Damon I. Papac

Characterization of the Cellular and Antitumor Effects of MPI-0479605, a Small-Molecule Inhibitor of the Mitotic Kinase Mps1

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605, a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM- and RAD3-related-dependent activation of the p53–p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics. Mol Cancer Ther; 10(12); 2267–75. ©2011 AACR.

Abstract 3237: Evaluation of the pharmacokinetics and efficacy of a novel pro-drug of the HSP90 inhibitor, MPC-3100, designed with improved solubility

MPC-3100 is a synthetic, orally bioavailable HSP90 inhibitor in clinical development. The low solubility of this compound requires a solubility enhancing agent to enable uniform oral bioavailability. A pro-drug of MPC-3100 with enhanced aqueous solubility was synthesized and evaluated for its physical-chemical and pharmacokinetic properties, and anti-tumor activity in mice. The alanine ester of MPC-3100 was synthesized by esterifying a hydroxyl group on the active compound, MPC-3100. Solubility and permeability were determined over the pH range of 5 – 7.5 using the PIon solubility analyzer and the double sink PAMPA method. To investigate esterase-mediated activation of the pro-drug, the pro-drug was incubated with biological matrices known to contain esterases (mouse and human plasma and liver microsomes). The pharmacokinetics of the active component of the pro-drug was determined at 360 mg/kg in female CD-1 mice after administration as an oral suspension in 2% carboxymethylcellulose (CMC). Plasma concentrations of the active compound, MPC-3100, were quantified by LC-ESI-MS/MS. Five million NCI-N87 human gastric carcinoma cells were implanted subcutaneously into athymic mice (nu/nu) to produce a mouse xenograft model. Efficacy was determined in this model using once daily oral dosing in 2% CMC for 21 days. The kinetic solubility of the alanine ester pro-drug of MPC-3100 at pH 6.5 was 536 µg/mL compared to 10.2 µg/mL for MPC-3100. The pro-drug had an apparent permeability of 2.7 × 10-6 cm/sec at pH 6.2; whereas, the apparent permeability of MPC-3100 was 420 × 10-6 cm/sec. The low permeability of the pro-drug suggests conversion to active would be required to occur in the lumen prior to MPC-3100 absorption. The pro-drug was converted to MPC-3100 (half-life The alanine ester pro-drug of MPC-3100 had a > 50-fold increase in aqueous solubility and was converted rapidly by mouse plasma and mouse and human liver microsomes into active MPC-3100 in vitro and in vivo. These improved properties resulted in pro-drug that was efficacious in a xenograft tumor model, when dosed without a solubility enhancing agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3237. doi:10.1158/1538-7445.AM2011-3237

Abstract B137: Pharmacokinetics, antitumor activity, and therapeutic index of Nampt inhibitor MPC-8640 in mice.

