The Pharmacokinetics, Metabolism, and Clearance Mechanisms of Ritlecitinib, a Janus Kinase 3 and Tyrosine-Protein Kinase Family Inhibitor, in Humans.

Ritlecitinib is an oral once-daily irreversible inhibitor of Janus kinase 3 and tyrosine-protein kinase family being developed for the treatment of moderate-to-severe alopecia areata. This study examined the disposition of ritlecitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite profiles. The results indicated ritlecitinib had a systemic clearance of 43.7 L/h, a steady state volume of distribution of 73.8 L, extent of absorption of 89%, time to maximum plasma concentration of ∼0.5 hours, and absolute oral bioavailability of 64%. An observed long terminal half-life of total radioactivity was primarily attributed to ritlecitinib binding to plasma albumin. Ritlecitinib was the main circulating drug species in plasma (∼30%), with one major pharmacologically inactive cysteine conjugated metabolite (M2) at >10%. Oxidative metabolism (fractional clearance 0.47) and glutathione-related conjugation (fractional clearance 0.24) were the primary routes of elimination for ritlecitinib with the greatest disposition of radioactivity shown in the urine (∼71%). In vitro phenotyping indicated ritlecitinib cytochrome P450 (CYP) fraction of metabolism assignments of 0.29 for CYP3A, 0.09 for CYP2C8, 0.07 for CYP1A2, and 0.02 for CYP2C9. In vitro phenotyping in recombinant human glutathione S-transferases indicated ritlecitinib was turned over by a number of cytosolic and microsomal enzyme isoforms. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of ritlecitinib, a JAK3 and TEC family kinase inhibitor for alopecia areata in humans, as well as characterization of clearance pathways and pharmacokinetics of ritlecitinib and its metabolites. As an AMS-based ADME study design, we have expanded on reporting the standard ADME endpoints, providing key pharmacokinetic parameters, such as clearance, volume of distribution, and bioavailability, allowing for a more comprehensive understanding of drug disposition.

The Metabolism and Disposition of Brepocitinib in Humans and Characterization of the Formation Mechanism of an Aminopyridine Metabolite.

Brepocitinib is an oral once-daily Janus kinase 1 and Tyrosine kinase 2 selective inhibitor currently in development for the treatment of several autoimmune disorders. Mass balance and metabolic profiles were determined using accelerator mass spectrometry in six healthy male participants following a single oral 60 mg dose of 14C-brepocitinib (∼300 nCi). The average mass balance recovery was 96.7% ± 6.3%, with the majority of dose (88.0% ± 8.0%) recovered in urine and 8.7% ± 2.1% of the dose recovered in feces. Absorption of brepocitinib was rapid, with maximal plasma concentrations of total radioactivity and brepocitinib achieved within 0.5 hours after dosing. Circulating radioactivity consisted primarily of brepocitinib (47.8%) and metabolite M1 (37.1%) derived from hydroxylation at the C5' position of the pyrazole ring. Fractional contributions to metabolism via cytochrome P450 enzymes were determined to be 0.77 for CYP3A4/5 and 0.14 for CYP1A2 based on phenotyping studies in human liver microsomes. However, additional clinical studies are required to understand the potential contribution of CYP1A1. Approximately 83% of the dose was eliminated as N-methylpyrazolyl oxidative metabolites, with 52.1% of the dose excreted as M1 alone. Notably, M1 was not observed as a circulating metabolite in earlier metabolic profiling of human plasma from a multiple ascending dose study with unlabeled brepocitinib. Mechanistic studies revealed that M1 was highly unstable in human plasma and phosphate buffer, undergoing chemical oxidation leading to loss of the 5-hydroxy-1-methylpyrazole moiety and formation of aminopyrimidine cleavage product M2. Time-dependent inhibition and trapping studies with M1 yielded insights into the mechanism of this unusual and unexpected instability. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of brepocitinib, a JAK1/TYK2 inhibitor for atopic dermatitis, in humans as well as characterization of clearance pathways and pharmacokinetics of brepocitinib and its metabolites.

Utilization of Rosuvastatin and Endogenous Biomarkers in Evaluating the Impact of Ritlecitinib on BCRP, OATP1B1, and OAT3 Transporter Activity.

