ZYBT1, a potent, irreversible Bruton's Tyrosine Kinase (BTK) inhibitor that inhibits the C481S BTK with profound efficacy against arthritis and cancer.

Bruton's tyrosine kinase (BTK) plays a central and pivotal role in controlling the pathways involved in the pathobiology of cancer, rheumatoid arthritis (RA), and other autoimmune disorders. ZYBT1 is a potent, irreversible, specific BTK inhibitor that inhibits the ibrutinib-resistant C481S BTK with nanomolar potency. ZYBT1 is found to be a promising molecule to treat both cancer and RA. In the present report we profiled the molecule for in-vitro, in-vivo activity, and pharmacokinetic properties. ZYBT1 inhibits BTK and C481S BTK with an IC50 of 1 nmol/L and 14 nmol/L, respectively, inhibits the growth of various leukemic cell lines with IC50 of 1 nmol/L to 15 µmol/L, blocks the phosphorylation of BTK and PLCγ2, and inhibits secretion of TNF-α, IL-8 and IL-6. It has favorable pharmacokinetic properties suitable for using as an oral anti-cancer and anti-arthritic drug. In accordance with the in-vitro properties, it demonstrated robust efficacy in murine models of collagen-induced arthritis (CIA) and streptococcal cell wall (SCW) induced arthritis. In both models, ZYBT1 alone could suppress the progression of the diseases. It also reduced the growth of TMD8 xenograft tumor. The results suggested that ZYBT1 has high potential for treating RA, and cancer.

Discovery of diaminopyrimidine-carboxamide derivatives as JAK3 inhibitors.

Selective inhibition of janus kinase (JAK) has been identified as an important strategy for the treatment of autoimmune disorders. Optimization at the C2 and C4-positions of pyrimidine ring of Cerdulatinib led to the discovery of a potent and orally bioavailable 2,4-diaminopyrimidine-5-carboxamide based JAK3 selective inhibitor (11i). A cellular selectivity study further confirmed that 11i preferentially inhibits JAK3 over JAK1, in JAK/STAT signaling pathway. Compound 11i showed good anti-arthritic activity, which could be correlated with its improved oral bioavailability. In the repeat dose acute toxicity study, 11i showed no adverse changes related to gross pathology and clinical signs, indicating that the new class JAK3 selective inhibitor could be viable therapeutic option for the treatment of rheumatoid arthritis.

Discovery of 1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-ones based novel, potent and PI3Kδ selective inhibitors.

PI3Kδ is implicated in various inflammatory and autoimmune diseases. For the effective treatment of chronic immunological disorders such as rheumatoid arthritis, it is essential to develop isoform selective PI3Kδ inhibitors. Structure guided optimization of an imidazo-quinolinones based pan-PI3K/m-TOR inhibitor (Dactolisib) led to the discovery of a potent and orally bioavailable PI3Kδ isoform selective inhibitor (10h), with an improved efficacy in the animal models.

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of ZYTP1, a novel poly (ADP-ribose) polymerase inhibitor.

ZYTP1 is a novel Poly (ADP-ribose) polymerase protein inhibitor being developed for cancer indications. The focus of the work was to determine if ZYTP1 had a perpetrator role in the in vitro inhibition of cytochrome P450 (CYP) enzymes to aid dosing decisions during the clinical development of ZYTP1. ZYTP1 IC50 for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 was determined using human liver microsomes and LC-MS/MS detection. CYP3A4/5 IC50 of depropylated metabolite of ZYTP1 was also determined. Time dependent inhibition of CYP3A4/5 by ZYTP1 was also assessed using substrates, testosterone and midazolam. The mean IC50 values of ZYTP1 were >100 µM for CYP1A2, 2B6 and 2D6, while 56.1, 24.5, 39.5 and 23.3-58.7 µM for CYP2C8, 2C9, 2C19 and 3A4/5, respectively. The CYP3A4/5 IC50 of depropylated metabolite was 11.95-24.51 µM. Time dependent CYP3A4/5 inhibition was noted for testosterone and midazolam with IC50 shift of 10.9- and 39.9-fold, respectively. With midazolam, the kinact and KI values of ZYTP1 were 0.075 min-1 and 4.47 µM for the CYP3A4/5 time dependent inhibition, respectively. Because of potent inhibition of CYP3A4/5, drugs that undergo metabolism via CYP3A4/5 pathway should be avoided during ZYTP1 therapy.

Combating Autoimmune Diseases With Retinoic Acid Receptor-Related Orphan Receptor-γ (RORγ or RORc) Inhibitors: Hits and Misses.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma (RORγ or RORc) is a key transcription factor for the production of pro-inflammatory cytokines implicated in the pathogenesis of autoimmune diseases. Recently, small molecule inhibitors of RORc drew the enormous attention of the research community worldwide as a possible therapy for autoimmune diseases, mediated by the IL-17 cytokine. With the clinical proof-of-concept inferred from a small molecule inhibitor VTP-43742 for psoriasis and recent inflow of several RORc inhibitors into the clinic for therapeutic interventions in autoimmune diseases, this field continues to evolve. This review briefly summarizes the RORc inhibitors disclosed in the literature and discusses the progress made by these inhibitors in combating autoimmune diseases.

Identification and preclinical characterization of a novel and potent poly (ADP-ribose) polymerase (PARP) inhibitor ZYTP1.

PURPOSE: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in the detection and repair of DNA damage. Studies have shown that inhibition of PARP and Tankyrase (TNKS) has significant antitumor effect in several types of cancers including BRCA-negative breast cancers. METHODS: Identification of ZYTP1, a novel PARP inhibitor, through a battery of in vitro assays and in vivo studies. PARP and TNKS inhibitory activity of ZYTP1 was assessed in cell-free kinase assay. In vitro cell killing potency of ZYTP1 was tested in a panel of cell lines including BRCA-negative cells. ZYTP1 was also tested in xenograft models in combination with temozolomide (TMZ). The pharmacokinetic profile of ZYTP1 was determined in rodent and non-rodent preclinical species. Safety of ZYTP1 was assessed in Wistar rats and Beagle dogs upon repeated dosing. RESULTS: ZYTP1 inhibited PARP1, PARP2, Tankyrase-1 and Tankyrase-2 with IC50 of 5.4, 0.7, 133.3 and 289.8 nM, respectively, and additionally trapped PARP1 onto damaged DNA. It also potentiated MMS-mediated killing of different cancer cell lines. Compound demonstrated good Caco-2 cell permeability. The oral bioavailability of ZYTP1 in mice, rats and dogs ranged between 40 and 79% and demonstrated efficacy in colon cancer xenograft model at a dose of 1-10 mg/kg in combination with TMZ. In a 28-day repeat dosing, oral toxicity study in rats, it was found to show > 10× safety margin. CONCLUSIONS: ZYTP1 is a novel PARP inhibitor that showed potential for development as a treatment for various solid tumors.

Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of Anemia Associated with Chronic Diseases.

Chronic kidney disease, cancer, chronic inflammatory disorders, nutritional, and genetic deficiency can cause anemia. Hypoxia causes induction of hypoxia-inducible factor (HIF), which stimulates erythropoietin (EPO) synthesis. Prolyl hydroxylase domain (PHD) enzyme inhibition can stabilize hypoxia-inducible factor (HIF). HIF stabilization also decreases hepcidin, a hormone of hepatic origin, which regulates iron homeostasis. PHD inhibitors represent a novel pharmacological treatment of anemia associated with chronic diseases. Many orally active PHD inhibitors like roxadustat, molidustat, vadadustat, and desidustat are in late phase clinical trials. This review discusses the role of PHD inhibitors in the treatment of anemia associated with chronic diseases.

Influence of acute and chronic kidney failure in rats on the disposition and pharmacokinetics of ZYAN1, a novel prolyl hydroxylase inhibitor, for the treatment of chronic kidney disease-induced anemia.

1. ZYAN1 is a prolyl hydroxylase inhibitor in clinical development for treatment of anemia associated with chronic kidney disease (CKD). We evaluated the effect of acute and chronic kidney impairment on the pharmacokinetics of ZYAN1 in rat models. 2. Cisplatin (2.5, 5 and 7.5 mg/kg) was used to induce acute kidney injury (AKI), and five-sixth and total nephrectomy was used to induce chronic kidney injury (CKI) in male Wistar rats. All groups received a single 15 mg/kg oral dose of ZYAN1. Blood/urine samples were analyzed for ZYAN1 to assess peak concentration (Cmax), area under the concentration-time curve (AUCinf), total body clearance (CL/F) and elimination half-life (T1/2). 3. Cmax and AUCinf were not significantly different in the various AKI groups or in five-sixth nephrectomized rats, as compared to control rats. Recovery of ZYAN1 in urine was reduced; the impact on the CL/F was minimal. There was a 2-fold increase in AUCinf with reduction in CL/F in total nephrectomized rats. T1/2 was longer for ZYAN1 in the severe AKI/five-sixth nephrectomy rats and total nephrectomy rats as compared to control rats. 4. Based on the rodent data it may be inferred that PK of ZYAN1 in CKD patients may be minimally affected.

A sensitive assay for ZYAN1 in human whole blood and urine utilizing positive LC-MS/MS electrospray ionization.

AIM: A sensitive LC-MS/MS method was developed and validated for estimation of ZYAN1 in human blood/urine. METHODS: An analog internal standard IOX2 along with ZYAN1 was quantified using selective reaction monitoring in positive mode. The chromatographic separation was performed by gradient elution with C18 analytical column (3 µm, 50 mm × 2.0 mm) with 4-min run time using an acidified mobile phase consisting of ammonium formate and acetonitrile. Protein precipitation enabled extraction of analytes from diluted blood/urine. RESULTS: Calibration curve of ZYAN1 was linear (2-5000 ng/ml). The recovery of ZYAN1 and IOX2 was between 87 and 104%. Interday and intraday accuracy and precision was found well within the acceptance criteria. CONCLUSION: The validated assay was applied for clinical pharmacokinetics of ZYAN1 in healthy volunteers.

ZY15557, a novel, long acting inhibitor of dipeptidyl peptidase-4, for the treatment of Type 2 diabetes mellitus.

BACKGROUND AND PURPOSE: Dipeptidyl peptidase (DPP)-4 inhibitors increase levels of glucagon-like peptide-1 (GLP-1) and provide clinical benefit in the treatment of type 2 diabetes mellitus. As longer acting inhibitors have therapeutic advantages, we developed a novel DPP-4 inhibitor, ZY15557, that has a sustained action and long half-life. EXPERIMENTAL APPROACH: We studied the potency, selectivity, efficacy and duration of action of ZY15557, in vitro, with assays of DPP-4 activity. In vivo, the pharmacodymamics and pharmacokinetics of ZY15557 were studied, using db/db mice and Zucker fatty rats, along with normal mice, rats, dogs and non-human primates. KEY RESULTS: ZY15557 is a potent, competitive and long acting inhibitor of DPP-4 (Ki 5.53 nM; Koff 3.2 × 10-4 ·s-1 , half-life 35.8 min). ZY15557 treatment inhibited DPP-4 activity, and enhanced active GLP-1 and insulin in mice and rats, providing dose-dependent anti-hyperglycaemic effects. Anti-hyperglycaemic effects were also observed in db/db mice and Zucker fatty rats. Following oral dosing, ZY15557 significantly inhibited plasma DPP-4 activity, determined ex vivo, in mice and rats for more than 48 h, and for up to 168 h in dogs and non-human primates. Allometric scaling predicts a half-life for ZY15557 in humans of up to 60 h. CONCLUSIONS AND IMPLICATIONS: ZY15557 is a potent, competitive and long acting DPP-4 inhibitor. ZY15557 showed similar DPP-4 inhibition across different species. ZY15557 showed excellent oral bioavailability in preclinical species. It showed a low plasma clearance (CL) and large volume of distribution (Vss ) across species, resulting in an extended half-life.

Is the Inhibition of Dipeptidyl Peptidase-4 (DDP-4) Enzyme Route Dependent and/or Driven by High Peak Concentration?- Seeking Answers with ZYDPLA1, a Novel Long Acting DPP-4 Inhibitor, in a Rodent Model.

