Sanjeev Redkar

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-X(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction.

Mechanisms of cytotoxicity to pim kinase inhibitor, SGI-1776, in acute myeloid leukemia

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound, SGI-1776 inhibits Pim-1, Pim-2 and Pim-3, and was evaluated in AML-cell line, -xenograft model, and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets, c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47), were both decreased in actively cycling AML cell lines MV-4-11, MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2, Bcl-x(L), XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data, xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly, SGI-1776 was also cytotoxic in AML primary cells, irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment.

Pharmacologic inhibition of Pim kinases alters prostate cancer cell growth and resensitizes chemoresistant cells to taxanes

The serine/threonine family of Pim kinases function as oncogenes and have been implicated in prostate cancer progression, particularly in hormone-refractory prostate disease, as a result of their antiapoptotic function. In this study, we used a pharmacologic inhibitor targeting the Pim family members, SGI-1776, to determine whether modulation of Pim kinase activity could alter prostate cancer cell survival and modulate chemotherapy resistance. Extensive biochemical characterization of SGI-1776 confirmed its specificity for the three isoforms of the Pim family. Treatment of prostate cancer cells with SGI-1776 resulted in a dose-dependent reduction in phosphorylation of known Pim kinase substrates that are involved in cell cycle progression and apoptosis (p21Cip1/WAF1 and Bad). Consequently, SGI-1776 compromised overall cell viability by inducing G1 cell cycle arrest and triggering apoptosis. Overexpression of recombinant Pim-1 markedly increased sensitivity of SGI-1776–mediated prostate cancer cell apoptosis and p21Cip1/WAF1 phosphorylation inhibition, reinforcing the specificity of SGI-1776. An additional cytotoxic effect was observed when SGI-1776 was combined with taxane-based chemotherapy agents. SGI-1776 was able to reduce cell viability in a multidrug resistance 1 protein–based taxane-refractory prostate cancer cell line. In addition, SGI-1776 treatment was able to resensitize chemoresistant cells to taxane-based therapies by inhibiting multidrug resistance 1 activity and inducing apoptosis. These findings support the idea that inhibiting Pim kinases, in combination with a chemotherapeutic agent, could play an important role in prostate cancer treatment by targeting the clinical problem of chemoresistance. [Mol Cancer Ther 2009;8(10):2882–93]

S110, a 5-Aza-2′-Deoxycytidine–Containing Dinucleotide, Is an Effective DNA Methylation Inhibitor In vivo and Can Reduce Tumor Growth

Methylation of CpG islands in promoter regions is often associated with gene silencing and aberrant DNA methylation occurs in most cancers, leading to the silencing of some tumor suppressor genes. Reversal of this abnormal hypermethylation by DNA methylation inhibitors is effective in reactivating methylation-silenced tumor suppressor genes both in vitro and in vivo. Several DNA methylation inhibitors have been well studied; the most potent among them is 5-aza-2′-deoxycytidine (5-Aza-CdR), which can induce myelosuppression in patients. S110 is a dinucleotide consisting of 5-Aza-CdR followed by a deoxyguanosine, which we previously showed to be effective in vitro as a DNA methylation inhibitor while being less prone to deamination by cytidine deaminase, making it a promising alternative to 5-Aza-CdR. Here, we show that S110 is better tolerated than 5-Aza-CdR in mice and is as effective in vivo in inducing p16 expression, reducing DNA methylation at the p16 promoter region, and retarding tumor growth in human xenograft. We also show that S110 is effective by both i.p. and s.c. deliveries. S110 therefore is a promising new agent that acts similarly to 5-Aza-CdR and has better stability and less toxicity. Mol Cancer Ther; 9(5); 1443–50. ©2010 AACR.

Transbuccal delivery of 5-aza-2 -deoxycytidine: effects of drug concentration, buffer solution, and bile salts on permeation.

Delivery of 5-aza-2′-deoxycytidine (decitabine) across porcine buccal mucosa was evaluated as an alternative to the complex intravenous infusion regimen currently used to administer the drug. A reproducible high-performance liquid chromatography method was developed and optimized for the quantitative determination of this drug. Decitabine showed a concentration-dependent passive diffusion process across porcine buccal mucosa. An increase in the ionic strength of the phosphate buffer from 100 to 400 mM decreased the flux from 3.57±0.65 to 1.89±0.61 μg/h/cm2. Trihydroxy bile salts significantly enhanced the flux of decitabine at a 100 mM concentration (P>.05). The steady-state flux of decitabine in the presence of 100 mM of sodium taurocholate and sodium glycocholate was 52.65±9.48 and 85.22±7.61 μg/cm2/h, respectively. Two dihydroxy bile salts, sodium deoxytaurocholate and sodium deoxyglycocholate, showed better enhancement effect than did trihydroxy bile salts. A 38-fold enhancement in flux was achieved with 10 mM of sodium deoxyglycocholate.

S110, a novel decitabine dinucleotide, increases fetal hemoglobin levels in baboons (P. anubis)

BackgroundS110 is a novel dinucleoside analog that could have advantages over existing DNA methyltransferase (DNMT) inhibitors such as decitabine. A potential therapeutic role for S110 is to increase fetal hemoglobin (HbF) levels to treat β-hemoglobinopathies. In these experiments the effect of S110 on HbF levels in baboons and its ability to reduce DNA methylation of the γ-globin gene promoter in vivo were evaluated.MethodsThe effect of S110 on HbF and γ-globin promoter DNA methylation was examined in cultured human erythroid progenitors and in vivo in the baboon pre-clinical model. S110 pharmacokinetics was also examined in the baboon model.ResultsS110 increased HbF and reduced DNA methylation of the γ-globin promoter in human erythroid progenitors and in baboons when administered subcutaneously. Pharmacokinetic analysis was consistent with rapid conversion of S110 into the deoxycytosine analog decitabine that binds and depletes DNA.ConclusionS110 is rapidly converted into decitabine, hypomethylates DNA, and induces HbF in cultured human erythroid progenitors and the baboon pre-clinical model.

Structure–activity relationships for 4-anilinoquinoline derivatives as inhibitors of the DNA methyltransferase enzyme DNMT1

A series of 4-anilinoquinoline derivatives related to the known inhibitor SGI-1027, containing side chains of varying pK(a), were prepared by acid-catalysed coupling of the pre-formed side chains with 4-chloroquinolines. The compounds were evaluated for their ability to reduce the level of DNMT1 protein in HCT116 human colon carcinoma cells by Western blotting. With a very strongly basic N-methylpyridinium side chain, only NHCO-linked compounds were effective, whereas less strongly basic ((diaminomethylene)hydrazono)ethyl or 3-methylpyrimidine-2,4-diamine side chains allowed both NHCO- and CONH-linked compounds to show activity. In contrast, the pK(a) of the quinoline unit had little apparent influence on activity.

Abstract 4076: SGI-110, a novel subcutaneous (SQ) second generation DNA hypomethylating agent achieves improved pharmacodynamics (PD), safety and pharmacokinetics (PK) in comparison to IV decitabine in a non-human primate in vivo study

SGI-110, is a novel second generation DNA methylation inhibitor that is currently in Phase I/II clinical study for treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). SGI-110 is a dinucleotide of DAC and deoxyguanosine designed to be more stable than decitabine to deamination by cytidine deaminase, thus offering a promising alternative to current hypomethylating agents approved in MDS. We report here the results of a preclinical study in which safety, PK, and PD of different dosing regimens of SGI-110 SQ were compared to the clinical dose and regimen of DAC IV in 4 groups of male cynomolgus monkeys (n=4). The treatment groups consisted of: 1) Control group of DAC IV 1-h infusion at a dose equivalent to the clinically approved regimen of 20 mg/m2 x 5 (1.7 mg/kg daily x5); 2) 1.7 mg/kg SGI-110 SQ daily x 5 (molar equivalent to 42% of the clinical DAC dose); 3) 3.0 mg/kg SGI-110 SQ daily x 5 (molar equivalent to 75% of the clinical DAC dose); and 4) 3.0 mg/kg of SGI-110 SQ once weekly x3 (molar equivalent to 44% of the total clinical DAC dose). DAC and SGI-110 plasma levels were measured and monkeys were monitored for 28 days for hematological changes, and global DNA methylation (LINE-1). Reversible hematological changes included a reduction in leukocytes, red blood cells (RBCs), and neutrophil counts with the nadir counts generally occurring between D8 and D14 and recovery occurring D21 to D28. The DAC-treated group showed the greatest reduction and slowest recovery compared to all SGI-110 treated groups. Changes in methylation patterns of LINE-1 and miRNA-29b were evaluated in DNA extracted from monkey blood as PD markers of biological efficacy after treatment with SGI-110 or DAC. All groups achieved a decrease in LINE-1 methylation of approximately 15-20% from baseline between Day 8 and 21 with remethylation back to baseline levels by day 28. Both SGI-110 SQ dailyx5 regimens achieved slightly more hypomethylation than DAC IV on days 8 (group 3) and 11 (group 2). Overall, the magnitude and duration of the decrease in DNA methylation at lower molar equivalent doses of SQ SGI-110 were similar to or better than DAC IV. SGI-110 appeared to convert to DAC resulting in similar exposure window compared to IV DAC. The dose-adjusted plasma DAC exposures, on molar equivalence basis, were somewhat lower compared to the DAC group. The C max ranged 52-163 vs 215-525(DAC) ng/mL, while dose-adjusted AUCs were 21.6-51.6 vs 98.6-221(DAC) ng*hr/mL. In conclusion, these preclinical studies showed that SQ SGI-110 may represent a more convenient and tolerable option for delivering DAC where either the weeklyx3 or dailyx5 regimens at a lower dose achieved DNA hypomethylating effects that were similar to or better than seen with DAC IV and with less myelosuppressive effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4076. doi:1538-7445.AM2012-4076

