Gary Mather

Discovery of N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a Potent Apoptosis Inducer and Efficacious Anticancer Agent with High Blood Brain Barrier Penetration

As a continuation of our structure-activity relationship (SAR) studies on 4-anilinoquinazolines as potent apoptosis inducers and to identify anticancer development candidates, we explored the replacement of the 2-Cl group in our lead compound 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (6b, EP128265, MPI-0441138) by other functional groups. This SAR study and lead optimization resulted in the identification of N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine (6h, EP128495, MPC-6827) as an anticancer clinical candidate. Compound 6h was found to be a potent apoptosis inducer with EC(50) of 2 nM in our cell-based apoptosis induction assay. It also has excellent blood brain barrier penetration, and is highly efficacious in human MX-1 breast and other mouse xenograft cancer models.

Discovery of 2-Chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (EP128265, MPI-0441138) as a Potent Inducer of Apoptosis with High In Vivo Activity

Using a live cell, high-throughput caspase-3 activator assay, we have identified a novel series of 4-anilinoquinazolines as inducers of apoptosis. In this report, we discuss the discovery of 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine, compound 2b (EP128265, MPI-0441138) as a highly active inducer of apoptosis (EC50 for caspase activation of 2 nM) and as a potent inhibitor of cell proliferation (GI50 of 2 nM) in T47D cells. Compound 2b inhibited tubulin polymerization, was effective in cells overexpressing ABC transporter Pgp-1, and was efficacious in the MX-1 human breast and PC-3 prostate cancer mouse models. In contrast to the SAR of 4-anilinoquinazolines as EGFR kinase inhibitors, the methyl group on the nitrogen linker was essential for the apoptosis-inducing activity of 4-anilinoquinazolines and substitution in the 6- and 7-positions of the quinazoline core structure decreased potency.

Discovery of N-methyl-4-(4-methoxyanilino)quinazolines as potent apoptosis inducers. Structure-activity relationship of the quinazoline ring.

As a continuation of our efforts to discover and develop apoptosis inducing N-methyl-4-(4-methoxyanilino)quinazolines as novel anticancer agents, we explored substitution at the 5-, 6-, 7-positions of the quinazoline and replacement of the quinazoline by other nitrogen-containing heterocycles. A small group at the 5-position was found to be well tolerated. At the 6-position a small group like an amino was preferred. Substitution at the 7-position was tolerated much less than at the 6-position. Replacing the carbon at the 8-position or both the 5- and 8-positions with nitrogen led to about 10-fold reductions in potency. Replacement of the quinazoline ring with a quinoline, a benzo[d][1,2,3]triazine, or an isoquinoline ring showed that the nitrogen at the 1-position is important for activity, while the carbon at the 2-position can be replaced by a nitrogen and the nitrogen at the 3-position can be replaced by a carbon. Through the SAR study, several 5- or 6-substituted analogs, such as 2a and 2c, were found to have potencies approaching that of lead compound N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine (1g, EP128495, MPC-6827, Azixa).

Abstract A96: Phase 1 study of HSP90 inhibitor MPC-3100 in subjects with refractory or recurrent cancer.

Background: MPC-3100 is an orally-bioavailable, fully-synthetic inhibitor of the molecular chaperone HSP90. HSP90 is important for post-translational protein folding, stabilization, and function of so-called client proteins, many of which are necessary for growth and survival of cancer cells. In preclinical studies, MPC-3100 has demonstrated client protein modulation and antitumor activity against a broad range of tumor types. Methods: In this first-in-human, open label, dose escalating, 3+3 design with accelerated titration, multiple-dose study, the safety and tolerability of single agent MPC-3100 were assessed in subjects with recurrent or refractory cancer. Secondary objectives were to characterize the pharmacokinetic parameters (PK) of MPC-3100, assess antitumor activity, and evaluate pharmacodynamic (PD) biomarkers. Subjects received oral MPC-3100 once daily for 21 days followed by 7 days off at doses of 50, 100, 165, 245, or 340 mg/m2 (Cohorts 1–5, respectively) or for 28 days continuously at total daily doses of 480 mg or 640 mg administered as 240 mg or 320 mg Q12H (Cohorts 6 and 7, respectively) per 28-day cycle. Clinical examinations, blood draws for PK and PD, and tumor assessments were performed at pre-specified times during the study. Results: MPC-3100 was administered to 26 subjects [13 M, 13 F; median age 63.5 yr, range 45–85 yr; ECOG performance status 0 (n=13), 1 (n=11) and 2 (n=2) at screening; most-represented primary cancer types colon (n=6), prostate (6), and breast (3); median of 4 (range 0 to 16) prior chemotherapies]. The total number of cycles of MPC-3100 administered to all subjects was 44 (median 1, range Conclusions: MPC-3100 appears to be safe and tolerable when administered orally at doses below 600 mg per day to subjects with recurrent or refractory cancer. Side-effects were generally manageable or reversible upon discontinuation of MPC-3100. Biomarker modulation indicates appropriate HSP90 inhibition. Nearly half of the subjects attained stable disease. 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 A96.

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 4386: Administration of nicotinic acid reduces or prevents adverse effects of MPC-9528, a potent and selective Nampt inhibitor

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Inhibition of Nampt in cancer cell lines decreases NAD levels and induces cell death. We evaluate the potential toxicity of MPC-9528, a Nampt inhibitor, and investigate the effects of coadministration of nicotinic acid (NA), the substrate for an alternate pathway leading to NAD formation in normal tissues but lacking in many cancer cells. Methods: SD rats 8/sex/group were administered MPC-9528 at doses of 0, 10, or 15 mg/kg/day, or 15 mg/kg/day + 200 mg/kg/day of NA. Three additional rats were used to evaluate recovery after 7 days without treatment. Pathology was assessed at scheduled necropsies and PK parameters were determined, organ weights recorded, and selected tissues examined microscopically. Subsequently, CD-1 mice were given single oral doses of MPC-9528 at levels known to be efficacious in xenografts or lethal (75 or 300 mg/kg, respectively). NAD levels and white cell counts were determined. Female rats (n=6) were administered MPC-9528 (15 mg/kg/day) with or without pretreatment with NA (200 mg/kg/day) for 9 days. NAD levels were quantified by LC/MS/MS. Results: Cmax and AUC in females were approximately five times those in male rats. Two females died in the 10 mg/kg group and 8/11 females in the 15 mg/kg group. There were no deaths in male rats or in females dosed with MPC-9528 and NA. Leukocyte counts were reduced (36.4-77.0%) for all groups compared to controls. The reduction for rats treated concurrently with NA was less than groups treated with MPC-9528 alone. Leukocyte counts partially recovered 7 days off drug. There were no significant changes in clinical chemistry at any dose in male rats. AST and CPK were increased in a single female compared to controls. Albumin and total protein were reduced in females treated at 10 or 15 mg/kg/day. Thymus weights were reduced in all treated groups and spleen weights were reduced in females. These changes were not observed at the recovery sacrifice. Testis weight was reduced at both terminal and recovery in MPC-9528 treated males. Lymphoid depletion was noted on histopath. Clinical pathology, organ weight, and histopath changes were either reduced in severity or prevented by concurrent NA. NAD levels in mice given a single dose of 75 mg/kg were reduced >95% and white counts were reduced from a mean of 2.2 × 106/mL to 0.2 × 106/mL by Day 6. Lethality of a 300 mg/kg single dose in mice was completely prevented by coadministration of 1000 mg/kg NA. Similarly, NAD levels in rat blood decreased >50% by Day 6 in females administered MPC-9528 (15 mg/kg/day), however, with concurrent NA, NAD levels were reduced less than 30%. Conclusion: Coadministration of NA prevented or reduced the severity of adverse effects associated with daily administration of higher doses of MPC-9528. These data suggest that coadministration of NA has the potential to increase the therapeutic margin and to abrogate adverse clinical events in future clinical studies of MPC-9528. 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 4386. doi:10.1158/1538-7445.AM2011-4386

Abstract 2617: MPC-3100, a synthetic Hsp90 inhibitor, induces biomarker changes in vitro and in vivo

Introduction: MPC-3100 is a fully synthetic, orally bioavailable, Hsp90 inhibitor in clinical development. It is broadly active in xenograft models with anti-tumor activity ranging from tumor regression to tumor growth inhibition in many cancer types including colon, gastric, ovarian, prostate, breast, lung and myeloid leukemia. Here we evaluate the effect of MPC-3100 on stability of client proteins in cells and xenograft tumors. We also determine the effect on Hsp70 protein levels, a biomarker of Hsp90 inhibition, in peripheral blood mononuclear cells (PBMCs) from cancer patients receiving MPC-3100. Methods: Her2, Akt, Cdk4, c-Raf and Hsp70 client protein levels were monitored in cell culture with protein immunoblots. Formalin-fixed, paraffin-embedded sections of xenograft tumors from mice dosed orally with MPC-3100 were analyzed by immunohistochemistry (IHC) to monitor changes in Her2, Akt and Hsp70 protein levels. To monitor changes in Hsp70 in cancer patients treated with MPC-3100, PBMCs were collected prior to drug administration, 8 and 24 hours post-dose on Day 1 and 24 hours post-dose on Days 7 and 21 of the first treatment cycle. Hsp70 protein levels were determined by ELISA. Results: Exposure of HCT-116, NCI-N87 and DU-145 cells to MPC-3100 in vitro resulted in a time-dependent reduction in client protein levels with maximal reduction by 24 hours. The IC 50 values for client protein reduction ranged from 0.1 μM to 0.5 μM, comparable to the cellular cytotoxicity values of MPC-3100 at 72 hours for the various cell lines. IHC revealed reduction in Her2 and Akt protein in N87 xenografts in mice given a single oral dose of 200 mg/kg MPC-3100 relative to tumors from animals dosed with vehicle. Healthy volunteer PBMCs exposed to 1 μM MPC-3100 for 24 hours ex vivo revealed a reduction in Akt, c-Raf and Cdk4 protein levels ranging from 50% to 90%. PBMCs from cancer patients receiving MPC-3100 showed an increase of 28 to 589 ng of Hsp70 protein per mg total protein over baseline by Day 8. The increase in Hsp70 expression was seen as early as 8 hours after the first dose and sustained through at least Day 22. Conclusions: The changes in client proteins and biomarkers observed in cells and tumor xenografts exposed to MPC-3100 confirm that the cellular cytotoxic activity and anti-tumor activity in xenografts are a result of Hsp90 inhibition. The consistent increase in Hsp70 expression in PBMCs from cancer patients receiving MPC-3100 indicates that Hsp90 function is inhibited in patients at doses that have been well tolerated 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 2617. doi:10.1158/1538-7445.AM2011-2617

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 C230: MPC‐6827 is safely combined with temozolomide for the treatment of patients with metastatic melanoma

Purpose: MPC‐6827 (4‐arylaminoquinazoline) is a microtubule destabilizing agent with in vitro cytotoxic and in vivo vascular disrupting characteristics. We determined the dose‐limiting toxicity (DLT) and the most tolerable doses of MPC‐6827 in combination with daily administration of temozolomide. Experimental Design: Metastatic melanoma patients were treated weekly with a 2‐hour intravenous infusion of MPC‐6827 for 6 or 3 consecutive weeks during 8‐ or 4‐week cycles, respectively. Temozolomide was administered orally either at 75 mg/m2 daily during the first 6 weeks of an 8‐week cycle or at 85 mg/m2 daily during the first 3 weeks of a 4‐week cycle. Therapy was repeated every 56 days in the 8‐week cycle and every 28 days in the 4‐week cycle. Three pre‐selected doses of MPC‐6827 were tested: 2.1, 2.7, and 3.3 mg/m2. Patients in the 2.1 mg/m2 dose level were treated at 8‐week cycles. Due to the inconveniences of 8‐week cycles, patients enrolled in the 2.7 and 3.3 mg/m2 dose levels were treated at 4‐week cycles. Patients were observed for DLTs during the first cycle. Plasma samples were obtained at various timepoints after MPC‐6827 infusion to evaluate concentrations of MPC‐6827 and temozolomide. A conventional “3+3” design was used for dose escalation. Results: Twenty‐two patients (median age, 59 years; range, 35–81; median number of prior therapies,2; range 1–5) with stage IV melanoma (3 M1b, 19 M1c) were enrolled, two patients are still receiving therapy. Twenty patients (91 %) had an ECOG score 0–1 and ten (45%) patients had brain metastases. Eight patients (36%) received temozolomide/dacarbazine therapy previously. Four patients (18%) experienced a grade 3 or greater MPC‐6827‐related SAE, which include an increased Troponin I level (n=1), cerebral hemorrhage (n=2), and cerebral infarction associated with a brain metastasis (n=1). One of the cerebral hemorrhages was deemed a DLT. Median MPC‐6827 half‐life ranged from 2.9–4.0 hours. Cmax and AUC increased proportionally with dose. The highest dose tested was 3.3 mg/m2 once weekly with temozolomide 85 mg/m2 daily for 3 weeks of a 4‐week cycle. Employing modified RECIST criteria, two patients achieved partial response (durations of 6 and 4 months). Another nine patients achieved stable disease (duration range: 1 – 6 months). Conclusion: The combination of MPC‐6827 and temozolomide is safe and well tolerated in patients with refractory metastatic melanoma, including brain metastases. Responses are durable. A dose reduction of MPC‐6827 is not required when combined with temozolomide. Incidences of cerebral hemorrhage, deep venous thrombosis and/or pulmonary emboli were similar to those in previous reports. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C230.