ZYDPLA1 is a long acting enzyme dipeptidyl peptidase-4 (DPP-4) inhibitor. The comparative effect of DPP-4 inhibition after intravenous (IV) and oral administration of ZYDPLA1 in a rat model was evaluated to answer the question of route dependency and/or the need of high plasma levels of ZYDPLA1. The study was conducted using parallel design in male Wistar rats for IV/oral route (n=9 and 6, for IV and oral respectively). A single 30 mg/kg dose of ZYDPLA1 was administered. Plasma samples were analysed for ZYDPLA1 concentration and DPP-4 inhibition. Pharmacokinetic analysis was carried out to assess peak concentration, area under the concentration-time curve, total body clearance, elimination half-life, and mean residence time. The PK/PD correlation was performed using standard sigmoidal Emax modelling to derive; maximum effect (Emax) and concentration to exert 50% Emax effect (EC50). ZYDPLA1 showed rapid absorption, high volume of distribution, low clearance, and complete oral bioavailability. The Emax derived after both routes and corresponding PK/PD profile showed comparable DDP-4 inhibition. The EC50 for IV (0.021 µg/mL) was comparable to the oral route (0.019 µg/mL). ZYDPLA1 showed full DPP-4 inhibition without regard to the route of administration. Higher systemic peak levels showed no bearing on the DDP-4 inhibition.

Identification of an Orally Efficacious GPR40/FFAR1 Receptor Agonist.

GPR40/FFAR1 is a G protein-coupled receptor predominantly expressed in pancreatic ß-cells and activated by long-chain free fatty acids, mediating enhancement of glucose-stimulated insulin secretion. A novel series of substituted 3-(4-aryloxyaryl)propanoic acid derivatives were prepared and evaluated for their activities as GPR40 agonists, leading to the identification of compound 5, which is highly potent in in vitro assays and exhibits robust glucose lowering effects during an oral glucose tolerance test in nSTZ Wistar rat model of diabetes (ED50 = 0.8 mg/kg; ED90 = 3.1 mg/kg) with excellent pharmacokinetic profile, and devoid of cytochromes P450 isoform inhibitory activity.

Discovery of a Potent and Orally Efficacious TGR5 Receptor Agonist.

TGR5 is a G protein-coupled receptor (GPCR), activation of which promotes secretion of glucagon-like peptide-1 (GLP-1) and modulates insulin secretion. The 2-thio-imidazole derivative 6g was identified as a novel, potent, and selective TGR5 agonist (hTGR5 EC50 = 57 pM, mTGR5 = 62 pM) with a favorable pharmacokinetic profile. The compound 6g was found to have potent glucose lowering effects in vivo during an oral glucose tolerance test in DIO C57 mice with ED50 of 7.9 mg/kg and ED90 of 29.2 mg/kg.

Pharmacological characterization of ZYDPLA1, a novel long-acting dipeptidyl peptidase-4 inhibitor.

OBJECTIVE: Dipeptidyl peptidase-4 (DPP-4) is responsible for degradation of glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), the endogenous incretins that stimulate glucose-dependent insulin secretion. The objective was to evaluate preclinical profile of a novel DPP-4 inhibitor ZYDPLA1. METHODS: In vitro inhibition potency and selectivity were assessed using recombinant enzymes and/or plasma. In vivo efficacy was determined in oral glucose tolerance test or mixed meal tolerance test in C57BL/6J mice, db/db mice and Zucker fatty rats. Pharmacokinetics/pharmacodynamics was studied in mice, rats, dogs, and non-human primates. RESULTS: ZYDPLA1 is a potent, competitive and long acting inhibitor of DPP-4 (Ki 0.0027 µM; Koff 2.3 × 10(-4 ) s(-1) ). ZYDPLA1 was more than 7000-fold selective for recombinant DPP-4 relative to DPP-8 and DPP-9, and more than 60 000-fold selective relative to fibroblast activation protein (FAP) in vitro. DPP-4 inhibition was comparable across species. In vivo, oral ZYDPLA1 elevated circulating GLP-1 and insulin levels in mice and rats and showed dose-dependent anti-hyperglycemic effect. Anti-hyperglycemic effect was also observed in db/db mice and Zucker fatty rats. ZYDPLA1 showed low clearance, large volume of distribution, and a long half-life with excellent oral bioavailability in all species. It significantly inhibited plasma DPP-4 activity in mice and rats for more than 48 h, and for up to 168 h in dogs and non-human primates. Allometric scaling predicted a half-life in humans of 53 to 166 h. CONCLUSION: ZYDPLA1 is a potent, selective, long-acting oral DPP-4 inhibitor with potential to become once-a-week therapy for treatment of type 2 diabetes mellitus.

Design and synthesis of potent and subtype-selective PPARalpha agonists.

Beginning with a moderately potent PPARgamma agonist 9, a series of potent and highly subtype-selective PPARalpha agonists was identified through a systematic SAR study. Based on the results of the efficacy studies in the hamster and dog models of dyslipidemia and the desired pharmacokinetic data, the optimized compound 39 was selected for further profiling.

In vitro metabolism of a new oxazolidinedione hypoglycemic agent utilizing liver microsomes and recombinant human cytochrome P450 enzymes.

The compound, 5-{4-[3-(4-cyclohexyl-2-propylphenoxy)propoxy]phenyl}-1,3-oxazolidine-2,4-dione (compound A) is a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist. PPARgamma agonists have proven useful in the treatment of type 2 diabetes, which is characterized by hyperglycemia, insulin resistance and/or abnormal insulin secretion. The metabolism of this oxazolidinedione (OZD) was investigated in male rat, dog, monkey and human liver microsomes, and recombinant human cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in the presence of NADPH. Routes of metabolism included monohydroxylation of the cyclohexane ring at multiple positions, monohydroxylation of the n-propyl side chain or the tether linkage, and OZD ring opening, giving rise to the keto amide and alcohol amide entities. Liver microsomes showed subtle qualitative and quantitative metabolic differences among rat, dog, monkey and human preparations. Further, CYP2C8 and CYP2C19 did not display different regioselectivity for hydroxylation on the cyclohexane ring with both of them giving rise to C-3 and C-4 hydroxy metabolites, but they did display different stereoselectivity with CYP2C8 preferring cyclohexane hydroxylation in equatorial positions and CYP2C19 in axial positions.

(2R)-2-ethylchromane-2-carboxylic acids: discovery of novel PPARalpha/gamma dual agonists as antihyperglycemic and hypolipidemic agents.

A series of chromane-2-carboxylic acid derivatives was synthesized and evaluated for PPAR agonist activities. A structure-activity relationship was developed toward PPARalpha/gamma dual agonism. As a result, (2R)-7-(3-[2-chloro-4-(4-fluorophenoxy)phenoxy]propoxy)-2-ethylchromane-2-carboxylic acid (48) was identified as a potent, structurally novel, selective PPARalpha/gamma dual agonist. Compound 48 exhibited substantial antihyperglycemic and hypolipidemic activities when orally administered in three different animal models: the db/db mouse type 2 diabetes model, a Syrian hamster lipid model, and a dog lipid model.

Aryloxazolidinediones: identification of potent orally active PPAR dual alpha/gamma agonists.

A series of novel aryloxazolidine-2,4-diones was synthesized. A structure-activity relationship study of these compounds led to the identification of potent, orally active PPAR dual alpha/gamma agonists. Based on the results of efficacy studies in the db/db mice model of type 2 diabetes and the desired pharmacokinetic parameters, compound 12 was selected for further profiling.

5-aryl thiazolidine-2,4-diones: discovery of PPAR dual alpha/gamma agonists as antidiabetic agents.

A novel series of 5-aryl thiazolidine-2,4-diones based dual PPARalpha/gamma agonists was identified. A number of highly potent and orally bioavailable analogues were synthesized. Efficacy study results of some of these analogues in the db/db mice model of type 2 diabetes showed them superior to rosiglitazone in correcting hyperglycemia and hypertriglyceridemia.