PURPOSE: Ritlecitinib, an inhibitor of Janus kinase 3 and tyrosine kinase expressed in hepatocellular carcinoma family kinases, is in development for inflammatory diseases. This study assessed the impact of ritlecitinib on drug transporters using a probe drug and endogenous biomarkers. METHODS: In vitro transporter-mediated substrate uptake and inhibition by ritlecitinib and its major metabolite were evaluated. Subsequently, a clinical drug interaction study was conducted in 12 healthy adult participants to assess the effect of ritlecitinib on pharmacokinetics of rosuvastatin, a substrate of breast cancer resistance protein (BCRP), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporter 3 (OAT3). Plasma concentrations of coproporphyrin I (CP-I) and pyridoxic acid (PDA) were assessed as endogenous biomarkers for OATP1B1 and OAT1/3 function, respectively. RESULTS: In vitro studies suggested that ritlecitinib can potentially inhibit BCRP, OATP1B1 and OAT1/3 based on regulatory cutoffs. In the subsequent clinical study, coadministration of ritlecitinib decreased rosuvastatin plasma exposure area under the curve from time 0 to infinity (AUCinf) by ~ 13% and maximum concentration (Cmax) by ~ 27% relative to rosuvastatin administered alone. Renal clearance was comparable in the absence and presence of ritlecitinib coadministration. PK parameters of AUCinf and Cmax for CP-I and PDA were also similar regardless of ritlecitinib coadministration. CONCLUSION: Ritlecitinib does not inhibit BCRP, OATP1B1, and OAT3 and is unlikely to cause a clinically relevant interaction through these transporters. Furthermore, our findings add to the body of evidence supporting the utility of CP-I and PDA as endogenous biomarkers for assessment of OATP1B1 and OAT1/3 transporter activity.

A phase 1 study to investigate the absorption, distribution, metabolism and excretion of brepocitinib in healthy males using a 14 C-microdose approach.

AIMS: Brepocitinib is a tyrosine kinase 2/Janus kinase 1 inhibitor being investigated for the treatment of several autoimmune diseases. This study assessed the absorption, distribution, metabolism and excretion of oral brepocitinib, and the absolute oral bioavailability (F) and fraction absorbed (Fa ) using a 14 C microtracer approach. METHODS: This was a phase 1 open-label, nonrandomized, fixed sequence, two-period, single-dose study of brepocitinib in healthy male participants. Participants received a single oral 60 mg dose of 14 C brepocitinib (~300 nCi) in Period A, then an unlabelled oral 60 mg dose followed by an intravenous (IV) 30 µg dose of 14 C labelled brepocitinib (~300 nCi) in Period B. Mass balance, pharmacokinetic parameters and safety were assessed. RESULTS: Six participants were enrolled. Brepocitinib was absorbed rapidly following oral administration. In Period A, total recovery of the oral dose was 96.7% ± 6.3% (88.0% ± 8.0% in urine, 8.7% ± 2.1% in faeces). In Period B, a small fraction (6.0% of the oral dose) was recovered unchanged in urine. F and Fa were 74.6% (90% confidence interval 67.3%, 82.8%) and 106.9%, respectively. Brepocitinib demonstrated an acceptable safety profile and was well tolerated following oral or oral then IV administrations. No deaths, serious adverse events or discontinuations were reported. CONCLUSION: Intestinal absorption of brepocitinib was essentially complete after oral administration, with F ~75%. Drug-related material recovery was high, with the majority excreted in urine. The major route of elimination of brepocitinib was renal excretion as metabolites, whereas urinary elimination of unchanged brepocitinib was minor. NCT: NCT03770039.

Evaluation of the Impact of Ritlecitinib on Organic Cation Transporters Using Sumatriptan and Biomarkers as Probes.

Ritlecitinib, an inhibitor of Janus kinase 3 and hepatocellular carcinoma family kinases, is in development as potential treatment for several inflammatory diseases. In vitro studies presented ritlecitinib as an inhibitor of hepatic organic cation transporter (OCT) 1, renal transporters OCT2 and multidrug and toxin extrusion (MATE) proteins 1/2K using multiple substrates, and ritlecitinib's major inactive metabolite M2, as an inhibitor of OCT1. A clinical interaction study with an OCT1 drug probe (sumatriptan) and relevant probe biomarkers for OCT/MATE was conducted to assess the effect of ritlecitinib on these transporters in healthy adult participants. The selectivity of sumatriptan for OCT1 was confirmed through a series of in vitro uptake assays. A simple static model was used to help contextualize the observed changes in sumatriptan area under the plasma concentration-time curve (AUC). Coadministration of a single 400-mg dose of ritlecitinib increased sumatriptan AUC from time 0 to infinity (AUCinf ) by ≈30% relative to a single 25-mg sumatriptan administration alone. When administered 8 hours after a ritlecitinib dose, sumatriptan AUCinf increased by ≈50% relative to sumatriptan given alone. Consistent with OCT1 inhibition, the AUC from time 0 to 24 hours of isobutyryl-L-carnitine decreased by ≈15% after ritlecitinib. Based on the evaluation of the renal clearance of N1 -methylnicotinamide, ritlecitinib does not exert clinically meaningful inhibition on renal OCT2 or MATE1/2K. This study confirmed that ritlecitinib and M2 are inhibitors of OCT1 but not OCT2 or MATE1/2K in healthy adults.

Evaluation of the Effect of Abrocitinib on Drug Transporters by Integrated Use of Probe Drugs and Endogenous Biomarkers.

Abrocitinib is an oral Janus kinase 1 (JAK1) inhibitor currently approved in the United Kingdom for the treatment of moderate-to-severe atopic dermatitis (AD). As patients with AD may use medications to manage comorbidities, abrocitinib could be used concomitantly with hepatic and/or renal transporter substrates. Therefore, we assessed the potential effect of abrocitinib on probe drugs and endogenous biomarker substrates for the drug transporters of interest. In vitro studies indicated that, among the transporters tested, abrocitinib has the potential to inhibit the activities of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporter 3 (OAT3), organic cation transporter 1 (OCT1), and multidrug and toxin extrusion protein 1 and 2K (MATE1/2K). Therefore, subsequent phase I, two-way crossover, open-label studies in healthy participants were performed to assess the impact of abrocitinib on the pharmacokinetics of the transporter probe substrates dabigatran etexilate (P-gp), rosuvastatin (BCRP and OAT3), and metformin (OCT2 and MATE1/2K), as well as endogenous biomarkers for MATE1/2K (N1 -methylnicotinamide (NMN)) and OCT1 (isobutyryl-L -carnitine (IBC)). Co-administration with abrocitinib was shown to increase the plasma exposure of dabigatran by ~ 50%. In comparison, the plasma exposure and renal clearance of rosuvastatin and metformin were not altered with abrocitinib co-administration. Similarly, abrocitinib did not affect the exposure of NMN or IBC. An increase in dabigatran exposure suggests that abrocitinib inhibits P-gp activity. By contrast, a lack of impact on plasma exposure and/or renal clearance of rosuvastatin, metformin, NMN, or IBC suggests that BCRP, OAT3, OCT1, and MATE1/2K activity are unaffected by abrocitinib.

Ritlecitinib and brepocitinib demonstrate significant improvement in scalp alopecia areata biomarkers.

BACKGROUND: Janus kinase (JAK) inhibitors have shown encouraging results in the treatment of alopecia areata (AA), an autoimmune form of hair loss, in small, uncontrolled studies and case reports. OBJECTIVE: We conducted a biopsy substudy during the randomized, double-blind, placebo-controlled first 24 weeks of a phase 2a clinical trial that evaluated the efficacy and safety of ritlecitinib, an inhibitor of JAK3 and the tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase family, and brepocitinib, an inhibitor of tyrosine kinase 2 (TYK2)/JAK1 in the treatment of AA. METHODS: Change in biomarkers in lesional scalp biopsy samples between baseline and weeks 12 and 24 was an exploratory end point, and 46 patients participated from the ritlecitinib (n = 18), brepocitinib (n = 16), and placebo (n = 12) groups. Correlations of biomarkers with hair regrowth, measured using the Severity of Alopecia Tool (SALT) score, were also evaluated. CLINICAL TRIAL REGISTRATION: NCT02974868. RESULTS: At week 24, both ritlecitinib and brepocitinib demonstrated improvement exceeding 100% in the lesional scalp transcriptome toward a nonlesional profile. At week 12, the improvements in scalp tissue were greater with brepocitinib than ritlecitinib; however, at week 24, the improvements were greater with ritlecitinib. CONCLUSIONS: For both ritlecitinib and brepocitinib, improvement in the SALT scores was positively associated with expression of TH1 markers and negatively associated with expression of hair keratins. Larger, long-term clinical trials are warranted.

Demonstration of In Vitro to In Vivo Translation of a TYK2 Inhibitor That Shows Cross Species Potency Differences.

Translation of modulation of drug target activity to therapeutic effect is a critical aspect for all drug discovery programs. In this work we describe the profiling of a non-receptor tyrosine-protein kinase (TYK2) inhibitor which shows a functionally relevant potency shift between human and preclinical species (e.g. murine, dog, macaque) in both biochemical and cellular assays. Comparison of the structure and sequence homology of TYK2 between human and preclinical species within the ATP binding site highlights a single amino acid (I960 → V) responsible for the potency shift. Through TYK2 kinase domain mutants and a TYK2 980I knock-in mouse model, we demonstrate that this single amino acid change drives a functionally relevant potency difference that exists between human and all evaluated preclinical species, for a series of TYK2 inhibitors which target the ATP binding site.

Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors.

Ongoing interest in the discovery of selective JAK3 inhibitors led us to design novel covalent inhibitors that engage the JAK3 residue Cys909 by cyanamide, a structurally and mechanistically differentiated electrophile from other cysteine reacting groups previously incorporated in JAK3 covalent inhibitors. Through crystallography, kinetic, and computational studies, interaction of cyanamide 12 with Cys909 was optimized leading to potent and selective JAK3 inhibitors as exemplified by 32. In relevant cell-based assays and in agreement with previous results from this group, 32 demonstrated that selective inhibition of JAK3 is sufficient to drive JAK1/JAK3-mediated cellular responses. The contribution from extrahepatic processes to the clearance of cyanamide-based covalent inhibitors was also characterized using metabolic and pharmacokinetic data for 12. This work also gave key insights into a productive approach to decrease glutathione/glutathione S-transferase-mediated clearance, a challenge typically encountered during the discovery of covalent kinase inhibitors.

Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune Diseases: Discovery of (( S)-2,2-Difluorocyclopropyl)((1 R,5 S)-3-(2-((1-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700841).

Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).

Clearance Prediction of Targeted Covalent Inhibitors by In Vitro-In Vivo Extrapolation of Hepatic and Extrahepatic Clearance Mechanisms.

The concept of target-specific covalent enzyme inhibitors appears attractive from both an efficacy and a selectivity viewpoint considering the potential for enhanced biochemical efficiency associated with an irreversible mechanism. Aside from potential safety concerns, clearance prediction of covalent inhibitors represents a unique challenge due to the inclusion of nontraditional metabolic pathways of direct conjugation with glutathione (GSH) or via GSH S-transferase-mediated processes. In this article, a novel pharmacokinetic algorithm was developed using a series of Pfizer kinase selective acrylamide covalent inhibitors based on their in vitro-in vivo extrapolation of systemic clearance in rats. The algorithm encompasses the use of hepatocytes as an in vitro model for hepatic clearance due to oxidative metabolism and GSH conjugation, and the use of whole blood as an in vitro surrogate for GSH conjugation in extrahepatic tissues. Initial evaluations with clinical covalent inhibitors suggested that the scaling algorithm developed from rats may also be useful for human clearance prediction when species-specific parameters, such as hepatocyte and blood stability and blood binding, were considered. With careful consideration of clearance mechanisms, the described in vitro-in vivo extrapolation approach may be useful to facilitate candidate optimization, selection, and prediction of human pharmacokinetic clearance during the discovery and development of targeted covalent inhibitors.

Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition.

PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. In vitro, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and in vivo it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1ß production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases.

How current understanding of clearance mechanisms and pharmacodynamics of therapeutic proteins can be applied for evaluation of their drug-drug interaction potential.

Increasing use of therapeutic proteins (TPs) in polypharmacy settings calls for more in-depth understanding of the biological interactions that can lead to increased toxicity or loss of pharmacological effect. Factors such as patient population, medications that are likely to be coadministered in that population, clearance mechanisms of a TP, and concomitant drugs have to be taken into account to determine the potential for drug-drug interactions (DDIs). The most well documented TP DDI mechanism involves cytokine-mediated changes in drug-metabolizing enzymes. Because of the limitations of the current preclinical models for addressing this type of DDI, clinical evaluation is currently the most reliable approach. Other DDI mechanisms need to be addressed on a case-by-case basis. These include altered clearance of TPs resulting from the changes in the target protein levels by the concomitant medication, displacement of TPs from binding proteins, modulation of Fcγ receptor expression, and others. The purpose of this review is to introduce the approach used by Pfizer scientists for evaluation of the DDI potential of novel TP products during drug discovery and development.

Discovery of an Oral Potent Selective Inhibitor of Hematopoietic Prostaglandin D Synthase (HPGDS).

Hematopoietic prostaglandin D synthase (HPGDS) is primarly expressed in mast cells, antigen-presenting cells, and Th-2 cells. HPGDS converts PGH2 into PGD2, a mediator thought to play a pivotal role in airway allergy and inflammatory processes. In this letter, we report the discovery of an orally potent and selective inhibitor of HPGDS that reduces the antigen-induced response in allergic sheep.

Aminomethyl tetrahydronaphthalene ketopiperazine MCH-R1 antagonists--Increasing selectivity over hERG.

A direct correlation between hERG binding and QTc prolongation was established for a series of aminomethyl tetrahydronaphthalene ketopiperazine MCH-R1 antagonists. Compounds within this class with greater selectivity over hERG were developed. Compound 4h proved to have the best profile, with MCH-R1 Ki = 16 nm and hERG IC50 = 25 microM.