Background: MPI-0487316 is a potent and orally bioavailable small molecule inhibitor of nicotinamide phosphoribosyltransferase (Nampt), an enzyme which catalyzes the rate-limiting step for synthesis of NAD from nicotinamide. Inhibition of Nampt by MPI-0487316 results in cell death as a consequence of NAD depletion and inhibition of ATP synthesis. We have previously reported that MPI-0487316 can induce regressions in a xenograft model. MPC-8640, a prodrug of MPI-0487316, was developed to increase solubility for improved formulation. Myrexis has recently initiated preclinical development of this prodrug and here we present data on the pharmacokinetic and anti-tumor activity of MPC-8640 in mice. Methods: MPI-0487316 concentration in plasma was measured using LC-MS/MS. For xenograft studies, HT1080 human fibrosarcoma cells were implanted subcutaneously into nude mice and mice were administered vehicle or MPC-8640 by oral gavage at the times and doses indicated. Results: Oral administration of MPC-8640 to mice resulted in substantial plasma concentrations of the active moiety MPI-0487316 with increasing AUC and Cmax over a wide range of doses. MPC-8640 concentration itself was negligible in plasma when dosed orally at 300 mg/kg. MPC-8640 demonstrated strong activity in the HT1080 human fibrosarcoma xenograft model when dosed qd or bid for one to two weeks. After bid dosing for one week, complete tumor growth inhibition (TGI) was observed at 6 mg/kg and substantial regression at 10 mg/kg. There was no difference between responses after seven or 14 doses bid. For qd dosing, complete tumor growth inhibition required 20 mg/kg MPC-8640 and ≥24 mg/kg for tumor regression. The anti-tumor response seen at the end of seven days of qd dosing was subsequently maintained for at least one week. TGI was observed with three or four doses qd, but with lesser potency than for five or more consecutive qd doses. In studies to determine maximum tolerated dose, 98% of mice survived up to 90 mg/kg MPC-8640 qd for one week, whereas only 60% survived doses >90 mg/kg. Conclusions: Oral MPC-8640 is an effective prodrug in mice for systemic delivery of its active moiety Nampt inhibitor MPI-0487316. The lack of significant plasma concentrations of MPC-8640 indicates that the prodrug is effectively converted to active moiety in the gut or immediately after absorption and that anti-tumor activity of MPC-8640 against subcutaneous xenografts is through its active moiety. A one week on/one week off, daily dosing schedule appears optimal, since one week of dosing, either qd or bd, was maximally effective and the anti-tumor response was sustained for at least one week after the end of dosing. Tumor regressions were induced at doses of MPC-8640 well below its maximum tolerated dose, providing promise that MPC-8640 may have anti-tumor activity in the clinic at well-tolerated doses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B137.

Abstract 3233: Comparative in vitro and in vivo metabolism of MPC-3100, an oral HSP90 inhibitor, in rat, dog, monkey and human

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL MPC-3100, an 8, 9-disubstituted purine, is an orally bioavailable HSP90 inhibitor currently in Phase 1 clinical development. The objectives of these studies were to compare the metabolism of MPC-3100 in preclinical species to select the species most appropriate for toxicological testing and to identify the major phase I and II metabolites formed both in vitro and in vivo in rats, dogs, monkeys and humans. MPC-3100 was incubated with liver microsomes from rats, dogs, monkeys, and humans. In addition, urine, feces, and bile were collected from rats dosed with MPC-3100 intravenously (5 mg/kg) or orally (50 mg/kg), and urine was collected from dogs (2 mg/kg) and cynomolgus monkeys (2.5 mg/kg) dosed intravenously. Metabolites were identified by liquid chromatography electrospray-ionization mass spectrometry. Quantitative analysis was performed with an AB Sciex 4000 Q-trap and qualitative analysis was conducted on a high resolution Agilent Q-TOF 6520 mass spectrometer. Six authentic standards were synthesized and used to confirm structural identity. In human liver microsomes, four distinct peaks were observed following chromatographic analysis. Three of these were conclusively identified using synthetic standards, accurate mass, and chromatographic retention time. The most abundant metabolite in all species was the catechol. In human, monkey, and dog liver microsomes, the next most abundant metabolite was formed by oxidation of the 2-hydroxypropan-1-one moiety to propane-1, 2-dione. A third metabolite present in all incubations was the de-amidated product of MPC-3100. It was formed in microsomes in the absence of NADPH suggesting that its formation was due to pH-mediated hydrolysis. A fourth metabolite formed by the addition of oxygen (+16 Da) within the methylenedioxyphenyl ring was assigned based solely upon its product ion spectrum. Following intravenous administration of MPC-3100 to rats, fourteen metabolites were observed in the feces; whereas, only 6 metabolites were observed in urine. No glucuronides were found in either the urine or feces. Less than 1% of the dose was recovered in rat urine; whereas, up to 40% of the dose was recovered as MPC-3100 and metabolites in feces over a 24 hour period. As many as 25 different metabolites were observed in the bile based upon differences in their retention time and molecular weight. Most of the metabolites in the bile resulted from either glucuronidation or sulfation, some of which were conclusively identified with authentic standards. Rat, dog, and monkey liver microsomes all produced the four major metabolites formed in human liver microsomes. Three of these metabolites formed in human microsomes were conclusively identified by comparison to authentic synthetic standards. Although MPC-3100 and several metabolites were found in rat, dog and monkey urine, the primary route of elimination of MPC-3100 was through biliary excretion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3233. doi:10.1158/1538-7445.AM2011-3233

Abstract 2628: Antitumor activity of MPC-3100, a synthetic Hsp90 inhibitor, in combination with erlotinib and sorafenib

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: MPC-3100 is a fully synthetic, orally bioavailable, Hsp90 inhibitor in clinical development. It is active as a single agent in xenograft models with many cancer types including colon, gastric, ovary, prostate, breast, lung and myeloid leukemia. We evaluate here the activity of MPC-3100 in combination with erlotinib or sorafenib in xenograft models. Methods: Three to five million cells, depending on cell-type, were implanted subcutaneously into athymic mice (nu/nu) for tumor studies. Dosing was initiated when median tumor volume was >100 mm3. All compounds were dosed orally, once daily. MPC-3100 (100 mg/kg) and erlotinib (100 mg/kg) were dosed on Days 1-21 and sorafenib (60 mg/kg) was dosed on Days 1-9. Results: Anti-tumor activity for the combination of MPC-3100 and erlotinib was compared to that of the single agents in a lung cancer (A549) xenograft model sensitive to EGFR inhibition. Administration of MPC-3100 or erlotinib resulted in 52% and 64% tumor growth inhibition (TGI), respectively relative to vehicle by the end of dosing on Day 19. By contrast, the combination of MPC-3100 and erlotinib resulted in 30% tumor regression over the same period. Thus the combination of MPC-3100 and erlotinib was more effective at inhibiting tumor growth than either agent alone (p<0.05). The combined anti-tumor activity of MPC-3100 and sorafenib was compared to administration of the single agents in a xenograft model with the melanoma cell line A375 that harbors the activating B-raf mutation, V600E. The median tumor volume (MTV) of the cohorts dosed with vehicle, MPC-3100 or sorafenib as single agents was comparable and increased by 4.8-, 6.4- and 8.2-fold, respectively by the end of dosing on study Day 20, whereas MTV of the cohort dosed with a combination of MPC-3100 and sorafenib increased by only 1.6-fold. The combination of MPC-3100 and sorafenib resulted in 66% tumor growth inhibition relative to vehicle on Day 20 and was more effective at inhibiting tumor growth than single agent (p<0.05). Conclusions: The combination of MPC-3100 with erlotinib or sorafenib shows greater anti-tumor activity than either agent alone. Thus, in addition to its broad activity in xenograft models as a single agent, MPC-3100 has the potential to be combined with other targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2011-2628

Abstract 3526: Coadministration of nicotinic acid with the Nampt inhibitor MPC-9528 enhances antitumor activity in Naprt deficient cancer cells in culture and in xenografts

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: The tumoricidal small molecule MPC-9528 is a picomolar inhibitor of nicotinamide phosphoribosyltransferase (Nampt). Nampt catalyzes the first and rate-limiting step in NAD synthesis from nicotinamide. Nicotinic acid phosphoribosyltransferase (Naprt) catalyzes the first and rate-limiting step in an alternate pathway of NAD synthesis from nicotinic acid (NA). Cancer cells are particularly dependent on NAD and many cancer cell lines, but not most normal tissues, are deficient in Naprt activity. Therefore administration of NA could prevent MPC-9528-induced NAD depletion in normal tissues, but not in Naprt-deficient tumors, resulting in greater therapeutic index and efficacy. Methods: Cell viability was determined based on ATP levels. Naprt protein expresson was quantified by western blot and qRT-PCR. NAD was acid-extracted from cells and quantified by a coupled reaction based on fluorescent resorufin. Xenograft studies were performed in nu/nu mice. Results: In 44 out of 153 cancer cell lines surveyed, NA did not prevent MPC-9528-induced cell death, which correlated with low to undetectable levels of Naprt. MPC-9528-induced NAD depletion and cell death in HCT116 colon carcinoma cells were prevented by the addition of NA, consistent with high Naprt expression. A single dose of MPC-9528 at the maximum-tolerated dose (MTD) of 75 mg/kg caused tumor regression in HCT116 xenografts and NA coadministration completely blocked this effect. NA also completely blocked mortality in mice induced by 300 mg/kg MPC-9528, consistent with the finding that most mouse tissues have high Naprt expression. In Naprt-deficient MIA PaCa-2 xenografts, NA coadministration allowed tolerance of 200 mg/kg MPC-9528 with a substantially increased anti-tumor response relative to the MTD of 75 mg/kg MPC-9528 alone. Conclusions: Low Naprt expression correlates with the lack of effect of NA on MPC-9528 tumoricidal activity. Because Naprt deficiency is prevalent in cancer cell lines and in primary tumor specimens, but not in normal tissues, NA coadministration with MPC-9528 should increase the tolerability and efficacy of MPC-9528 in patients with Naprt-deficient tumors. A companion diagnostic designed to measure Naprt expression or activity in tumors could be used to identify tumors that would most likely benefit from such combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3526. doi:10.1158/1538-7445.AM2011-3526

Abstract 577: Basal NAD levels and Nampt expression correlate with in vitro and in vivo sensitivity of tumor cell lines to the Nampt inhibitor MPC-9528

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: MPC-9528 is a potent and selective inhibitor of the NAD biosynthetic enzyme nicotinamide phosphoribosyltransferase (Nampt). Inhibition of Nampt by MPC-9528 causes depletion of cellular NAD followed by a decrease in ATP and cell death. Cancer cells develop dependence on Nampt due to increased metabolic demands and the elevated activity of enzymes such as poly(ADP-ribose) polymerases (Parps) that consume NAD. MPC-9528 has shown anti-tumor activity, ranging from no response to complete regression in a variety of xenograft models. Materials and Methods: In vitro Nampt activity and cellular NAD levels were measured in coupled biochemical reactions. Cellular Parp activity was measured by immunofluorescent detection of poly(ADP-ribose) (PAR). Enzyme protein and mRNA levels were quantified by western blot and qRT-PCR, respectively. Mechanism of cell death was determined by Caspase 3/7 activity, Caspase 3 and Parp1 cleavage, and SytoxGreen staining. Cell viability was based on ATP levels. Xenografts were performed in nu/nu mice. Results: MPC-9528 inhibited Nampt activity in vitro with an average IC50 of 40 pM and suppressed cellular NAD levels and nuclear PAR levels, with potencies of 170 pM and 120 pM, respectively. In a screen of 93 cancer cell lines of diverse origin, MPC-9528 had a median TC50 of 2.8 nM with a range of 100 pM to 62 nM. Similar to cultured cells, a range of tumor responses was observed in six different xenograft models. In HCT116 colon carcinoma and HT1080 fibrosarcoma xenografts, oral administration of MPC-9528 at 75 mg/kg intermittently resulted in tumor regressions. In contrast, similar treatment of MIA PaCa-2 pancreatic cancer, N87 gastric carcinoma or HCC827 and NCI-H460 lung cancer xenografts led to partial tumor growth inhibition or no response. The effects in xenografts correlated with TC50 values for MPC-9528 for these cell lines in culture, which ranged from 260 pM to 24 nM. The TC50 values also correlated well with basal cellular NAD levels, IC50 values for MPC-9528-induced NAD depletion, and Nampt protein expression, but not with expression of three other enzymes involved in NAD metabolism – Naprt, Qprt or Parp1. The mechanism of cell death induced by MPC-9528 was cell type dependent and did not correlate with MPC-9528 potency in culture. Conclusions: NAD levels in cancer cell lines are primarily dependent upon the Nampt pathway. The differential sensitivity of tumor cells to the Nampt inhibitor MPC-9528 is likely due to the magnitude of NAD production, which is a function of Nampt expression. MPC-9528 has the greatest effect on tumor cell lines with lower Nampt expression; therefore, a companion diagnostic based upon Nampt expression in primary tumor specimens could be used to select patients most likely to respond to MPC-9528 monotherapy in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 577. doi:10.1158/1538-7445.AM2011-577

Abstract LB-393: The cancer metabolism inhibitor MPC-9528 induces tumor regression in xenograft models with multiple dosing schedules by causing rapid and sustained reduction in tumor NAD

Introduction : MPC-9528 is a potent, selective, orally bioavailable inhibitor of nicotinamide phosphoribosyltransferase (Nampt), which catalyzes the rate-limiting step for NAD biosynthesis from nicotinamide. Inhibition of Nampt by MPC-9528 results in cell death as a consequence of NAD depletion and inhibition of ATP synthesis. MPC-9528 is a potent tumoricidal agent of cancer cell lines of diverse origin and shows anti-tumor activity in multiple xenograft models. Here we explore the determinants of efficacy in xenograft models by comparing the effects of MPC-9528 oral dosing schedules in terms of both tumor NAD depletion and survival. Methods : HT1080 human fibrosarcoma cells were implanted subcutaneously into athymic mice (nu/nu) for tumor studies. Dosing was initiated when median tumor volume was >100 mm 3 . MPC-9528 was dosed orally, either once weekly, once daily, or twice daily, for a total of two or three weeks. For pharmacodynamic (PD) studies, animals were dosed with MPC-9528 and tumors were collected at various times post-dosing for NAD determination by LC-MS/MS. Results : MPC-9528 (75 mg/kg) dosed once weekly for three weeks in a HT1080 xenograft model resulted in 75% tumor regression on study Day 23, 8 days after the last dose. The ED 50 for anti-tumor activity with this schedule was 44 mg/kg and doses at or below 35 mg/kg showed no anti-tumor activity. All doses were equally well-tolerated with Conclusions : MPC-9528 administration results in regression of tumors when plasma concentrations of compound are maintained above a threshold. MPC-9528 shows comparable efficacy when given intermittently, on a weekly schedule, or continuously on a once, or twice daily schedule. This unique attribute of MPC-9528 reflects its mechanism of action as a cell metabolism inhibitor that functions by inhibiting NAD synthesis in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-393. doi:10.1158/1538-7445.AM2011-LB-393

Abstract LB-288: MPI-0486348: A novel inhibitor of nicotinamide phosphoribosyltransferase (Nampt) induces tumor regression in a preclinical model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first step in the recycling of nicotinamide into NAD. Cancer cells develop dependence on Nampt due to increased energy requirements and the elevated activity of NAD consuming enzymes such as sirtuins and mono and poly(ADP-ribose) polymerases (PARPs). Identification of Nampt as the target of an orphan cytotoxic compound using chemical proteomics spawned a Nampt drug discovery program at Myriad. We have developed a potent and selective inhibitor of Nampt that inhibits the growth of cancer cells in culture and induces tumor regression in a mouse xenograft model. Materials and Methods: In vitro Nampt activity was measured in a coupled biochemical assay based on the production of fluorescent resorufin. Viability was determined by measuring cellular ATP levels at 72 hrs following addition of compound. Nampt cellular activity was assayed by measuring levels of NAD and NAD-dependent poly (ADP)-ribosylation (PAR). Pharmacokinetic studies were conducted in female CD-1 mice. Anti-tumor activity was evaluated in an HCT116 human colon carcinoma xenograft in nu/nu mice. Results: MPI-0486348 inhibited Nampt activity in vitro with an average IC50 value of 40 pM. Depending upon the time of compound exposure, MPI-0486348 showed TC50 values of 180 pM to 20 nM in HCT116 cells. MPI-0486348 treatment also reduced cellular NAD levels and nuclear PAR levels, with IC50 values of 170 pM and 120 pM, respectively. These cellular effects of MPI-0486348 were prevented by coadministration of nicotinic acid, demonstrating on target mechanism of action. In mouse pharmacokinetic studies, absolute oral bioavailability of 47% was determined using 40% Captisol as the vehicle at an oral dose of 10 mg/kg. The same dose in rats gave an absolute oral bioavailability of 46%. Finally, in HCT116 tumor-bearing mice, a single oral dose of MPI-0486348 (50 or 75 mg/kg) resulted in 31% and 67% regression, respectively. Conclusions : MPI-0486348 is an orally bioavailable small molecule inhibitor of Nampt and shows promising preclinical results in a human xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-288.