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.

Leprosy drug clofazimine activates peroxisome proliferator-activated receptor-γ and synergizes with imatinib to inhibit chronic myeloid leukemia cells.

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38- progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.

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.

Evaluation of the oncolytic potential of R2B Mukteshwar vaccine strain of Newcastle disease virus (NDV) in a colon cancer cell line (SW-620).

Virotherapy is emerging as an alternative treatment of cancer. Among the candidate oncolytic viruses (OVs), Newcastle disease virus (NDV) has emerged as a promising non-engineered OV. In the present communication, we explored the oncolytic potential of R2B Mukteshwar strain of NDV using SW-620 colon cancer cells. SW-620 cells were xenografted in nude mice and after evaluation of the safety profile, 1 x 107 plaque forming units (PFU) of NDV were inoculated as virotherapeutic agent via the intratumoral (I/T) and intravenous (I/V) route. Tumor growth inhibition was compared with their respective control groups by gross volume and histopathological evaluation. Antibody titer and virus survival were measured by hemagglutination inhibition (HI)/serum neutralization test (SNT) and real-time PCR, respectively. During the safety trial, the test strain did not produce any abnormal symptoms nor weight loss in BALB/c mice. Significant tumor lytic activity was evident when viruses were injected via the I/T route. There was a 43 and 57% tumor growth inhibition on absolute and relative tumor volume basis, respectively, compared with mock control. On the same basis, the I/V route treatment resulted in 40 and 16% of inhibition, respectively. Histopathological examination revealed that the virus caused apoptosis, followed by necrosis, but immune cell infiltration was not remarkable. The virus survived in 2/2 mice until day 10 and in 3/6 mice by day 19, with both routes of administration. Anti-NDV antibodies were generated at moderate level and the titer reached a maximum of 1:32 and 1:64 via the I/T and I/V routes, respectively. In conclusion, the test NDV strain was found to be safe and showed oncolytic activity against the SW-620 cell line in mice.

Effect of pioglitazone on metabolic features in endotoxemia model in obese diabetic db/db mice.

BACKGROUND: Infectious diseases are more frequent in diabetic patients, leading to increased morbidity and mortality. Endotoxemia affects glucose metabolism and lipolytic capacity. The aims of the present study were to determine whether endotoxemia exacerbates metabolic features (adipose inflammation, adipogenesis, and insulin resistance [IR]) in an animal model of diabetes (i.e. db/db mice) after acute infection and the effects of pioglitazone. METHODS: Female db/db mice treated with pioglitazone (3 and 30 mg/kg, p.o.) for 14 days were challenged with lipopolysaccharide (LPS; 200 µg/kg), followed by an oral glucose tolerance test (OGTT). Quantitative real-time polymerase chain reaction (PCR) was used to evaluate the expression of genes in white adipose tissue (WAT) involved in: (i) adipogenesis (lipoprotein lipase [Lpl], fatty acid binding protein-4 [Ap2] and adiponectin [Adipoq]); (ii) insulin signaling (peroxisome proliferator-activated receptor gamma [Pparg], suppressor of cytokine signaling 3 [Socs3], solute carrier family 2 [facilitated glucose transporter], member 4 [Slc2a4]); and (iii) inflammation (tumor necrosis factor [Tnf], interleukin-6 [Il6], monocyte chemoattractant protein-1 [Ccl2], cyclo-oxygenase-2 [prostaglandin-endoperoxide synthase 2; Ptgs2]). RESULTS: Experimental endotoxemia downregulated mRNA expression of Pparg, Slc2a4, Adipoq, Lpl, and Ap2, which coincided with upregulation of Il6, Tnf, Ccl2, Ptgs2, and Socs3 expression. Pioglitazone dose-dependently decreased Tnf, Il6, Ccl2, Ptgs2, and Socs3 expression in WAT, in association with upregulation of Lpl, Ap2, Slc2a4, and Adipoq expression, indicating improvement in endotoxin-induced IR. CONCLUSIONS: The findings suggest that LPS challenge exacerbates IR in db/db mice by altering the expression of genes in WAT involved in adipogenesis and inflammation, which is effectively controlled by pioglitazone treatment.

Differential effects of dexamethasone and rosiglitazone in a sephadex-induced model of lung inflammation in rats: possible role of tissue inhibitor of metalloproteinase-3.

OBJECTIVES: To study the effects of two different classes of drugs in sephadex-induced lung inflammation using rats and explore the potential mechanism (s). MATERIALS AND METHODS: Effects of dexamethasone (0.3 mg/kg, p.o.) and rosiglitazone (10 mg/kg, p.o.) treatments were evaluated up to 3 days in sephadex challenged rats. 72 h postsephadex administration, broncho-alveolar lavage fluid (BALF) was collected for cell count and cytokine estimation. Lung tissues were harvested for gene expression and histopathology. RESULTS: Dexamethasone treatment resulted in significant inhibition of lymphocytes, monocytes, eosinophils and neutrophils, whereas rosiglitazone inhibited eosinophils and neutrophils only. Further, dexamethasone reduced the elevated levels of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) after sephadex challenge while rosiglitazone significantly reduced the PGE2 levels without altering LTB4 in the BALF. Hydroxyproline content in rat lung homogenate was significantly reduced with dexamethasone treatment but not with rosiglitazone. Both the drugs were found to suppress matrix metallo proteinase 9, whereas only dexamethasone showed inhibition of tumor necrosis factor-alpha and up-regulation of tissue inhibitor of metalloproteinase 3 (TIMP-3) expression and preserved the broncho-alveolar microstructure. CONCLUSIONS: Our results revealed that up-regulation of TIMP-3 corroborated well with dexamethasone mediated inhibition of collagen degradation and restoration of alveolar micro-architecture.

Chemopreventive effect of a novel, selective TACE inhibitor on DMBA- and TPA-induced skin carcinogenesis.

UNLABELLED: Abstract Context: Tumor necrosis factor (TNF)-α, a potent proinflammatory cytokine, plays a major role in the pathogenesis of cancer. TNF-α converting enzyme (TACE) mediates processing and release of biologically active TNF-α. OBJECTIVE: We aimed to investigate the effect of a novel, selective TACE inhibitor (compound 11p) on skin inflammation and associated tumorigenesis in mice. METHODS: Skin edema was induced in mice by dermal application 12-O-tetradecanoylphorbol-13-acetate (TPA) solution in acetone on to the ear and the effect of post-treatment of compound 11p (topical application) was evaluated. Edema and inflammation was assessed by measuring ear thickness, weight of skin punch and cytokine levels. Skin cancer in mice was initiated by single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by repeated TPA application for 20 weeks. The effect of compound 11p on papilloma incidence and multiplicity was evaluated. RESULTS: Treatment with compound 11p strongly suppressed TPA-induced elevation in skin thickness and weight. A dose-dependent suppression in TPA-mediated TNF-α, IL-6, IFN-γ, IL-17 and PGE2 levels which was associated with a decrease in infiltration of inflammatory cells was also observed with the treatment. Moreover, compound 11p treatment delayed the onset, markedly reduced the papilloma incidence and multiplicity persuaded by DMBA and TPA. DISCUSSION AND CONCLUSION: These findings suggest that selective blockade of TACE suppresses TPA-induced epidermal hyperplasia, inflammatory cell infiltration and cytokine level. Inhibition of inflammatory events related to tumor growth might have led to the anti-tumor effect in mouse skin cancer model induced by DMBA and TPA.

Involvement of TACE in colon inflammation: a novel mechanism of regulation via SIRT-1 activation.

TNF-α converting enzyme (TACE) processes the membrane TNF-α to release the bioactive soluble TNF-α. Several evidences suggest the involvement of TNF-α and TACE in inflammatory bowel disease (IBD). Tissue inhibitor of metalloproteinase (TIMP)-3, an endogenous inhibitor of TACE, is positively associated with silent information regulator (SIRT)-1. We aimed to study the expression of TACE, TIMP-3 and SIRT-1 at different stages of colitis and how TACE is regulated in response to SIRT-1 activation. Acute colitis was induced by 3.5% dextran sulfate sodium (DSS) in drinking water for 5days and levels of cytokines and mRNA expression of TACE, TIMP-3 and SIRT-1 were measured in colon at different time intervals. Next, the effect of SIRT-1 activator (resveratrol) or a selective TACE inhibitor (compound 11p) treatment was evaluated. Elevated levels of TNF-α, interleukin (IL)-6, IL-1ß, interferon (IFN)-γ and IL-17 were observed during DSS exposure phase which restored to the normal level after DSS removal. A significant increase in TACE and suppression in TIMP-3 and SIRT-1 mRNA level was observed during DSS exposure phase which reverts back to normal towards the remission phase. Treatment with resveratrol significantly elevated SIRT-1 and TIMP-3 and suppressed TACE mRNA expression and was associated with amelioration of disease. Furthermore, treatment with selective TACE inhibitor significantly suppressed body weight loss, disease activity index, colonic myeloperoxidase activity and the elevated levels of cytokines after DSS challenge. These results strongly emphasize the involvement of TACE in colon inflammation and inhibition of TACE directly or indirectly via SIRT-1 activation ameliorates colitis.

Selective inhibition of tumor necrosis factor-α converting enzyme attenuates liver toxicity in a murine model of concanavalin A induced auto-immune hepatitis.

Emerging evidence suggest that tumor necrosis factor (TNF)-α plays a major role in pathogenesis of auto-immune hepatitis (AIH) induced liver injury. Blockade of TNF-α synthesis or bio-activity protects against experimental AIH. TNF-α converting enzyme (TACE) is a member of the ADAM (a disintegrin and metalloproteinase) family which processes precursor TNF-α to release soluble TNF-α. We hypothesized that selective inhibition of TACE might protect AIH. To investigate this, we studied the effects of a selective TACE inhibitor DPC-333 on murine model of liver injury and fibrosis induced with concanavalin A (Con A). Pre-treatment with DPC-333 significantly suppressed plasma alanine transaminase, aspartate transaminase and cytokines such as TNF-α, interferon (IFN)-γ, interleukin (IL)-2 and IL-6 levels due to acute Con A challenge. Interestingly; DPC-333 inhibited liver poly (ADP-ribose) polymerase (PARP)-1 activity which was associated with reduced number of necrotic hepatocytes in histological examination and mortality associated with Con A. In fibrosis study, repeated Con A administration significantly up-regulated liver collagen deposition as assessed by measurement of hydroxyproline content which was further confirmed in liver histology with Masson's trichrome staining. Treatment with 30mg/kg of DPC-333 was able to suppress liver hydroxyproline and fibrous tissue proliferation which corroborated well with inhibition in expression of pro-fibrotic genes such as tissue inhibitor of metalloproteinase (TIMP)-1 and transforming growth factor (TGF)-ß1. These observations suggest that selective TACE inhibition is an effective approach for the treatment of both immune mediated hepatic inflammation and fibrosis.

Blockade of tumor necrosis factor-α converting enzyme (TACE) enhances IL-1ß and IFN-γ via caspase-1 activation: a probable cause for loss of efficacy of TACE inhibitors in humans?

TNF-α converting enzyme (TACE) is a member of the ADAM (a disintegrin and metalloproteinase) family and is known as ADAM17, which processes precursor TNF-α in order to release soluble TNF-α (sTNF-α). Inhibition of TACE has been effective as a strategy to inhibit arthritis in animal models; however, it has not been translated in the clinic due to lack of efficacy or toxicity. We hypothesized that inhibition of TACE may activate a different pro-inflammatory pathway in human. To investigate this, we studied the effect of TACE inhibitor DPC-333 on cytokine levels in concanavalin A (Con A) activated human peripheral blood mononuclear cells (hPBMC). We have also studied the effects of DPC-333 on Con A induced cytokine levels in mice in vivo or in vitro in whole blood assay. DPC-333 treatment significantly up-regulated IL-1ß and IFN-γ in Con A activated hPBMC. In contrast, pre-treatment with DPC-333 effectively suppressed IL-1ß and IFN-γ in mice in vivo or in vitro. Interestingly, DPC-333 was found to up-regulate mRNA expression of caspase-1 in hPBMC in a dose dependent fashion and selective caspase-1 inhibitor completely restored DPC-333 induced IL-1ß and IFN-γ. Furthermore, selective IL-1ß receptor antagonist (anakinra) prevented DPC-333 induced IFN-γ. In conclusion, our data demonstrates that blockade of TACE enhances IL-1ß in a caspase-1 dependent manner in vitro in hPBMC and the elevation of IFN-γ is secondarily mediated via IL-1ß. This novel finding might explain the possible cause behind the loss of efficacy of TACE inhibitors in human.

Retinol-binding protein 4 : a possible role in cardiovascular complications.

BACKGROUND AND PURPOSE: Retinol-binding protein 4 (RBP4) is an adipocyte-secreted hormone proposed to link obesity with insulin resistance. However, the role of RBP4 in cardiovascular complications is yet to be fully understood. The present study is aimed to decipher the association between RBP4 with pro-inflammatory cytokines and low-density lipoprotein (LDL) cholesterol in diet-induced obese and hyperlipidaemic mice. To understand the correlation, rimonabant, an anti-obesity drug, has been used to relieve the atherosclerotic predisposition. EXPERIMENTAL APPROACH: Adipose and/or aortic tissue expressions of RBP4, pro-inflammatory cytokine genes and circulating LDL levels were measured in high fat (HF)-fed female C57BL/6 and high cholesterol (HC)-fed apolipoprotein E3 (ApoE3) Leiden mice. KEY RESULTS: Mice fed a HF diet had a significantly increased adipose expression of RBP4, TNF-α and monocyte chemoattractant protein 1 (MCP-1) and down-regulated adiponectin mRNA levels. A significant increase in aortic RBP4 and MCP-1 expression and circulating levels of LDL and C-reactive protein (CRP) was found in the ApoE3 mice fed a HC diet. Interestingly, rimonabant treatment lowered the elevated aortic RBP4, MCP-1 expressions and significantly reduced the serum levels of LDL, CRP, RBP4 and MCP-1. CONCLUSION AND IMPLICATIONS: Our results indicate that RBP4 is positively associated with markers of inflammation in obese and pro-atherogenic conditions and could play a role in a predisposition to atherosclerosis. Furthermore, our results indicate that rimonabant may improve vascular function by modulating RBP4 along with pro-inflammatory cytokines.

Involvement of adipokines in rimonabant-mediated insulin sensitivity in ob/ob mice.

OBJECTIVES: It has been recently reported that blockade of type 1 cannabinoid (CB1) receptors by specific antagonists or genetic manipulation alleviates dyslipidaemia, hyperglycaemia and insulin resistance in animal models of obesity and type 2 diabetes. However, the precise role of adipokines in the insulin-sensitising effects of the CB1 antagonist rimonabant is not clear. METHODS: ob/ob mice were treated with different doses of rimonabant and then subjected to an oral glucose tolerance test. The expression of different adipokines in white adipose tissue was analysed by quantitative real-time PCR. KEY FINDINGS: Rimonabant (30 mg/kg) significantly inhibited body weight and fat pad weight gain (P < 0.05) and improved glucose tolerance. Gene expression analysis indicated that tumour necrosis factor-alpha, visfatin and retinol binding protein-4 were downregulated in the adipose tissue of ob/ob mice treated with rimonabant compared with controls, whereas adiponectin was significantly upregulated. CONCLUSIONS: Rimonabant-mediated alteration of adipokines in white adipose tissues may play a role in improving insulin sensitivity in obese animals.

Subtherapeutic dose of pioglitazone reduces expression of inflammatory adipokines in db/db mice.

Agonists of the thiazolidinedione class of peroxisome proliferator-activated receptor-gamma exhibit both insulin-sensitizing and anti-inflammatory effects. We hypothesized that pioglitazone might be able to exert its anti-inflammatory properties at a lower dose than that required for its insulin-sensitizing effect. In order to investigate this hypothesis, we evaluated the effects of pioglitazone on inflammatory as well as metabolic biomarkers in serum and white adipose tissue (WAT) at 2 different doses. Female db/db mice were treated orally with therapeutic (30 mg/kg) and subtherapeutic (3 mg/kg) doses of pioglitazone for 14 days followed by an oral glucose tolerance test. Other parameters measured were inflammatory markers such as tumor necrosis factor (TNF)-alpha, interleukin-6 (IL-6) and metabolic biomarkers in serum (insulin, glucose and adiponectin). Moreover, adiponectin, fatty acid-binding protein (aP2) and lipoprotein lipase (LPL) mRNA expression in WAT were determined by real-time PCR. A subtherapeutic dose of pioglitazone significantly suppresses the expression of TNF-alpha and IL-6 mRNA in WAT, but does not alter the serum glucose, insulin and WAT expression of adiponectin, adipocyte aP2 and LPL. A therapeutic dose of pioglitazone improves insulin sensitivity, enhances LPL, aP2 and adiponectin expression, and also suppresses TNF-alpha and IL-6 expression. In conclusion, the current study indicates that the anti-inflammatory effect of pioglitazone is produced at a subtherapeutic dose, which is considerably lower than the dose needed to produce any desired metabolic effects. Anti-inflammatory effects of pioglitazone may precede its insulin-sensitizing effects in db/db mice.