclinical toxicity studies has been a difficult problem for the pharmaceutical industry for decades. Drugs associated with vascular injury include several classes of antibacterial agents, antimalarials, antivirals, antiinflammatory agents, and molecules manipulating multiple mechanisms for neurologic diseases, cardiovascular diseases, and diabetes mellitus. While some mechanisms of action (e.g., dopamine receptor agonists or antagonists, phosphodiesterase inhibitors, adrenergic agonists and antagonists, endothelin antagonists, and compounds that modulate endothelial nitric oxide synthase) would be expected to have vasoactive properties, other compounds can produce vascular injury with no obvious mechanism. Acute or multidose cardiovascular safety pharmacology studies in animals often show no systemic effects on blood pressure, heart rate, electrocardiographic data, or cardiac contractility for chemicals associated with vascular injury. Changes in vascular tone in regional arterial beds often are suspected but are difficult to demonstrate. The relevance of these findings in rats, dogs, or monkeys for human risk assessment is uncertain, and drug-induced vascular injury in humans related to vasoactive properties of chemicals is sparsely reported and poorly characterized. Human risk assessment is further complicated because vascular injury is difficult to detect or monitor in patients. Historically, drug-induced vascular injury has been a problem for small molecule pharmaceuticals, but more recently some monoclonal antibodies, other protein therapeutics, and oligonucleotide therapies have produced vascular injury in animal species through mechanisms that are likely different from those for small molecules. Further complicating risk assessment for vascular injury observed in nonclinical studies is the infrequent and sporadic nature of the findings in animals treated with some chemicals. Spontaneous arterial injury (arteriopathy) in control animals is uncommon, but not rare. The incidences of vascular injury in control rats, dogs, and cynomolgus monkeys from 2008 to 2013 in studies sponsored by Pfizer Inc. are shown in Table 1. Although the incidences vary considerably from year to year, it is unusual to find more than 1 control animal with vascular injury in any particular study, and very often the vascular injury in control animals is minimal in severity and limited to 1 or very few blood vessels. Unfortunately, vascular injury in the treated groups often is similarly limited to 1 or a few animals and only a few blood vessels. Based on the authors’ review of nonclinical toxicity studies over a 5-year period, vascular injury was reported in studies representing 114 small molecule drug candidates across many different mechanisms of action. Fifty-five of these compounds had vascular injury reported in only a single animal in just 1 study, and for 17 of these 55 molecules the vascular injury was limited to the nonrodent species with no vascular findings in rats. When the number of animals and number of blood vessels affected are very low in a study, the determination of whether these effects are test article related or incidental background noise can be difficult, particularly when there are limited historical data for the compound. When the interpretation of small exploratory studies is equivocal, one option is to move forward into larger studies to help provide more definitive information. However, due to the difficulties in differentiating spontaneous from drug-induced vascular injury, some sponsors may halt development of a drug candidate even though it may have benefit without causing vascular injury in patients. The pharmaceutical industry has been looking for decades for reliable noninvasive biomarkers of vascular injury in body fluids, other methods to detect or visualize vascular injury in humans, tools to identify mechanisms of vascular injury, and in vitro models that accurately predict human vascular injury. Progress has been frustratingly slow. Investigators representing many pharmaceutical companies are collaborating through the Vascular Injury Working Group within the Critical Path Institute’s Predictive Safety Testing Consortium (PSTC) to summarize the mechanisms of arterial injury in animals associated with vasodilation and vasoconstriction, establish a recommended histomorphologic lexicon based on anatomic site of vascular injury, propose a strategy to identify and qualify vascular injury biomarkers, and initiate early testing of multiple proposed biomarkers (see Mikaelian et al. in this issue). The Safer and Faster Evidence-based Translation (SAFE-T) collaboration within the Innovative Medicines Initiative sponsored by the European Commission and the The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The author(s) received no financial support for the research, authorship, and/or publication of this article. Address correspondence to: Daniel Morton, Pfizer Inc., 200 Cambridge Park Drive, T4017C, Cambridge, MA 02140, USA; e-mail: dan.g.morton@pfizer.com. Abbreviations: EFPIA, European Federation of Pharmaceutical Industries and Associations; PSTC, Predictive Safety Testing Consortium; SAFE-T, Safer and Faster Evidence-based Translation.

Mechanisms of Skin Toxicity Associated with Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators

Cutaneous reactions represent one of the most common adverse drug effects observed in clinical trials leading to substantial compound attrition. Three negative allosteric modulators (NAMs) of metabotropic glutamate receptors (mGluRs), which represent an important target for neurological diseases, developed by Pfizer, were recently failed in preclinical development due to delayed type IV skin hypersensitivity observed in non-human primates (NHPs). Here we employed large-scale phenotypic profiling in standardized panels of human primary cell/co-culture systems to characterize the skin toxicity mechanism(s) of mGluR5 NAMs from two different series. Investigation of a database of chemicals tested in these systems and transcriptional profiling suggested that the mechanism of toxicity may involve modulation of nuclear receptor targets RAR/RXR, and/or VDR with AhR antagonism. The studies reported here demonstrate how phenotypic profiling of preclinical drug candidates using human primary cells can provide insights into the mechanisms of toxicity and inform early drug discovery and development campaigns.

Using Histopathologic Evidence to Differentiate Reproductive Senescence from Xenobiotic Effects in Middle-aged Female Sprague-Dawley Rats:

The female reproductive cycle is orchestrated by cyclical and coordinated hormonal changes under the direction of the hypothalamic–pituitary–gonadal (HPG) axis. Any disruption of the HPG axis may lead to functional and structural alterations in the female reproductive system. Test article–related disturbances in the estrous cycle can be recognized in nonclinical toxicity studies by staging the cycle based on microscopic evaluation of female reproductive organs. In chronic rat toxicity studies, an additional complication is the development of reproductive senescence, which is associated with natural alterations in the reproductive cycle leading to changes in the female reproductive system that can potentially be confused with test article effects. The current article describes the features of persistent estrus, one stage of reproductive senescence, in middle-aged Sprague-Dawley rats and discusses elements to help differentiate senescence from induced effects.

Two-year Carcinogenicity Study in Rats with a Nonnucleoside Reverse Transcriptase Inhibitor

Administration of lersivirine, a nonnucleotide reverse transcriptase inhibitor, daily by oral gavage to Sprague-Dawley rats for up to 2 yr was associated with decreased survival, decreased body weights, and an increase in neoplasms and related proliferative lesions in the liver, thyroid, kidney, and urinary bladder. Thyroid follicular adenoma and carcinoma, the associated thyroid follicular hypertrophy/hyperplasia, hepatocellular adenoma/adenocarcinoma, altered cell foci, and hepatocellular hypertrophy were consistent with lersivirine-related induction of hepatic microsomal enzymes. Renal tubular adenoma and renal tubular hyperplasia were attributed to the lersivirine-related exacerbation of chronic progressive nephropathy (CPN), while urinary bladder hyperplasia and transitional cell carcinoma in the renal pelvis and urinary bladder were attributed to urinary calculi. Renal tubular neoplasms associated with increased incidence and severity of CPN, neoplasms of transitional epithelium attributed to crystalluria, and thyroid follicular and hepatocellular neoplasms related to hepatic enzyme induction have low relevance for human risk assessment.

Effects of Lersivirine on Canine and Rodent Thyroid Function

Lersivirine is a nonnucleoside reverse transcriptase inhibitor (NNRTI) being developed for the treatment of HIV-1 infection. Like other NNRTIs, lersivirine is a potent enzyme inducer in rodents capable of inducing a number of hepatic enzymes including those involved in its own metabolism. Preclinically lersivirine has been associated with hepatocellular hypertrophy and thyroid gland follicular cell hypertrophy in rats, mice, and dogs. In rodents, we show that development of thyroid hypertrophy is related to the classic mechanism, namely increased thyroxine (T4) clearance secondary to induction of uridine-diphosphoglucuronosyltransferase (UDPGT) in the liver and a resulting increase in thyroid-stimulating hormone. Similarly, lersivirine-exposed dogs exhibit a significant increase in hepatic UDPGT enzyme activity along with increased T4 clearance although clear effects on serum thyroid hormone levels were less apparent. These effects on thyroid hormonal clearance in the dog suggest that thyroid gland hypertrophy in this species is due to the same mechanism shown to occur in rodents although, as expected, dogs better adapt to these effects and therefore maintain relatively normal thyroid hormonal balance. It is also notable that the minimal thyroid follicular hypertrophy that occurs in dogs does not progress as is seen in rodents. As is the case with rodents, these adaptive changes in the dog are not considered indicative of a human health risk.

Design and Synthesis of Clinical Candidate PF-06751979: A Potent, Brain Penetrant, β-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Lacking Hypopigmentation

A major challenge in the development of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimers disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.