Reversible effects on female rat fertility with abrocitinib, a Janus kinase 1 inhibitor.

BACKGROUND: Abrocitinib is a Janus kinase (JAK) 1 selective inhibitor approved for the treatment of atopic dermatitis. Female reproductive tissues were unaffected in general toxicity studies, but an initial female rat fertility study resulted in adverse effects at all doses evaluated. A second rat fertility study was conducted to evaluate lower doses and potential for recovery. METHODS: This second study had 4 groups of 20 females each administered abrocitinib (0, 3, 10, or 70 mg/kg/day) 2 weeks prior to cohabitation through gestation day (GD) 7. In addition, 2 groups of 20 rats (0 or 70 mg/kg/day) were dosed for 3 weeks followed by a 4-week recovery period before mating. All mated females were evaluated on GD 14. RESULTS: No effects were observed at ≤10 mg/kg/day. At 70 mg/kg/day (29x human exposure), decreased pregnancy rate, implantation sites, and viable embryos were observed. All these effects reversed 4 weeks after the last dose. CONCLUSIONS: Based on these data and literature on the potential role of JAK signaling in implantation, we hypothesize that these effects may be related to JAK1 inhibition and, generally, that peri-implantation effects such as these, in the absence of cycling or microscopic changes in nonpregnant female reproductive tissues, are anticipated to be reversible.

Novel JAK Inhibitors to Reduce Graft-Versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation in a Preclinical Mouse Model.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.

Tofacitinib and peficitinib inhibitors of Janus kinase for autoimmune disease treatment: a quantum biochemistry approach.

Autoimmune inflammatory diseases, such as rheumatoid arthritis (RA) and ulcerative colitis, are associated with an uncontrolled production of cytokines leading to the pronounced inflammatory response of these disorders. Their therapy is currently focused on the inhibition of cytokine receptors, such as the Janus kinase (JAK) protein family. Tofacitinib and peficitinib are JAK inhibitors that have been recently approved to treat rheumatoid arthritis. In this study, an in-depth analysis was carried out through quantum biochemistry to understand the interactions involved in the complexes formed by JAK1 and tofacitinib or peficitinib. Computational analyses provided new insights into the binding mechanisms between tofacitinib or peficitinib and JAK1. The essential amino acid residues that support the complex are also identified and reported. Additionally, we report new interactions, such as van der Waals; hydrogen bonds; and alkyl, pi-alkyl, and pi-sulfur forces, that stabilize the complexes. The computational results revealed that peficitinib presents a similar affinity to JAK1 compared to tofacitinib based on their interaction energies.

Pharmacokinetic characteristics of golidocitinib, a highly selective JAK1 inhibitor, in healthy adult participants.

Background: Golidocitinib is an orally available, potent and highly selective JAK (Janus kinase)-1 inhibitor of JAK/STAT3 signaling under clinical development for the treatment of cancer and autoimmune diseases. The objectives of the two reported studies were to investigate the pharmacokinetics (PK), safety, and tolerability of golidocitinib in healthy Chinese participants as compared to those healthy Western participants, as well as the food effect exploration. Methods: Two phase I studies (JACKPOT2 and JACKPOT3) were conducted in USA and China, respectively. In JACKPOT2 study, participants were randomized into placebo or golidocitinib arm in single-ascending dose cohorts (5 - 150 mg) and multiple-ascending dose cohorts (25 - 100 mg, once daily) for 14 days. In the food effect cohort, golidocitinib (50 mg) was administrated shortly after a high-fat meal (fed conditions) as compared to under fasting conditions. In JACKPOT3 study conducted in China, participants were randomized to placebo or golidocitinib arm in single-ascending dose cohorts (25 - 150 mg). Results: Exposure of golidocitinib generally increased in a dose-proportional manner across a dose range of 5 mg to 150 mg (single dose) and 25 mg to 100 mg (once daily). High-fat food did not alter the PK of golidocitinib with statistical significance. Low plasma clearance and extensive volume of distribution characterizes PK of golidoctinib, and long half-life across the dose levels supported once daily dosing. The inter-ethnic difference in primary PK parameters was evaluated. The result suggested slightly higher peak plasma concentrations (Cmax) but comparable area under the plasma concentration-time curve (AUC) was observed in Asian (Chinese) subjects as compared to Caucasian and/or Black subjects, while it was not considered clinically relevant. Golidocitinib was well tolerated without Common Terminology Criteria for Adverse Events (CTCAE) grade 3 or higher drug-related treatment emergent adverse events (TEAE) reported. Conclusion: No noticeable inter-ethnic difference was observed among Asian, Black, and Caucasian healthy subjects in anticipation of the favorable PK properties of golidocitinib. The effect of food on the bioavailability of golidocitinib was minor following a single oral administration of 50 mg. These data guided to use the same dose and regimen for multinational clinical development. Clinical trial registrations: https://clinicaltrials.gov/ct2/show/NCT03728023?term=NCT03728023&draw=2&rank=1, identifier (NCT03728023); http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml, identifier (CTR20191011).

Theoretical Studies on Selectivity of HPK1/JAK1 Inhibitors by Molecular Dynamics Simulations and Free Energy Calculations.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T cell receptor, which has been regarded as a potential target for immunotherapy. Yu et al. observed the off-target effect of the high-throughput screening HPK1 kinase inhibitor hits on JAK1 kinase. The off-target effect is usually due to the lack of specificity of the drug, resulting in toxic side effects. Therefore, exploring the mechanisms to selectively inhibit HPK1 is critical for developing effective and safe inhibitors. In this study, two indazole compounds as HPK1 inhibitors with different selectivity towards JAK1 were used to investigate the selectivity mechanism using multiple computational methods, including conventional molecular dynamics simulations, binding free energy calculations and umbrella sampling simulations. The results indicate that the salt bridge between the inhibitor and residue Asp101 of HPK1 favors their selectivity towards HPK1 over JAK1. Information obtained from this study can be used to discover and design more potent and selective HPK1 inhibitors for immunotherapy.

Identification of TUL01101: A Novel Potent and Selective JAK1 Inhibitor for the Treatment of Rheumatoid Arthritis.

Janus kinase 1 (JAK1) is a potential target for the treatment of rheumatoid arthritis (RA). In this study, the introduction of a spiro ring with a difluoro-substituted cyclopropionamide resulted in the identification of TUL01101 (compound 36) based on a triazolo[1,5-a]pyridine core of filgotinib. It showed excellent potency on JAK1 with an IC50 value of 3 nM and exhibited more than 12-fold selectivity for JAK2 and TYK2. Whole blood assay also demonstrated the high activity and selectivity (37-fold for JAK2). At the same time, TUL01101 also demonstrated excellent metabolic stability and pharmacokinetics (PK) profiles were assayed in three species (mouse, rat, and dog). Moreover, it has been validated for effective activity in the treatment of RA both in collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA) models, with low dose and low toxicity. Now, TUL01101 has progressed into phase I clinical trials.

Induction of T cell exhaustion by JAK1/3 inhibition in the treatment of alopecia areata.

Alopecia areata (AA) is an autoimmune disease caused by T cell-mediated destruction of the hair follicle (HF). Therefore, approaches that effectively disrupt pathogenic T cell responses are predicted to have therapeutic benefit for AA treatment. T cells rely on the duality of T cell receptor (TCR) and gamma chain (γc) cytokine signaling for their development, activation, and peripheral homeostasis. Ifidancitinib is a potent and selective next-generation JAK1/3 inhibitor predicted to disrupt γc cytokine signaling. We found that Ifidancitinib robustly induced hair regrowth in AA-affected C3H/HeJ mice when fed with Ifidancitinib in chow diets. Skin taken from Ifidancitinib-treated mice showed significantly decreased AA-associated inflammation. CD44+CD62L- CD8+ T effector/memory cells, which are associated with the pathogenesis of AA, were significantly decreased in the peripheral lymphoid organs in Ifidancitinib-treated mice. We observed high expression of co-inhibitory receptors PD-1 on effector/memory CD8+ T cells, together with decreased IFN-γ production in Ifidancitinib-treated mice. Furthermore, we found that γc cytokines regulated T cell exhaustion. Taken together, our data indicate that selective induction of T cell exhaustion using a JAK inhibitor may offer a mechanistic explanation for the success of this treatment strategy in the reversal of autoimmune diseases such as AA.

The Pharmacokinetics, Metabolism, and Clearance Mechanisms of Abrocitinib, a Selective Janus Kinase Inhibitor, in Humans.

Abrocitinib is an oral once-daily Janus kinase 1 selective inhibitor being developed for the treatment of moderate-to-severe atopic dermatitis. This study examined the disposition of abrocitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite (M) profiles. The results indicated abrocitinib had a systemic clearance of 64.2 L/h, a steady-state volume of distribution of 100 L, extent of absorption >90%, time to maximum plasma concentration of ∼0.5 hours, and absolute oral bioavailability of 60%. The half-life of both abrocitinib and total radioactivity was similar, with no indication of metabolite accumulation. Abrocitinib was the main circulating drug species in plasma (∼26%), with 3 major monohydroxylated metabolites (M1, M2, and M4) at >10%. Oxidative metabolism was the primary route of elimination for abrocitinib, with the greatest disposition of radioactivity shown in the urine (∼85%). In vitro phenotyping indicated abrocitinib cytochrome P450 fraction of metabolism assignments of 0.53 for CYP2C19, 0.30 for CYP2C9, 0.11 for CYP3A4, and ∼0.06 for CYP2B6. The principal systemic metabolites M1, M2, and M4 were primarily cleared renally. Abrocitinib, M1, and M2 showed pharmacology with similar Janus kinase 1 selectivity, whereas M4 was inactive. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of abrocitinib, a Janus kinase inhibitor for atopic dermatitis, in humans, as well as characterization of clearance pathways and pharmacokinetics of abrocitinib and its metabolites.

Identification of a Novel 2,8-Diazaspiro[4.5]decan-1-one Derivative as a Potent and Selective Dual TYK2/JAK1 Inhibitor for the Treatment of Inflammatory Bowel Disease.

In this study, we described a series of 2,8-diazaspiro[4.5]decan-1-one derivatives as selective TYK2/JAK1 inhibitors. Systematic exploration of the structure-activity relationship through the introduction of spirocyclic scaffolds based on the reported selective TYK2 inhibitor 14l led to the discovery of the superior derivative compound 48. Compound 48 showed excellent potency on TYK2/JAK1 kinases with IC50 values of 6 and 37 nM, respectively, and exhibited more than 23-fold selectivity for JAK2. Compound 48 also demonstrated excellent metabolic stability and more potent anti-inflammatory efficacy than tofacitinib in acute ulcerative colitis models. Moreover, the excellent anti-inflammatory effect of compound 48 was mediated by regulating the expression of related TYK2/JAK1-regulated genes, as well as the formation of Th1, Th2, and Th17 cells. Taken together, these findings suggest that compound 48 is a selective dual TYK2/JAK inhibitor, deserving to be developed as a clinical candidate.

Design, Synthesis and Structure-Activity Relationship Studies of Meridianin Derivatives as Novel JAK/STAT3 Signaling Inhibitors.

Hyperactivation of Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling is an attractive therapeutic target for tumor therapy. Herein, forty-eight novel meridianin derivatives were designed and synthesized, and their antitumor activity was evaluated in vitro both for activity optimization and structure-activity relationship (SAR) study. The results indicated that most derivatives exhibited significantly improved antitumor activity, especially for compound 6e. The compound 6e contains an isothiouronium linked by an alkyl chain consisting of six carbon atoms with IC50 ranging from 1.11 to 2.80 µM on various cancer cell lines. Consistently, the 6e dose dependently induced the apoptosis of A549 and DU145 cells, in which STAT3 is constitutively active. Western blotting assays indicated that the phosphorylation levels of JAK1, JAK2 and STAT3 were inhibited by 6e at 5 µM without significant change in the total STAT3 level. Moreover, 6e also suppressed the expression of STAT3 downstream genes, including c-Myc, Cyclin D1 and Bcl-XL at 10 µM. An additional in vivo study revealed that 6e at the dose of 10 mg/kg could potently inhibit the DU145 xenograft tumor without obvious body weight loss. These results clearly indicate that 6e could be a potential antitumor agent by targeting the JAK/STAT3 signaling pathway.

Adherence to ruxolitinib, an oral JAK1/2 inhibitor, in patients with myelofibrosis: interim analysis from an Italian, prospective cohort study (ROMEI).

ROMEI, a prospective, observational study in patients with myelofibrosis receiving the oral JAK1/2 inhibitor ruxolitinib in real-world practice, assesses treatment adherence based on the 8-item Morisky Medication Adherence Scale (MMAS-8). Here, we present MMAS-8 results at week 24. Overall, 101 of 188 evaluable patients completed the questionnaire at every visit (full completers). Mean (±standard deviation) total MMAS-8 scores remained stable from week 4 to week 24 in the overall population (7.54 ± 0.77 and 7.67 ± 0.70, respectively) and full completers (7.53 ± 0.79 and 7.67 ± 0.73, respectively). Rates of low (MMAS-8 ˂6) or medium (MMAS-8 ≥ 6 to ˂8) adherence were 25-40% and 26-36%, respectively. Fifty-five full completers (54%) reported ≥1 change in adherence category (improvement and/or worsening), most of which were associated with unintentional behavior. The data suggest that one-third of patients receiving ruxolitinib may be undertreated due to non-adherence, potentially undermining disease control, and indicate a need for better interventions addressing noncompliance to oral therapies.

Cerebrospinal fluid neopterin as a biomarker of treatment response to Janus kinase inhibition in Aicardi-Goutières syndrome.

Janus kinase (JAK) 1 inhibition represents a precision medicine approach in the treatment of Aicardi-Goutières syndrome (AGS), through targeting of type I interferon-mediated cell signalling. Blood interferon mRNAseq has been proposed as a biomarker of disease with utility in therapeutic monitoring. Objective cerebrospinal fluid (CSF) biomarkers tracking treatment efficacy are currently lacking. Here, we report a retrospective case series of 13 patients (median age 6y, range 2y 6mo-17y; five females, eight males) with AGS demonstrating significantly elevated CSF neopterin levels at first sampling (median 200nmol/L, range 45-2024nmol/L), compared to 13 age-matched controls with non-inflammatory neurological conditions (median 23nmol/L, range 5-34nmol/L, p<0.001). Five patients with AGS treated with JAK inhibitors demonstrated a median 81.5% reduction of CSF neopterin (range -36% to -88% change from baseline), compared to eight untreated patients with AGS demonstrating a median 7% reduction in CSF neopterin (range -63% to +117% change) (p=0.047). Our data indicate a biological effect of JAK inhibitors, and the potential role of CSF neopterin as a biomarker of treatment response.

Synthesis and structure-activity relationship studies of 1,5-isomers of triazole-pyrrolopyrimidine as selective Janus kinase 1 (JAK1) inhibitors.

JAK inhibitors have been considered as useful targets for the treatment of related diseases. However, first-generation JAK inhibitors have side effects such as anemia, thrombocytopenia, neutropenia and headaches which have been suggested to result from high JAK2 inhibition. Second-generation JAK inhibitors with more specific JAK isozyme inhibition have been studied to eliminate these adverse effects. In this study, novel 4-(1,5- or 2,5-triazole)-pyrrolopyrimidine derivatives with aromatic moieties were synthesized as JAK1 inhibitors, and an in vitro enzyme assay was used to evaluate the JAK inhibitory effects. Among these JAK1 inhibitors, the compound 23a showed an IC50 level of 72 nM, as well as being selective against other JAKs by 12 times or more: the results of molecular docking studies suggested that the high JAK1 selectivity resulted from a key interaction between the iodine atom of compound 23a and His-885 of hJAK1.

A Functional Precision Medicine Pipeline Combines Comparative Transcriptomics and Tumor Organoid Modeling to Identify Bespoke Treatment Strategies for Glioblastoma.

Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.