Abstract 2096: Bozitinib, a highly selective inhibitor of cMet, demonstrates robust activity in gastric, lung, hepatic and pancreatic in vivo models

Background: cMET is a receptor tyrosine kinase that is located on the cell surface and is activated by the binding of its ligand, hepatocyte growth factor (HGF). In cancer cells, MET can be aberrantly active and cause abnormal signaling, which leads to tumor growth, angiogenesis, and metastasis. In vitro studies have demonstrated that bozitinib (CBT-101, PLB-1001) is a highly selective and specific inhibitor (8 nM) of tumor cell proliferation. Methods: In-vivo PD studies of gastric (MKN45), lung (LUM858, LU1901, LU2503), hepatic (LIM0612, LIM0801), and pancreatic (KP4) were evaluated. These models covered both the HGF-dependent and HGF-independent mechanisms. Among these models, LUM858, LU1901, LU2503, LIM0612 and LIM0801 are PDX models. In particular, in the LU1901 model, bozitinib (BT) was compared to capmatinib (INC280). Groups included: BT at 1, 3 and 10 mg/kg QD×21 and INC280 at 1, 3, and 10 mg/kg QD×21 and 10 mg/kg BID×21 via IG, CDDP 5 mg/kg, Q7D×3 as a positive control via IP and the vehicle control (QD×21 via IG). Each group (n=8 mice) and the tumor volume was evaluated on D21. Results: In MKN45, LU2503, LIM0612 and LIM0801, the effect of BT seemed superior than that of crizotinib; in LUM858, its effect was higher than that of erlotinib; in LU1901, its effect was higher than that of crizotinib and INC280. In the LU1901 model, the strongest activity was observed at BT 10 mg/kg with a T/C ratio of 2%, compared to an equi-dose of INC280 (T/C of 22%). All doses of BT and INC280 were well tolerated; no mouse experienced weight loss. In MKN45 model, BT showed a PK/PD correlation and dose-dependence. BT inhibited the phosphorylation of c-Met protein; the rate of target inhibition exceeded 90% at >7 mg/kg. The plasma concentration for BT decreased over time with a significant decrease 16h after its administration, conferring at least 16h of phosphorylation inhibition of the c-Met protein. Conclusions: In conclusion, BT was well-tolerated, with no animal death nor major weight loss. The in vivo experiments demonstrated that BT is a viable candidate with effective anti-tumor activities. BT is currently under evaluation in cMet dysregulated NSCLC (NCT02896231) with additional trials planned. Citation Format: Joe Shih, Boyu Zhong, Hepeng Shi, David Xue, Gavin S. Choy, Sanjeev Redkar. Bozitinib, a highly selective inhibitor of cMet, demonstrates robust activity in gastric, lung, hepatic and pancreatic in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2017-2096

Expect the unexpected–screen your screens

Rubitecan (9-nitrocamptothecin) has been investigated in multiple phase 3 clinical trials for the treatment of pancreatic cancer that supported a new drug application (NDA) with US Food and Drug Administration. The drug product is an oral, hard gelatine capsule in two strengths, 0.5 and 1.25 mg, respectively. Capsules constitute a homogenous, loosely bound, dry, pale-yellow powder blend of rubitecan, lactose monohydrate, colloidal silicon dioxide, and magnesium stearate. A number of NDA qualification and engineering batches were manufactured before embarking on validating the process and generating the initial commercial supplies. Unexpected difficulties were encountered during blend manufacturing that nearly failed the validation effort. Described below are lessons learned during the ensued investigation. Rubitecan capsules are intended for oral administration and as the drug is poorly water soluble, drug particle size is reduced to impart better dissolution of drug and eventual higher drug uptake. Drug as is has a median diameter, D (v, 0.5) of 104 :m and an in-process specification of D (v, 0.5) 15–40 :m and D (v, 0.9) 50–125 :m was set based on manufacturing history of lots used in clinical studies. Because of this, micronization was not an option. Thus, a key step in the blend manufacturing was reduction of drug particle size by passing an initial blend of drug and lactose in 2:1 ratio through a rotating impeller, conical screen mill. Blend was sequentially screened twice through 152-:m screen and once through 228-:m screen. During the manufacturing of the first validation batch, rapid and severe clogging of the 152-:m screen that was not seen before was observed with barely 20% of the fed blend passing through. This led to the abortion of the batch and initiation of a crossfunctional and extensive investigation to determine