Shevan Silva

Synthesis and structure-activity relationships for 1-(4-(piperidin-1-ylsulfonyl)phenyl)pyrrolidin-2-ones as novel non-carboxylate inhibitors of the aldo-keto reductase enzyme AKR1C3

High expression of the aldo-keto reductase enzyme AKR1C3 in the human prostate and breast has implicated it in the development and progression of leukemias and of prostate and breast cancers. Inhibitors are thus of interest as potential drugs. Most inhibitors of AKR1C3 are carboxylic acids, whose transport into cells is likely dominated by carrier-mediated processes. We describe here a series of (piperidinosulfonamidophenyl)pyrrolidin-2-ones as potent (<100 nM) and isoform-selective non-carboxylate inhibitors of AKR1C3. Structure-activity relationships identified the sulfonamide was critical, and a crystal structure showed the 2-pyrrolidinone does not interact directly with residues in the oxyanion hole. Variations in the position, co-planarity or electronic nature of the pyrrolidinone ring severely diminished activity, as did altering the size or polarity of the piperidino ring. There was a broad correlation between the enzyme potencies of the compounds and their effectiveness at inhibiting AKR1C3 activity in cells.

Abstract 5358: The hypoxia-activated EGFR-TKI TH-4000 overcomes erlotinib-resistance in preclinical NSCLC models at plasma levels achieved in a Phase 1 clinical trial

Clinical data indicate that mutant EGFR NSCLC is often heterozygous (PLoS ONE 2013; 8: e54170; PLoS ONE 2009; 4: e7464) and the presence of wild type (WT) EGFR allele is associated with limited response to EGFR-tyrosine kinase inhibitor (TKI) therapy (Cancer Sci 2008; 99:929). Tumor hypoxia upregulates WT EGFR protein and its cognate ligand TGFα via several HIF-dependent mechanisms (reviewed in: Curr Pharm Des 2013; 19:907). NSCLC is known to be a hypoxic tumor, and thus hyperactivation of WT EGFR may be an important cause of resistance to EGFR-TKI therapy. TH-4000 (formerly called PR610) is a clinical-stage hypoxia-activated prodrug that releases an irreversible EGFR-TKI under hypoxic conditions and may overcome resistance to conventional TKI therapy. We tested this hypothesis using the heterozygous WT/Δ19 EGFR PC9 tumor model and found it to be resistant to clinically relevant doses of the EGFR-TKI erlotinib; 100% of tumors progressed during treatment with human matched plasma PK exposures of erlotinib. In contrast, the homozygous Δ19 mutant EGFR tumor HCC827 was readily controlled by erlotinib (100% tumor regression). TH-4000 (15 mg/kg) produced 100% tumor regressions in both models. In vitro, PC9 cells exposed to hypoxia had elevated EGFR protein and were more resistant to erlotinib as measured by EGFR phosphorylation. In nude mice, single-dose administration of 15 mg/kg TH-4000 achieved a plasma AUC equivalent to 32 mg/m2 in human subjects, one-fifth of the maximum tolerated dose (MTD) defined in the Phase 1 trial (MTD = 150 mg/m2/week; NCT01631279). A single dose of TH-4000 (15 mg/kg) cleared rapidly from mouse plasma (T½ = 0.37 h) but had durable residency in PC9 tumors (T½ = 39 h), releasing TKI above efficacious levels for 7 days (T½β = 84 h). Consistent with these PK properties, tumor shutdown of EGFR signalling was durable, with no recovery by day 7. To confirm the mechanism of action, TH-4000 was shown to be metabolized efficiently under hypoxia using a panel of human NSCLC cell lines (rate of TKI release 0.4-2.1 nmol/hr/106 cells), a process that was inhibited by oxygen (TKI release 80% (538 vs 99 nmol/kg; p Citation Format: Adam V. Patterson, Shevan Silva, Christopher Guise, Maria Abbattista, Matthew Bull, Huai-Ling Hsu, Charles Hart, Jessica Sun, Angus Grey, Amir Ashoorzadeh, Robert Anderson, Jeff B. Smaill. The hypoxia-activated EGFR-TKI TH-4000 overcomes erlotinib-resistance in preclinical NSCLC models at plasma levels achieved in a Phase 1 clinical trial. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5358. doi:10.1158/1538-7445.AM2015-5358

Abstract A67: Preclinical efficacy of tarloxotinib bromide (TH-4000), a hypoxia-activated EGFR/HER2 inhibitor: rationale for clinical evaluation in EGFR mutant, T790M-negative NSCLC following progression on EGFR-TKI therapy

Tarloxotinib bromide (T) is a prodrug that releases an irreversible EGFR/HER2 inhibitor (T-TKI) under hypoxic conditions. NSCLC is known to be a hypoxic disease and wild type (WT) EGFR is upregulated by multiple hypoxia-driven mechanisms (Curr Pharm Des, 19:907). Mutant EGFR NSCLC is commonly heterozygous and may result in maintenance of WT EGFR signalling (Can Sci, 103:1946; PloS One 8:e54170). Clinical studies indicate NSCLC patients harbouring WT/mut heterozygous EGFR have significantly poorer ORR, PFS and OS on treatment with EGFR-TKI (Can Sci, 99:929). Other mechanisms of resistance to EGFR-TKI include 50-60% with T790M EGFR mutation, 8-13% with HER2 amplification, while 15-20% lack identifiable mutation/amplification events (Nat Rev Clin Onc, 11:473). The combination of cetuximab/afatinib provides an ORR of 25% and PFS of 4.6 months in T790M-negative NSCLC suggesting the persistence of HER signalling plays a role in resistance. However the high proportion of Grade 3/4 toxicity seen with cetuximab/afatinib indicates an opportunity for dose-intensification with an improved therapeutic index (Can Discov, 4:1). In addition, early clinical data on resistance to the 3rd Gen (WT EGFR-sparing) TKI rociletinib, fails to identify further mutations by NGS in some patients and describes reversion to EGFR-WT (T790) status (Can Discov, 5:713). Collectively these data support the hypothesis that WT EGFR heterozygosity may be a mechanism of resistance to current EGFR-TKI. Current EGFR-TKI lack the therapeutic index to silence WT EGFR signalling in tumors due to on-target skin/GI toxicities (Ann Oncol 18:761). Therefore we sought to examine the potency of T-TKI relative to erlotinib, afatinib and AZD9291 in five human cancer cell lines expressing WT EGFR (H1838, H2073, H1648, H125 and A431). In antiproliferative assays T-TKI was more dose-potent than erlotinib (25- to 110-fold) afatinib (4- to 32-fold) and AZD9291 (120- to 71-fold). This activity correlated with inhibition of WT EGFR phosphorylation and downstream MAPK signalling. We used a prototypic WT EGFR driven xenograft model (A431) to benchmark T activity against each EGFR-TKI by ‘retrotranslation’ of reported plasma exposure for each agent in human subjects back to the xenograft model. Only treatment with clinically relevant doses and schedules of T was associated with tumor regression and durable inhibition of WT EGFR tumor phosphorylation. Consistent with these findings, T treatment can also regress the WT EGFR NSCLC tumor models H125 and H1648, demonstrating T provides the necessary therapeutic index to inhibit WT EGFR in vivo. The transfection of WT EGFR into mutant EGFR NSCLC line PC9 (vs GFP control) conferred TGFα dependent induction of p-EGFR that was supressed by T-TKI but resistant to inhibition by erlotinib, afatinib or AZD9291. This was associated with reduced antiproliferative activity for EGFR-TKIs. Collectively these data indicate T-TKI is a dose-potent inhibitor of WT EGFR signalling and the prodrug T may possess the therapeutic index to silence WT EGFR signalling in xenograft models at plasma exposure levels achieved in a human Ph1 trial. T is under investigation in a Phase 2 clinical trial for EGFR mutant, T790M-negative, NSCLC patients who have progressed on EGFR-TKI (NCT02454842). Citation Format: Shevan Silva, Victoria Jackson, Christopher Guise, Maria Abbattista, Matthew Bull, Angus Grey, Robert Anderson, Amir Ashoorzadeh, Charles Hart, Tillman Pearce, Adam V. Patterson, Jeff B. Smaill. Preclinical efficacy of tarloxotinib bromide (TH-4000), a hypoxia-activated EGFR/HER2 inhibitor: rationale for clinical evaluation in EGFR mutant, T790M-negative NSCLC following progression on EGFR-TKI therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A67.

Abstract A66: Preclinical rationale for the ongoing Phase 2 study of the hypoxia-activated EGFR-TKI tarloxotinib bromide (TH-4000) in patients with advanced squamous cell carcinoma of the head and neck (SCCHN) or skin (SCCS)

Tarloxotinib bromide (T), a hypoxia-activated prodrug of a potent irreversible EGFR tyrosine kinase inhibitor (T-TKI), was designed to provide clinical benefit in patients with advanced SCCHN and SCCS. In these clinical settings excessive wild type (WT) EGFR overexpression may underlie the limited activity seen with currently available EGFR-TKI where inadequate tumor signal inhibition is evident at recommended doses (Annals Oncol 2007, 18:761). In contrast to the systemic delivery of EGFR-TKI, our prodrug approach selectively targets the hypoxic microenvironment of tumors, with the goal of increasing tumor dose-intensification and thus improving the therapeutic index. Preclinical studies were conducted to test the efficacy of clinically relevant doses of T and the approved EGFR-targeted agents cetuximab and afatinib in preclinical models of SCCHN (FaDu) and SCCS (A431) that express wild type EGFR. Under varying hypoxic conditions, EGFR expression and signal transduction was assessed by western blotting (WB). Pharmacodynamic (PD) endpoints (p-EGFR, p-MAPK, p-AKT) were evaluated by WB or immunohistochemistry (IHC). Under hypoxic conditions, T was 13- and 16-fold more active than in normoxic conditions against FaDu and A431 cells, respectively, with hypoxia-specific release of T-TKI from T by LC/MS (294 and 176 pmol/h/106 cells). Notably, hypoxic exposure of FaDu cells induced a marked 25-fold induction in EGFR phosphorylation (Y1092) that was stable upon reoxygenation (≥5 h), and EGFR signalling in A431 cells was upregulated 2.3-fold post-anoxia. Dose-dependent inhibition of EGFR signalling was observed and correlated with antiproliferative activity. The SCCHN FaDu tumor xenograft had a mean hypoxic fraction (HF) of 14% as determined by IHC detection of pimonidazole binding. Treatment with 48 mg/kg (qw) T (plasma exposure = 150 mg/m2 IV in human subjects) resulted in 100% (8/8) response rate (RR). In contrast, 8 mg/kg (qw) cetuximab treatment (saturating dose) failed to control growth with 8/10 (80%) tumors progressing on treatment and 2/10 (20%) with stable disease. The SCCS A431 tumor xenograft exhibited a mean HF of 31%. Treatment with 30 mg/kg (qw) T (plasma exposure equivalent to 104 mg/m2 IV in human subjects) resulted in 100% (6/6) RR. In contrast, 5 mg/kg daily oral afatinib (plasma exposure = 33 mg daily in human subjects) failed to regress tumors with best response of stable disease in 4/6 (66%) during treatment, while 2/6 (33%) progressed during treatment. T efficacy was accompanied by marked shutdown of total tumor p-EGFR (Try1092) in both xenograft models as well as p-AKT (Ser473) and p-MAPK (Thr202/Tyr204), in the FaDu and A431 xenografts, respectively, which was not evident with the comparator treatments of cetuximab and afatinib. In preclinical models, T exhibited superior efficacy with a 100% response rate compared to cetuximab in SCCHN and afatinib in SCCS with no responses and superior pharmacodynamic effects of T on EGFR target modulation. Based on these models performed with clinically relevant plasma PK levels T may possess a superior therapeutic index relative to approved EGFR-targeted agents. These preclinical data support the weekly dose of 150 mg/m2 of T in the ongoing phase 2 clinical trial in SCCHN and SCCS (NCT02449681). Citation Format: Victoria Jackson, Shevan Silva, Maria Abbattista, Christopher Guise, Matthew Bull, Amir Ashoorzadeh, Charles Hart, Tillman Pearce, Jeff Smaill, Adam V. Patterson. Preclinical rationale for the ongoing Phase 2 study of the hypoxia-activated EGFR-TKI tarloxotinib bromide (TH-4000) in patients with advanced squamous cell carcinoma of the head and neck (SCCHN) or skin (SCCS). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A66.

Abstract B278: PR610: A novel hypoxia-selective tyrosine kinase inhibitor in phase I clinical trial.

PR610 is a hypoxia-selective irreversible Human Epidermal Growth Factor Receptor (HER) family inhibitor currently in phase I clinical trials in New Zealand and the USA (clinical trial ID NCT01631279). The prodrug PR610 releases the TKI (Tyrosine Kinase Inhibitor) PR610E, a picomolar irreversible inhibitor of EGFR (HER1), under oxygen-limiting conditions typically found in solid tumors. Human neoplastic cell lines exposed to PR610 show an anoxia-selective anti-proliferative response that is associated with G1 arrest and induction of apoptosis arising from inhibition of EGFR auto-phosphorylation and downstream silencing of associated signal transduction pathways. PR610 is optimized for long tumor residency (T½ >2 days); in a preclinical model of erlotinib-resistant NSCLC, a single injection of PR610 produces profound, global shutdown of signal transduction via EGFRT790M/L858R. Tumor PR610E concentrations were above cellular anti-proliferative IC50 concentrations for over 5 days, being more than sufficient to induce apoptosis via “oncogenic shock”. Pharmacokinetic (PK) studies of PR610 show significant differences in species toxicokinetics. Both the rat and dog preclinical toxicology models display considerable systemic conversion of PR610 to PR610E (6% - 30% and 24% - 29% of prodrug AUC, respectively) with attendant symptoms of EGFR inhibition including acneiform skin rash and diarrhea. In contrast NIH-III nude mice display minimal circulating TKI (PR610E) relative to PR610 (1.5% - 1.7% of AUC) consistent with a substantially improved tolerance as judged by PR610 plasma AUCinf at the maximum tolerated dose (MTD). Human subjects from the phase I clinical trial consistently experience the lowest systemic levels of PR610E (Mean 1.06% ± 0.69%; range 0.34% - 2.7%; n=20) across dose levels ranging from 10 - 150 mg/m2. Notably, a dose of 150 mg/m2 in human subjects produces a PR610 plasma AUCinf equivalent to that measured for 30 mg/kg PR610 in NIH-III mice, a dose that is active in several HER-dependent subcutaneous tumor xenograft models. Collectively, these data indicate that PR610 has the desirable characteristics of a deactivated prodrug in human subjects and preclinical models predict that an active dose range has been reached in the phase I trial. PR610 is a first-in-class hypoxia-selective EGFR/HER2 inhibitor with exciting clinical potential. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B278. Citation Format: Adam V. Patterson, Jagdish Jaiswal, Kendall Carlin, Maria R. Abbattista, Christopher P. Guise, Shevan Silva, Ho Lee, Guo-Liang Lu, Robert F. Anderson, Teresa J. Melink, John C. Gutheil, Jeff B. Smaill. PR610: A novel hypoxia-selective tyrosine kinase inhibitor in phase I clinical trial. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B278.