ABBV-319: a CD19-targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies.

ABSTRACT: Glucocorticoids are key components of the standard-of-care treatment regimens for B-cell malignancy. However, systemic glucocorticoid treatment is associated with several adverse events. ABBV-319 is a CD19-targeting antibody-drug conjugate engineered to reduce glucocorticoid-associated toxicities while possessing 3 distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) antibody-mediated delivery of a glucocorticoid receptor modulator (GRM) payload to activate apoptosis, (2) inhibition of CD19 signaling, and (3) enhanced fragment crystallizable (Fc)-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven antitumor activity against multiple malignant B-cell lines in vitro, as well as in cell line-derived xenografts and patient-derived xenografts (PDXs) in vivo. Remarkably, a single dose of ABBV-319 induced sustained tumor regression and enhanced antitumor activity compared with repeated dosing of systemic prednisolone at the maximum tolerated dose in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed antiproliferative activity in a subset of B-cell lymphoma cell lines through the inhibition of phosphoinositide 3-kinase signaling. Moreover, afucosylation of CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity. Notably, ABBV-319 displayed superior efficacy compared with afucosylated CD19 mAb in human CD34+ peripheral blood mononuclear cell-engrafted NSG-Tg(Hu-IL15) transgenic mice, demonstrating enhanced antitumor activity when multiple MOAs are enabled. ABBV-319 also showed durable antitumor activity across multiple B-cell lymphoma PDX models, including nongerminal center B-cell diffuse large B-cell lymphoma and relapsed lymphoma after R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 in a phase 1 clinical trial.

Synergistic therapeutic benefit by combining the antibody drug conjugate, depatux-m with temozolomide in pre-clinical models of glioblastoma with overexpression of EGFR.

PURPOSE: Depatux-m is an antibody drug conjugate (ADC) that targets and inhibits growth of cancer cells overexpressing the epidermal growth factor receptor (EGFR) or the 2-7 deletion mutant (EGFRvIII) in tumor models in vitro and in vivo. Treatment of patients suffering from relapsed/refractory glioblastoma (GBM) with a combination of depatux-m and temozolomide (TMZ) tended to increase overall survival. As a first step to understand the nature of the interaction between the two drugs, we investigated whether the interaction was synergistic, additive or antagonistic. METHODS: The efficacy of ADCs, antibodies, TMZ and radiation was tested in xenograft models of GBM, U-87MG and U-87MG EGFRvIII. Both models express EGFR. U-87MG EGFRvIII was transduced to express EGFRvIII. Changes in tumor volume, biomarkers of cell death and apoptosis after treatment were used to measure efficacy of the various treatments. Synergism of depatux-m and TMZ was verified in three-dimensional cultures of U-87MG and U-87MG EGFRvIII by the method of Chou and Talalay. RESULTS: Combined with TMZ and radiotherapy (RT), depatux-m inhibited xenograft growth of U-87MG and U-87MG EGFRvIII more than either treatment with depatux-m or TMZ + RT. Durability of the response to depatux-m + TMZ + RT or depatux-m + TMZ was more pronounced in U-87MG EGFRvIII than in U-87MG. Efficacy of depatux-m + TMZ was synergistic in U-87MG EGFRvIII and additive in U-87MG. CONCLUSION: Adding depatux-m enhances the efficacy of standard of care therapy in preclinical models of GBM. Durability of response to depatux-m + TMZ in vivo and synergy of the drug-drug interaction correlates with the amount of antigen expressed by the tumor cells.

Targeting Multiple EGFR-expressing Tumors with a Highly Potent Tumor-selective Antibody-Drug Conjugate.

ABBV-321 (serclutamab talirine), a next-generation EGFR-targeted antibody-drug conjugate (ADC) incorporates a potent pyrrolobenzodiazepine (PBD) dimer toxin conjugated to the EGFR-targeting ABT-806 affinity-matured AM1 antibody. ABBV-321 follows the development of related EGFR-targeted ADCs including depatuxizumab mafodotin (depatux-m, ABT-414), ABT-806 conjugated to monomethyl auristatin F (MMAF), and ABBV-221 (losatuxizumab vedotin), AM1 antibody conjugated to monomethyl auristatin E (MMAE). The distinct tumor selectivity of ABBV-321 differentiates it from many previous highly active antibody PBD conjugates that lack a therapeutic window. Potency of the PBD dimer, combined with increased binding of AM1 to EGFR-positive tumor cells, opens the possibility to target a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent antitumor activity in cellular and in vivo studies including xenograft cell line and patient-derived xenograft glioblastoma, colorectal, lung, head and neck, and malignant mesothelioma tumor models that are less sensitive to depatux-m or ABBV-221. Combination studies with ABBV-321 and depatux-m suggest a promising treatment option permitting suboptimal, and potentially better tolerated, doses of both ADCs while providing improved potency. Collectively, these data suggest that ABBV-321 may offer an extended breadth of efficacy relative to other EGFR ADCs while extending utility to multiple EGFR-expressing tumor indications. Despite its highly potent PBD dimer payload, the tumor selectivity of ABBV-321, coupled with its pharmacology, toxicology, and pharmacokinetic profiles, support continuation of ongoing phase I clinical trials in patients with advanced EGFR-expressing malignancies.

The Volume of Three-Dimensional Cultures of Cancer Cells InVitro Influences Transcriptional Profile Differences and Similarities with Monolayer Cultures and Xenografted Tumors.

Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro. Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of applying these models for drug development.

A "Prozone-Like" Effect Influences the Efficacy of the Monoclonal Antibody ABT-700 against the Hepatocyte Growth Factor Receptor.

ABT-700 is a therapeutic antibody against the hepatocyte growth factor receptor (MET). At doses or regimens that lead to exposures exceeding optimum in vivo, the efficacy of ABT-700 is unexpectedly reduced. We hypothesized that this reduction in efficacy was due to a "prozone-like" effect in vivo. A prozone-like effect, which is a reduction in efficacy beyond optimum exposure, is caused due a mechanism similar to the generation of false negative flocculation tests by excessive antibody titres. In vitro, we demonstrate that at higher ABT-700 concentrations, this "prozone-like" effect is mediated by a progressive conversion from bivalent to ineffective monovalent binding of the antibody. In vivo, the efficacy of ABT-700 is dependent on an optimum range of exposure as well. Our data suggest that the "prozone-like" effect is operative and independent of target expression. ABT-700 dose, regimen, exposure, and tumor burden are interdependent variables influencing the "prozone-like" effect and mediating and in vivo efficacy. By optimization of dosage and regimen we demonstrate that the "prozone-like" effect can be alleviated and ABT-700 efficacy at varying tumor loads can be further extended in combination with cisplatin. Our results suggest that optimization of exposure taking tumor burden into account may alleviate "prozone-like" effects without compromising efficacy.

ABBV-399, a c-Met Antibody-Drug Conjugate that Targets Both MET-Amplified and c-Met-Overexpressing Tumors, Irrespective of MET Pathway Dependence.

Purpose: Despite the importance of the MET oncogene in many malignancies, clinical strategies targeting c-Met have benefitted only small subsets of patients with tumors driven by signaling through the c-Met pathway, thereby necessitating selection of patients with MET amplification and/or c-Met activation most likely to respond. An ADC targeting c-Met could overcome these limitations with potential as a broad-acting therapeutic.Experimental Design: ADC ABBV-399 was generated with the c-Met-targeting antibody, ABT-700. Antitumor activity was evaluated in cancer cells with overexpressed c-Met or amplified MET and in xenografts including patient-derived xenograft (PDX) models and those refractory to other c-Met inhibitors. The correlation between c-Met expression and sensitivity to ABBV-399 in tumor and normal cell lines was assessed to evaluate the risk of on-target toxicity.Results: A threshold level of c-Met expressed by sensitive tumor but not normal cells is required for significant ABBV-399-mediated killing of tumor cells. Activity extends to c-Met or amplified MET cell line and PDX models where significant tumor growth inhibition and regressions are observed. ABBV-399 inhibits growth of xenograft tumors refractory to other c-Met inhibitors and provides significant therapeutic benefit in combination with standard-of-care chemotherapy.Conclusions: ABBV-399 represents a novel therapeutic strategy to deliver a potent cytotoxin to c-Met-overexpressing tumor cells enabling cell killing regardless of reliance on MET signaling. ABBV-399 has progressed to a phase I study where it has been well tolerated and has produced objective responses in c-Met-expressing non-small cell lung cancer (NSCLC) patients. Clin Cancer Res; 23(4); 992-1000. ©2016 AACR.

Expression Profile of BCL-2, BCL-XL, and MCL-1 Predicts Pharmacological Response to the BCL-2 Selective Antagonist Venetoclax in Multiple Myeloma Models.

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL-selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2(High)/BCL-XL (Low) In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132-44. ©2016 AACR.

Anti-c-Met monoclonal antibody ABT-700 breaks oncogene addiction in tumors with MET amplification.

BACKGROUND: c-Met is the receptor tyrosine kinase for hepatocyte growth factor (HGF) encoded by the MET proto-oncogene. Aberrant activation of c-Met resulting from MET amplification and c-Met overexpression is associated with poor clinical outcome in multiple malignancies underscoring the importance of c-Met signaling in cancer progression. Several c-Met inhibitors have advanced to the clinic; however, the development of inhibitory c-Met-directed therapeutic antibodies has been hampered by inherent agonistic activity. METHOD: We generated and tested a bivalent anti-c-Met monoclonal antibody ABT-700 in vitro for binding potency and antagonistic activity and in vivo for antitumor efficacy in human tumor xenografts. Human cancer cell lines and gastric cancer tissue microarrays were examined for MET amplification by fluorescence in situ hybridization (FISH). RESULTS: ABT-700 exhibits a distinctive ability to block both HGF-independent constitutive c-Met signaling and HGF-dependent activation of c-Met. Cancer cells addicted to the constitutively activated c-Met signaling driven by MET amplification undergo apoptosis upon exposure to ABT-700. ABT-700 induces tumor regression and tumor growth delay in preclinical tumor models of gastric and lung cancers harboring amplified MET. ABT-700 in combination with chemotherapeutics also shows additive antitumor effect. Amplification of MET in human cancer tissues can be identified by FISH. CONCLUSIONS: The preclinical attributes of ABT-700 in blocking c-Met signaling, inducing apoptosis and suppressing tumor growth in cancers with amplified MET provide rationale for examining its potential clinical utility for the treatment of cancers harboring MET amplification.

Clearance of systemic hematologic tumors by venetoclax (Abt-199) and navitoclax.

The Bcl-2 family inhibitors venetoclax and navitoclax demonstrated potent antitumor activity in chronic lymphocytic leukemia patients, notably in reducing marrow load and adenopathy. Subsequent trials with venetoclax have been initiated in non-Hodgkin's lymphoma and multiple myeloma patients. Traditional preclinical models fall short either in faithfully recapitulating disease progression within such compartments or in allowing the direct longitudinal analysis of systemic disease. We show that intravenous inoculation of engineered RS4;11 (acute lymphoblastic leukemia) and Granta 519 (mantle cell lymphoma) bioluminescent reporter cell lines result in tumor engraftment of bone marrow, with additional invasion of the central nervous system in the case of Granta 519. Importantly, apoptosis induction and response of these systemically engrafted tumors to Bcl-2 family inhibitors alone or in combination with standard-of-care agents could be monitored longitudinally with optical imaging, and was more accurately reflective of the observed clinical response.

Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets.

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

The Bcl-2 inhibitor ABT-263 enhances the response of multiple chemotherapeutic regimens in hematologic tumors in vivo.

PURPOSE: This study was designed to test the ability of the Bcl-2 family inhibitor ABT-263 to potentiate commonly used chemotherapeutic agents and regimens in hematologic tumor models. METHODS: Models of B-cell lymphoma and multiple myeloma were tested in vitro and in vivo with ABT-263 in combination with standard chemotherapeutic regimens, including VAP, CHOP and R-CHOP, as well as single cytotoxic agents including etoposide, rituximab, bortezomib and cyclophosphamide. Alterations in Bcl-2 family member expression patterns were analyzed to define mechanisms of potentiation. RESULTS: ABT-263 was additive with etoposide, vincristine and VAP in vitro in the diffuse large B-cell lymphoma line (DLBCL) DoHH-2, while rituximab potentiated its activity in SuDHL-4. Bortezomib strongly synergized with ABT-263 in the mantle cell lymphoma line Granta 519. Treatment of DoHH-2 with etoposide was associated with an increase in Puma expression, while bortezomib upregulated Noxa expression in Granta 519. Combination of ABT-263 with cytotoxic agents demonstrated superior tumor growth inhibition and delay in multiple models versus cytotoxic therapy alone, along with significant improvements in tumor response rates. CONCLUSIONS: Inhibition of the Bcl-2 family of proteins by ABT-263 enhances the cytotoxicity of multiple chemotherapeutics in hematologic tumors and represents a promising addition to the therapeutic arsenal for treatment of these diseases.

ABT-263 and rapamycin act cooperatively to kill lymphoma cells in vitro and in vivo.

ABT-263 is a potent, orally bioavailable inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-x(L), and Bcl-w, which is currently in phase I clinical trials. Previous work has shown that this compound has low nanomolar cell-killing activity in a variety of lymphoma and leukemia cell lines, many of which overexpress Bcl-2 through a variety of mechanisms. Rapamycin is a macrolide antibiotic that inhibits the mammalian target of rapamycin complex, leading to cell cycle arrest and inhibition of protein translation. Rapamycin (and its analogues) has shown activity in a variety of tumor cell lines primarily through induction of cell cycle arrest. Activity has also been shown clinically in mantle cell lymphoma and advanced renal cell carcinoma. Here, we show that treatment of the follicular lymphoma lines DoHH-2 and SuDHL-4 with 100 nmol/L rapamycin induces substantial G(0)-G(1) arrest. Addition of as little as 39 nmol/L ABT-263 to the rapamycin regimen induced a 3-fold increase in sub-G(0) cells. Combination of these agents also led to a significant increase in Annexin V staining over ABT-263 alone. In xenograft models of these tumors, rapamycin induced a largely cytostatic response in the DoHH-2 and SuDHL-4 models. Coadministration with ABT-263 induced significant tumor regression, with DoHH-2 and SuDHL-4 tumors showing 100% overall response rates. Apoptosis in these tumors was significantly enhanced by combination therapy as measured by staining with an antibody specific for cleaved caspase-3. These data suggest that combination of ABT-263 and rapamycin or its analogues represents a promising therapeutic strategy for the treatment of lymphoma.

Activity of the Bcl-2 family inhibitor ABT-263 in a panel of small cell lung cancer xenograft models.

PURPOSE: The purpose of this study was to characterize the activity of the Bcl-2 protein family inhibitor ABT-263 in a panel of small cell lung cancer (SCLC) xenograft models. EXPERIMENTAL DESIGN: A panel of 11 SCLC xenograft models was established to evaluate the efficacy of ABT-263. Single agent activity was examined on a continuous dosing schedule in each of these models. The H146 model was used to further evaluate dose and schedule, comparison to standard cytotoxic agents, and induction of apoptosis. RESULTS: ABT-263 exhibited a range of antitumor activity, leading to complete tumor regression in several models. Significant regressions of tumors as large as 1 cc were also observed. The efficacy of ABT-263 was also quite durable; in several cases, minimal tumor regrowth was noted several weeks after the cessation of treatment. Antitumor effects were equal or superior to that of several clinically approved cytotoxic agents. Regression of large established tumors was observed through several cycles of therapy and efficacy was retained in a Pgp-1 overexpressing line. Significant efficacy was observed on several dose and therapeutic schedules and was associated with significant induction of apoptosis. CONCLUSIONS: ABT-263 is a potent, orally bioavailable inhibitor of Bcl-2 family proteins that has recently entered clinical trials. The efficacy data reported here suggest that SCLC is a promising area of clinical investigation with this agent.

A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo.

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.

Isoquinoline-pyridine-based protein kinase B/Akt antagonists: SAR and in vivo antitumor activity.

The structure-activity relationships of a series of isoquinoline-pyridine-based protein kinase B/Akt antagonists have been investigated in an effort to improve the major short-comings of the lead compound 3, including poor pharmacokinetic profiles in several species (e.g., mouse i.v. t(1/2) = 0.3 h, p.o. F = 0%). Chlorination at C-1 position of the isoquinoline improved its pharmacokinetic property in mice (i.v. t(1/2) = 5.0 h, p.o. F = 51%) but resulted in >500-fold drop in potency. In a mouse MiaPaCa-2 xenograft model, an amino analog 10y significantly slowed the tumor growth, however was accompanied by toxicity.

Discovery and SAR of oxindole-pyridine-based protein kinase B/Akt inhibitors for treating cancers.

We describe a series of potent and selective oxindole-pyridine-based protein kinase B/Akt inhibitors. The most potent compound 11n in this series demonstrated an IC(50) of 0.17nM against Akt1 and more than 100-fold selectivity over other Akt isozymes. The selectivity against other protein kinases was highly dependent on the C-3 substitutions at the oxindole scaffold, with unsubstituted 9e or 3-furan-2-ylmethylene (11n) more selective and 3-(1H-pyrrol-2-yl)methylene (11f) or 3-(1H-imidazol-2-yl)methylene (11k) less selective. In a mouse xenograft model, 9d, 11f, and 11n inhibited tumor growth but with accompanying toxicity.

Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo.

Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.

Optimal classes of chemotherapeutic agents sensitized by specific small-molecule inhibitors of akt in vitro and in vivo.

Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor-induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654). Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment.