- NVS and PFE both announced unexpected eye toxicity in their HSP90 inhibitors in 2h2010. AUY922 (NVS) exhibited 89% ocular toxicity in the MTD cohort. This is caused by the drugs inducing apoptosis in a specific cell layer of the retina known as the outer nuclear layer (Slide 13)
- Slide 19: HSP90 inhibitors target the chaperone protein rather than a specific oncoprotein. This means we need to ask 2 separate questions: 1) How addicted is tumor to oncoprotein? 2) How addicted to HSP90 is the oncoprotein?
- Highly addicted client protein require the constant presence of HSP90- believe these situations are driving single agent ganetespib activity seen to date (ie, inhibition of HSP90 leads to instability of oncoprotein and increased cell death). Combination of HSP90 inhibitor with other agents results in sensitization of the cells to these agents- this is rationale for combo trial with docetaxel (Slide 23)
- When they took the poll as to the best path forward, the most votes went to pursuing single agent and combo paths to registration in parallel (but they didn't really announce the detailed results on the webcast- were displayed on screen at the event..)
- SNTA has talked to large pharma individuals- some of these folks have said that the data and promise of ganetespib warrant proceeding directly into large phase 3 program. However, due to the size of SNTA, a derisked strategy of the p2b/p3 trial design is being employed
- Slide 33- four chemotherapy regimens for NSCLC, all give indistinguishable results
- Goal in NSCLC is to deliver a HR of 0.80 or so
- At this point, the histological subtype determines which first-line therapy the NSCLC pt gets
- Slide 64- shows the progress made in RR and survival since 1975, but also that there is such a long way to go in this field
- Slide 83 - conclusions from second speaker. "There is no blockbuster targeted drug for adenocarcinoma" instead, there can be blockbuster drugs for small % subsets of NSCLC pts
- Ganetespib diarrhea side effect- the pts that had grade 3 AE were largely those who did not pre-treated with medicine such as immodium to optimally manage this side effect. If well managed, generally is grade 1 diarrhea for only 24 hrs or so
- SNTA is "well underway" in characterizing the correlation between clinical activity and specific pt genetic profiles
- Slide 92 mentions some of these mutations such as ALK-EML4 (ganetespib and other HSP90 inhibitors), Her2-neu, PDGRFA-alpha (unique for GIST), BRAF (melanoma, lung, gastric cancers)
- Safety profile of phase 2 trial very similar to what was seen in phase 1
- Longest pt on therapy in NSCLC trial so far is 14 months
- 4th speaker- covered rationale for combining gantespib with docetaxel
- Docetaaxel has been approved in NSCLC for over 10 years, is now approved for first-line in combination with Pt-based therapies, is equally effective for all histological subtypes
- In refractory disease, docetaxel, pematrexib, and erlotinib produce similar results (Slide 107): response rate <10%, median PFS of 3 months, median survival of 6-8 months
- Slide 109- in several trials, lower HSP90 levels have been correlated with increased survival
- Docetaxel dose-limiting toxicity is bone marrow-related, so not overlapping with ganetespib, important for combination therapy
- Taxanes such as docetaxel induce mitotic arrest and apoptosis- cells induce AKT pathway to inhibit apoptosis- this is an HSP90 client protein, so inhibition of HSP90 in combination helps the anti-cancer activity of taxanes
- A phase 1 trial of 17-AAG with paclitaxel was completed- was well tolerated but not enough efficacy to move forward- ganetespib is more potent so hope would perform better- this trial was done by the same investigator who is leading current gan-doc combo trial
- Phase 1 ongoing of gantespib (150 mg/m2) plus docetaxel (75 mg/m2) combo, 13 pts so far, 7 still on study, expanding at the 150/75 dose level (Slide 119)
- Have seen activity in combo trial in a variety of tumors including NSCLC, majority of pts have seen some benefit
- Slide 121 shows p2b trial design for NSCLC
- Most competing HSP90 inhibitors are analogs of 17-AAG (including the Infinity compound). Ganetespib is based on a pharmacophore, designed for max inhibition of target with minimal toxicity
- Now that several competitors have failed (and even another one Motesanib from AMGN and Takeda since the presentation- see link), first panelist (Langer) thinks should take the time to maximize therapeutic window of sta-9090 and find out where it can be applied
- Patients are much more willing now to enroll in clinical trials requiring biopsy than used to be (see success of targeted therapies requiring genetic profiling)
- Ganetespib as single agent is closer to phase 3 than the combo (I didn't think much of this as the comment was made, but since the phase 3 portion of the docetaxel combo could start 1h2012, that means SNTA is confident they have/will identify a subpopulation with superior efficacy and could start a trial shortly)
- Moderators did not orally discuss the "after" poll on the preferred path forward of the attendees
- Question- why not 200 mg dose in combo? Answer: 150 gives basically the same efficacy, only used 200 dose in single agent trials due to PK variability
- Q: note importance of a comprehensive genetic profile at start of trial before randomization- or else imbalance in some unknown genotype could seriously confound results
- SNTA mentioned that they are looking at another set of biomarkers associated with response to ganetespib that are not genetic mutations- but not wiling to discuss these yet
- Partnering interest and form may depend on the elucidation of genotypes that the drug works best for (makes sense, this would determine the market size and time to approval)
- Pharma companies are very interested in the potential to develop combination therapies for cancer from within their own pipelines- this is reflected in partnering discussions
- The use of docetaxel in 2nd line NSCLC is actually increasing- this is because first line is moving away from docetaxel, so when pts fail the newer drugs (such as pem), they are more often going onto docetaxel
- Docetaxel will shortly go off patent, which is bad for SNY, but good for SNTA in developing the combo
- The expert panel does not foresee the approval of a new single agent therapy in the next 1-2 years. Several phase 3 trials on single agents in 2nd line vs docetaxel have failed recently
SNTA hosted a webcast to discuss non-small cell lung cancer (NSCLC) treatments and landscape generally and in regards to their HSP90 inhibitor ganetespib (aka STA-9090) specifically. Click here for the link to the very informative and highly recommended webcast as well as slides from each of the presenters. I'll copy my notes from the talks below- I don't intend to summarize everything that was said, just items of notes in my opinion. I'll make reference to particular slide #'s in the PDF file (126 slides of information total!)
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"
J. Jay Boniface, Vijay R. Baichwal, Daniel M. Cimbora, Lynn DeMie, Tracey C. Fleischer, Andrew D. Gassman, Siavash Ghaffari, Jeremy P. Green, Jeff S. Flick, Ryan T. Terry-Lorenzo, Damon I. Papac, Ashley Peterson, Rosann Robinson, Robert O. Carlson. Myrexis, Inc., Salt Lake City, UT
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.
Abstract #2551"The Nampt inhibitor MPC-9528 synergizes with DNA damaging agents"
Ryan T. Terry-Lorenzo, Thomas B. Douce, Harry A. Austin, J. Jay Boniface, Daniel M. Cimbora, Tracey C. Fleischer, Robert O. Carlson. Myrexis, Inc., Salt Lake City, UT
Background: MPC-9528 reduces cellular NAD levels and causes cell death, by blocking the NAD salvage pathway through inhibition of nicotinamide phosphoribosyltransferase (Nampt). Many DNA damaging agents also reduce cellular NAD levels, by activating the NAD consuming enzyme poly(ADP-ribose) polymerase (Parp). We hypothesized that the combination of a Nampt inhibitor and a DNA damaging agent would synergize in killing cancer cells, due to a combined effect on NAD levels through two independent mechanisms.
Methods: Cellular NAD was measured using a coupled enzymatic assay. Drug combination experiments were performed in HCT116 colon carcinoma cells, using measurement of ATP levels as a cell viability endpoint. Synergy, antagonism, or additivity was assessed using the MacSynergy II program.
Results: In HCT116 cells, saturating doses of MPC-9528 induced depletion of NAD with a half-life of 5 hours and a decrease in ATP that was delayed approximately 14 hours relative to NAD. Lower, sublethal concentrations of MPC-9528 induced partial NAD depletion without a concomitant ATP loss. At these sublethal concentrations, MPC-9528 was found to synergize with the DNA alkylating agents temozolomide and streptozotocin, which are known to activate Parp. Additionally, MPC-9528 was found to synergize with two structurally different thymidylate synthase inhibitors, 5-fluorouracil (5-FU) and raltitrexed, neither of which have been reported to activate Parp. Individually, 5-FU and raltitrexed each caused NAD depletion in HCT116 cells, which was enhanced by combination with MPC-9528. Furthermore, both 5-FU- and raltitrexed-mediated NAD depletion and synergy with MPC-9528 were completely blocked by the Parp inhibitor olaparib.
Conclusions: Parp activation induced by the alkylating agents temozolomide and streptozotocin, or by the thymidylate synthase inhibitors 5-FU and raltitrexed, is the basis for tumoricidal synergy with the Nampt inhibitor MPC-9528. This synergy is a direct consequence of the NAD depletion resulting from Parp activation coupled with the inhibition of NAD synthesis due to Nampt inhibition. These results provide a basis for clinical combination of MPC-9528 with the agents studied here or with related agents that induce Parp activation.
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"
Tracey C. Fleischer, Vijay R. Baichwal, J. Jay Boniface, Daniel M. Cimbora, Lynn DeMie, Thomas B. Douce, Andrew D. Gassman, Damon I. Papac, Ashley Peterson, Rosann Robinson, Ryan T. Terry-Lorenzo, Robert O. Carlson. Myrexis, Inc., Salt Lake City, UT
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.
Abstract #4386 "Administration of nicotinic acid reduces or prevents adverse effects of MPC-9528, a potent and selective Nampt inhibitor"
Gary G. Mather, Valerie L. Belcher, Anna Costa, Lynn DeMie, Orvelin Roman, Lori Fotheringham, Chad Bradford. Myrexis, Inc., Salt Lake City, UT
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 x 106/mL to 0.2 x 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.
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"
Vijay R. Baichwal, Adam J. Willardsen, Jeff W. Lockman, Brett J. Murphy, Ruth Gordillo, Tracey C. Fleischer, Chad L. Bradford, Damon I. Papac, Gary G. Mather, Robert O. Carlson. Myrexis, Inc., Salt Lake City, UT
--this is a late-breaking abstract and the full text abstract results are not yet available online
Abstract #2617 "MPC-3100, a synthetic Hsp90 inhibitor, induces biomarker changes in vitro and in vivo"
Vijay R. Baichwal, Brita Brown, Rosann Robinson, Daniel Cimbora, Daniel Wettstein, Andrew P. Beelen, Gary G. Mather, Robert O. Carlson. Myrexis Inc., Salt Lake City, UT
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 IC50 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.
Abstract #2628 "Antitumor activity of MPC-3100, a synthetic Hsp90 inhibitor, in combination with erlotinib and sorafenib" Vijay R. Baichwal, Brita Brown, Daniel Wettstein, Damon I. Papac, Gary G. Mather, Robert O. Carlson. Myrexis Inc., Salt Lake City, UT
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.
Abstract #3233 "Comparative in vitro and in vivo metabolism of MPC-3100, an oral HSP90 inhibitor, in rat, dog, monkey and human"Damon I. Papac1, J. Scott Patton1, Leslie Reeves1, Katrina Bulka2, Lynn DeMie1, Orvelin Roman, Jr.1, Chad Bradford1, Se-Ho Kim1, Rajendra Tangallapally1, Richard Trovato1, Benjamin Markovitz3, Ashok Bajji1, Daniel Wettstein1, Vijay Baichwal1, Gary Mather1. 1Myrexis, Salt Lake City, UT; 2Myriad Genetics, Salt Lake City, UT;3Nease Corporation, Cincinnati, OH
Introduction: 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.
Methods: 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.
Results: 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.
Abstract #3237 "Evaluation of the pharmacokinetics and efficacy of a novel pro-drug of the HSP90 inhibitor, MPC-3100, designed with improved solubility"Damon Papac, J. Scott Patton, Leslie Reeves, Lynn DeMie, Chad Bradford, Brian Hachey, Brian Clemetson, Christin Christensen, Se-Ho Kim, Rajendra Tangallapally, Daniel Parker, Richard Trovato, In Chul Kim, Daniel Wettstein, Vijay R. Baichwal, Ashok Bajji. Myrexis, Salt Lake City, UTMPC-3100 is a synthetic, orally bioavailable
Introduction: 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.
Methods: 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.
Results: 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 x 10-6 cm/sec at pH 6.2; whereas, the apparent permeability of MPC-3100 was 420 x 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 < 3 minutes) in mouse plasma and in mouse and human NADPH-deficient liver microsomes, but was converted slowly in human plasma with a half-life of 105 minutes, similar to the rate of non-enzymatic conversion in phosphate buffer (pH 7.4). An oral dose of 360 mg/kg of the pro-drug yielded a Cmax for MPC-3100 of 21.7 ug/mL and an AUC(0-inf) of 91.0 hr*ug/mL, which was comparable to exposures seen at efficacious doses of MPC-3100 formulated with solubility-enhancing agents. At this same dose, the pro-drug demonstrated a 42% tumor regression in the N-87 mouse xenograft model. This anti-tumor effect observed with the pro-drug was comparable to the 80% tumor regression observed with MPC-3100 at 200 mg/kg formulated with solubility-enhancing agents.
ConcluThe 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.
While I haven't yet loaded all my research notes, I pasted a few slides from the Roth conference presentation over on the SNMX research page. Here are my notes from the talk: