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Overexpression of the antiapoptotic protein B-cell lymphoma-extra large (BCL-X L ) is associated with drug resistance and disease progression in numerous cancers. The compelling nature of this protein as a therapeutic target prompted efforts to develop selective small-molecule BCL-X L inhibitors. Although efficacious in preclinical models, we report herein that selective BCL-X L inhibitors cause severe mechanism-based cardiovascular toxicity in higher preclinical species. To overcome this liability, antibody-drug conjugates were constructed using altered BCL-X L -targeting warheads, unique linker technologies, and therapeutic antibodies. The epidermal growth factor receptor-targeting antibody-drug conjugate AM1-15 inhibited growth of tumor xenografts and did not cause cardiovascular toxicity nor dose-limiting thrombocytopenia in monkeys. While an unprecedented BCL-X L -mediated toxicity was uncovered in monkey kidneys upon repeat dosing of AM1-15, this toxicity was mitigated via further drug-linker modification to afford AM1-AAA (AM1-25). The AAA drug-linker has since been incorporated into mirzotamab clezutoclax, the first selective BCL-X L -targeting agent to enter human clinical trials.

Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. Although a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug ( 3 , ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing., (©2021 American Association for Cancer Research.)

Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi 1-5 . Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice 6 , the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions 7-9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported 10,11 , suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment 10-13 . Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-075 14 . Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.

In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.

Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (https://openscienceprobes.sgc-frankfurt.de/). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project., Competing Interests: SM, SA, CA, JB, CB, PB, DD, DD, AE, JE, SF, AI, TH, SL, BM, BR, NS, AS, PT, CW, TW, WZ, SK No competing interests declared, MB employee of Bayer AG, JB employee of Vertex Pharmaceuticals, JB employee of Boehringer Ingelheim, MB Mark E Bunnage: employee of Vertex Pharmaceuticals, AC Adrian J Carter: employee of Boehringer Ingelheim, VD Reviewing editor, eLife, JE employee of Janssen Pharmaceutical Research and Development LLC, CF employee of Merck & Co., Inc. AG Andreas Gollner: employee of Boehringer Ingelheim, CG employee of Ched Grimshaw Consulting, LLC, IH Ingo V Hartung: employee of Bayer AG, SH employee of Takeda California Inc. TH employee of J&J Innovation Centre, TH Terry V Hughes: employee of J&J Innovation Centre, VL Volkhart MJ Li: employee of Bayer AG, SL employee of Pfizer, HM employee of Takeda Pharmaceutical Company Ltd, JM John Mathias: employee of Pfizer, RO Ronan C O'Hagan: employee of Merck & Co., Inc.,, DO Dafydd R Owen: employee of Pfizer, VP employee of Janssen-Pharmaceutical Companies of Johnson & Johnson, DR Daniel Rauh received consulting and lecture fees (Sanofi-Aventis, Takeda, Novartis, Pfizer, LDC) as well as research support (Novartis, J&J, Bayer, Merck, MSD). SR employee of AbbVie, CS Cora Scholten: employee of Bayer AG, KS Kumar Singh Saikatendu: employee of Takeda California Inc, MT Masayuki Takizawa: employee of Takeda Pharmaceutical Company Ltd. CT Chris Tse: employee of AbbVie, DT employee of Eurofins DiscoverX, AV Amélia YI Viana: employee of Boehringer Ingelheim, AM Anke Mueller-Fahrnow: employee of Bayer AG, (© 2018, Müller et al.)

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.

Cancer cells are highly reliant on NAD + -dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD + salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD + , and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD + depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR ., (©2017 American Association for Cancer Research.)

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G 1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro / in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976-89. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.

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.

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 ability of a cancer cell to avoid apoptosis is crucial to tumorigenesis and can also contribute to chemoresistance. The Bcl-2 family of prosurvival proteins (Bcl-2, Bcl-X(L), Bcl-w, Mcl-1, and A1) plays a key role in these processes. We previously reported the discovery of ABT-263 (navitoclax), a potent small-molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w. While navitoclax exhibits single-agent activity in tumors dependent on Bcl-2 or Bcl-X(L) for survival, the expression of Mcl-1 has been shown to confer resistance to navitoclax, most notably in solid tumors. Thus, therapeutic agents that can downregulate or neutralize Mcl-1 are predicted to synergize potently with navitoclax. Here, we report the activity of navitoclax in combination with 19 clinically relevant agents across a panel of 46 human solid tumor cell lines. Navitoclax broadly enhanced the activity of multiple therapeutic agents in vitro and enhanced efficacy of both docetaxel and erlotinib in xenograft models. The ability of navitoclax to synergize with docetaxel or erlotinib corresponded to an altered sensitivity of the mitochondria toward navitoclax, which was associated with the downmodulation of Mcl-1 and/or upregulation of Bim. These data provide a rationale to interrogate these combinations clinically.

ABT-263 inhibits the antiapoptotic proteins Bcl-2, Bcl-x(L), and Bcl-w and has single-agent efficacy in numerous small cell lung carcinoma (SCLC) and leukemia/lymphoma cell lines in vitro and in vivo. It is currently in clinical trials for treating patients with SCLC and various leukemia/lymphomas. Identification of predictive markers for response will benefit the clinical development of ABT-263. We identified the expression of Bcl-2 family genes that correlated best with sensitivity to ABT-263 in a panel of 36 SCLC and 31 leukemia/lymphoma cell lines. In cells sensitive to ABT-263, expression of Bcl-2 and Noxa is elevated, whereas expression of Mcl-1 is higher in resistant cells. We also examined global expression differences to identify gene signature sets that correlated with sensitivity to ABT-263 to generate optimal signature sets predictive of sensitivity to ABT-263. Independent cell lines were used to verify the predictive power of the gene sets and to refine the optimal gene signatures. When comparing normal lung tissue and SCLC primary tumors, the expression pattern of these genes in the tumor tissue is most similar to sensitive SCLC lines, whereas normal tissue is most similar to resistant SCLC lines. Most of the genes identified using global expression patterns are related to the apoptotic pathway; however, all but Bcl-rambo are distinct from the Bcl-2 family. This study leverages global expression data to identify key gene expression patterns for sensitivity to ABT-263 in SCLC and leukemia/lymphoma and may provide guidance in the selection of patients in future clinical trials.

Purpose: ABT-888, currently in phase 2 trials, is a potent oral poly(ADP-ribose) polymerase inhibitor that enhances the activity of multiple DNA-damaging agents, including temozolomide (TMZ). We investigated ABT-888+TMZ combination therapy in multiple xenograft models representing various human tumors having different responses to TMZ., Experimental Design: ABT-888+TMZ efficacy in xenograft tumors implanted in subcutaneous, orthotopic, and metastatic sites was assessed by tumor burden, expression of poly(ADP-ribose) polymer, and O(6)-methylguanine methyltransferase (MGMT)., Results: Varying levels of ABT-888+TMZ sensitivity were evident across a broad histologic spectrum of models (55-100% tumor growth inhibition) in B-cell lymphoma, small cell lung carcinoma, non-small cell lung carcinoma, pancreatic, ovarian, breast, and prostate xenografts, including numerous regressions. Combination efficacy in otherwise TMZ nonresponsive tumors suggests that TMZ resistance may be overcome by poly(ADP-ribose) polymerase inhibition. Profound ABT-888+TMZ efficacy was seen in experimental metastases models that acquired resistance to TMZ. Moreover, TMZ resistance was overcome in crossover treatments, indicating that combination therapy may overcome acquired TMZ resistance. Neither tumor MGMT, mismatch repair, nor poly(ADP-ribose) polymer correlated with the degree of sensitivity to ABT-888+TMZ., Conclusions: Robust ABT-888+TMZ efficacy is observed across a spectrum of tumor types, including orthotopic and metastatic implantation. As many TMZ nonresponsive tumors proved sensitive to ABT-888+TMZ, this novel combination may broaden the clinical use of TMZ beyond melanoma and glioma. Although TMZ resistance may be influenced by MGMT, neither MGMT nor other mechanisms of TMZ resistance (mismatch repair) precluded sensitivity to ABT-888+TMZ. Underlying mechanisms of TMZ resistance in these models are not completely understood but likely involve mechanisms independent of MGMT.

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Poly(ADP-ribose) polymerase (PARP) senses DNA breaks and facilitates DNA repair via the polyADP-ribosylation of various DNA binding and repair proteins. We explored the mechanism of potentiation of temozolomide cytotoxicity by the PARP inhibitor ABT-888. We showed that cells treated with temozolomide need to be exposed to ABT-888 for at least 17 to 24 hours to achieve maximal cytotoxicity. The extent of cytotoxicity correlates with the level of double-stranded DNA breaks as indicated by gammaH2AX levels. In synchronized cells, damaging DNA with temozolomide in the presence of ABT-888 during the S phase generated high levels of double-stranded breaks, presumably because the single-stranded DNA breaks resulting from the cleavage of the methylated nucleotides were converted into double-stranded breaks through DNA replication. As a result, treatment of temozolomide and ABT-888 during the S phase leads to higher levels of cytotoxicity. ABT-888 inhibits poly(ADP-ribose) formation in vivo and enhances tumor growth inhibition by temozolomide in multiple models. ABT-888 is well tolerated in animal models. ABT-888 is currently in clinical trials in combination with temozolomide.

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.

ABT-888 is a potent, orally bioavailable PARP-1/2 inhibitor shown to potentiate DNA damaging agents. The ability to potentiate temozolomide (TMZ) and develop a biological marker for PARP inhibition was evaluated in vivo. Doses/schedules that achieve TMZ potentiation in the B16F10 syngeneic melanoma model were utilized to develop an ELISA to detect a pharmacodynamic marker, ADP ribose polymers (pADPr), after ABT 888 treatment. ABT-888 enhanced TMZ antitumor activity, in a dose-proportional manner with no observed toxicity (44-75% tumor growth inhibition vs. TMZ monotherapy), but did not show single agent activity. Extended ABT-888 dosing schedules showed no advantage compared to simultaneous TMZ administration. Efficacy correlated with plasma/tumor drug concentrations. Intratumor drug levels correlated with a dose-proportional/time-dependent reduction in pADPr. Potentiation of TMZ activity by ABT-888 correlated with drug levels and inhibition of PARP activity in vivo. ABT-888 is in Phase 1 trials using a validated ELISA based on the assay developed here to assess pharmacological effect.

Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654), a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G(2)/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

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.

Overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1) is commonly associated with tumor maintenance, progression, and chemoresistance. We previously reported the discovery of ABT-737, a potent, small-molecule Bcl-2 family protein inhibitor. A major limitation of ABT-737 is that it is not orally bioavailable, which would limit chronic single agent therapy and flexibility to dose in combination regimens. Here we report the biological properties of ABT-263, a potent, orally bioavailable Bad-like BH3 mimetic (K(i)'s of <1 nmol/L for Bcl-2, Bcl-xL, and Bcl-w). The oral bioavailability of ABT-263 in preclinical animal models is 20% to 50%, depending on formulation. ABT-263 disrupts Bcl-2/Bcl-xL interactions with pro-death proteins (e.g., Bim), leading to the initiation of apoptosis within 2 hours posttreatment. In human tumor cells, ABT-263 induces Bax translocation, cytochrome c release, and subsequent apoptosis. Oral administration of ABT-263 alone induces complete tumor regressions in xenograft models of small-cell lung cancer and acute lymphoblastic leukemia. In xenograft models of aggressive B-cell lymphoma and multiple myeloma where ABT-263 exhibits modest or no single agent activity, it significantly enhances the efficacy of clinically relevant therapeutic regimens. These data provide the rationale for clinical trials evaluating ABT-263 in small-cell lung cancer and B-cell malignancies. The oral efficacy of ABT-263 should provide dosing flexibility to maximize clinical utility both as a single agent and in combination regimens.

Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.

Purpose: To evaluate the preclinical pharmacokinetics and antitumor efficacy of a novel orally bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor, ABT-888., Experimental Design: In vitro potency was determined in a PARP-1 and PARP-2 enzyme assay. In vivo efficacy was evaluated in syngeneic and xenograft models in combination with temozolomide, platinums, cyclophosphamide, and ionizing radiation., Results: ABT-888 is a potent inhibitor of both PARP-1 and PARP-2 with K(i)s of 5.2 and 2.9 nmol/L, respectively. The compound has good oral bioavailability and crosses the blood-brain barrier. ABT-888 strongly potentiated temozolomide in the B16F10 s.c. murine melanoma model. PARP inhibition dramatically increased the efficacy of temozolomide at ABT-888 doses as low as 3.1 mg/kg/d and a maximal efficacy achieved at 25 mg/kg/d. In the 9L orthotopic rat glioma model, temozolomide alone exhibited minimal efficacy, whereas ABT-888, when combined with temozolomide, significantly slowed tumor progression. In the MX-1 breast xenograft model (BRCA1 deletion and BRCA2 mutation), ABT-888 potentiated cisplatin, carboplatin, and cyclophosphamide, causing regression of established tumors, whereas with comparable doses of cytotoxic agents alone, only modest tumor inhibition was exhibited. Finally, ABT-888 potentiated radiation (2 Gy/d x 10) in an HCT-116 colon carcinoma model. In each model, ABT-888 did not display single-agent activity., Conclusions: ABT-888 is a potent inhibitor of PARP, has good oral bioavailability, can cross the blood-brain barrier, and potentiates temozolomide, platinums, cyclophosphamide, and radiation in syngeneic and xenograft tumor models. This broad spectrum of chemopotentiation and radiopotentiation makes this compound an attractive candidate for clinical evaluation.

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.

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.

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.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (K(i) = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses approximately 2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting.

Chk1 is the major mediator in the activation of cell-cycle checkpoints in response to a variety of genotoxic stresses. We have previously shown that inhibition of Chk1 sensitizes tumor cells to topoisomerase inhibitors such as camptothecin and doxorubicin through abrogation of cell-cycle arrest (S or G2/M checkpoints). However, it was not clear whether inhibition of Chk1 could potentiate antimetabolites, a mainstay of cancer therapy, which confer genotoxic stress through a different mechanism than topoisomerase inhibitors. 5-Fluorouracil (5-FU) is the most widely used antimetabolite in the treatment of colorectal, breast and other major types of cancers. Here we demonstrate that 5-FU activates Chk1 and induces an early S-phase arrest. Chk1 downregulation abrogates this arrest and dramatically sensitizes tumor cells to the cytotoxic effects of 5-FU. 5-FU confers S-phase arrest through Chk1-mediated Cdc25A proteolysis leading to inhibition of Cdk2. Chk1 elimination stabilizes the Cdc25A protein and results in the abrogation of the S checkpoint and resumption of DNA synthesis, which leads to excessive accumulation of double-stranded DNA breaks. As a result, downregulation of Chk1 potentiates 5-FU efficacy through induction of premature chromosomal condensation followed by apoptosis. Interestingly, the profiles of various cell-cycle markers indicate that cells progress to early M phase to induce apoptosis after checkpoint abrogation. Yet, cells fail to increase their DNA content to 4N as revealed by FACS analysis, probably due to the dramatic induction of double-stranded DNA breaks and chromosomal fragmentation. This is significantly different from the cell-cycle profiles observed in the potentiation of topoisomerase inhibitors by Chk1 siRNA, which showed mitotic progression with 4N DNA content leading to mitotic catastrophe after abrogation of the S or G2 checkpoint. Thus, our results illustrate a novel mode of checkpoint abrogation and cell death conferred by Chk1 inhibition. Additionally, we show that Chk1 deficiency potentiates 5-FU efficacy through the preferential induction of the caspase-8 pathway and subsequent caspase-3 activation. In conclusion, we have clearly demonstrated that inhibition of Chk1 not only potentiates the toxicity of conventional DNA-damaging agents such as ionizing radiation and topoisomerase inhibitors, but also enhances the toxicity of antimetabolites in cancer cell lines. This discovery reveals novel scope of checkpoint abrogation and will significantly broaden the potential application of Chk1 inhibitors in cancer therapy if they do not potentiate the toxicity of 5-FU in normal cells.

UV and ionizing radiation (IR) activate DNA damage checkpoints and induce Cdc25A degradation (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429; Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas J. (2001) Nature 410, 842-847). The degradation of Cdc25A is abrogated by caffeine, which implicates Chk1 as the potential mediator (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429). However, the involvement of Chk1 is far from clear, because caffeine is a rather nonspecific inhibitor of the ATR/Chk1 signaling pathway. Additionally, it is not known whether DNA-damaging drugs commonly used in chemotherapy, which may activate different signal transduction pathways than UV or IR, also confer Cdc25A degradation. Herein, we show that camptothecin and doxorubicin, two widely used topoisomerase inhibitors conferring S and G2 arrest, respectively, cause the degradation of Cdc25A. Using a small interfering RNA that enables the specific elimination of Chk1 expression, we show that the observed proteolysis of Cdc25A is mediated through Chk1. Moreover, Cdc25A overexpression abrogates the Chk1-mediated degradation and overcomes the doxorubicin-induced G2 arrest through dephosphorylation and activation of Cdc2/Cdk1 in a dose-dependent manner. These results suggest that: (a) Cdc25A is involved in the G2/M transition in addition to its commonly accepted effect on G1/S progression, and (b) Chk1 mediates both S and G2 checkpoint and is thus a more ubiquitous cell cycle checkpoint mediator than previously thought.

In this report, we describe the antitumor activity of A-289099, an indolyloxazoline derivative with antimitotic activity. A-289099 decreased the proliferation of a variety of cells with EC(50) values ranging from 5.1 to 12.8 nM in a P-glycoprotein-independent manner. In cultured cells, microtubules depolymerized in a time- and dose-dependent manner when treated with A-289099. In competition-binding assays, A-298099 competed with [(3)H]colchicine for binding to tubulin (K(i) = 0.65 micro M); however, it did not compete with [(3)H]paclitaxel or [(3)H]vincristine. There was an accumulation of cells in G(2)-M after treatment with A-289099 for 8 h and a subsequent increase in a subdiploid population and an increase in caspase-3 activity, indicative of apoptosis after treatment for 24 and 48 h. The antitumor activities of A-289099 were evaluated using the syngeneic M5076 murine reticulum sarcoma flank tumor model. Animals size-matched for established tumors ( approximately 350 mm(3)) were dosed p.o. (50 mg/kg every day) for 11 days starting on day 10 postinoculation. Tumors from A-289099-treated animals regressed throughout the 11-day dosing period with a percentage of the average treated-tumor-volume divided by the average vehicle-control-tumor-volume (% T/C) value of 11% after treatment for 7 days. Examination of tumor sections revealed an increase in internucleosomal DNA fragmentation or cell death within the central core after drug-treatment. A decrease in the perfusion of tumors was observed after drug-treatment that was localized primarily to the central core and closely associated with the regions of cell death. In summary, our findings indicate A-289099 is a promising, orally active tubulin-binding compound with antitumor activity in vivo.

Survivin, one of the most tumor-specific gene products, has been implicated in both anti-apoptosis and cytokinesis. However, the mechanism by which survivin regulates these two different processes is still elusive. Here, we show that survivin binds to the catalytic domain of Aurora-B. We demonstrate that in the presence of survivin, Aurora-B phosphorylates histone H3 much more efficiently than in the absence of survivin in a cell-free system. Furthermore, we confirm that cells lacking survivin due to survivin antisense oligonucleotide-treatment have lower Aurora-B kinase activity, whereas cells overexpressing survivin have higher Aurora-B kinase activity. We also provide evidence that depletion of survivin by survivin antisense oligonucleotide treatment causes significant reduction of endogenous phosphorylated histone H3 and mislocalization of Aurora-B. These results indicate that survivin stimulates Aurora-B kinase activity and helps correctly target Aurora-B to its substrates during the cell cycle, thus providing a mechanism as to how survivin exerts its function in human cells.

Drug resistance is a prevalent problem in the treatment of neoplastic disease, and the effectiveness of many clinically useful drugs is limited by the fact that they are substrates for the efflux pump, P-glycoprotein. Because there is a need for new compounds that are effective in treating drug-resistant tumors, we tested A-204197 (4-[4-acetyl-4,5-dihydro-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-N,N-dimethylbenzeneamine), a novel oxadiazoline derivative with antiproliferative properties, on cell lines that were either sensitive or resistant to known microtubule inhibitors. Cell lines that were resistant to paclitaxel, vinblastine, or colchicine were equally sensitive to A-204197 (proliferation IC50s ranging from 36 to 48 nM) despite their expression levels of P-glycoprotein. The effect of A-204197 on cell growth was associated with cell cycle arrest in G2-M, increased phosphorylation of select G2-M checkpoint proteins, and apoptosis. In competition-binding assays, A-204197 competed with [3H]-labeled colchicine for binding to tubulin (K(i) = 0.75 microM); however, it did not compete with [3H]-labeled paclitaxel. A-204197 prevented tubulin polymerization in a dose-dependent manner (IC50 = 4.5 microM) in vitro and depolymerized microtubules in a time-dependent manner in cultured cells. These findings indicate A-204197 is a promising new tubulin-binding compound with antimitotic activity that has potential for treating neoplastic diseases with greater efficacy than currently used antimitotic agents.

The serine/threonine kinases, Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma, play a critical role in preventing cancer cells from undergoing apoptosis. However, the function of individual Akt isoforms in the tumorigenicity of cancer cells is still not well defined. In the current study, we used an Akt1 antisense oligonucleotide (AS) to specifically downregulate Akt1 protein in both cancer and normal cells. Our data indicate that Akt1 AS treatment inhibits the ability of MiaPaCa-2, H460, HCT-15, and HT1080 cells to grow in soft agar. The treatment also induces apoptosis in these cancer cells as demonstrated by FACS analysis and a caspase activity assay. Conversely, Akt1 AS treatment has little effect on the cell growth and survival of normal human cells including normal human fibroblast (NHF), fibroblast from muscle (FBM), and mammary gland epithelial 184B5 cells. In addition, Akt1 AS specifically sensitizes cancer cells to typical chemotherapeutic agents. Thus, Akt1 is indispensable for maintaining the tumorigenicity of cancer cells. Inhibition of Akt1 may provide a powerful sensitization agent for chemotherapy specifically in cancer cells.

We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is detected in most common human cancers but not in adjacent normal cells. Previous studies suggest that survivin associates with the mitotic spindle and directly inhibits caspase activity. To further investigate the function of survivin, we used a survivin antisense (AS) oligonucleotide to downregulate survivin expression in normal and cancer cells. We found that inhibition of survivin expression increased apoptosis and polyploidy while decreasing colony formation in soft agar. Immunohistochemistry showed that cells without survivin can initiate the cleavage furrow and contractile ring, but cannot complete cytokinesis, thus resulting in multinucleated cells. These findings indicate that survivin plays important roles in a late stage of cytokinesis, as well as in apoptosis.

Peptidic renin inhibitors have been poorly absorbed across the intestine or rapidly eliminated by the liver and have been reported to have oral bioavailabilities of less than 2%. A peptide-based renin inhibitor, A-72517 (molecular mass of 706 daltons), was devised that has oral bioavailabilities of 8, 24, 32, and 53% in the monkey, rat, ferret, and dog, respectively. Dose-related reductions in blood pressure, plasma renin activity, and plasma angiotensin II in parallel with increased plasma drug concentrations were observed after oral administration of A-72517 to conscious, salt-depleted dogs. Thus, peptide-based molecules of sizable molecular mass can be absorbed intact into the systemic circulation of animals. These findings support the potential of peptide-based drugs for oral administration.

A series of nonpeptidic human renin inhibitors with a 4-methoxymethoxypiperidinylamide at the P4 position of the molecule exhibited slow tight binding to the enzyme. Replacement of the methoxymethoxy moiety on the piperidine ring with H, OH, methoxyethyl, propyloxy or n-butyl eliminated the effect. The inhibition was partially reversed by prolonged dialysis at 4 degrees C, arguing against formation of a covalent bond in the tightened complex.

In 150 retinoblastomas the authors found a uniform thickness of the cuff of viable retinoblastoma cells that surrounds blood vessels. The mean thickness was 98.7 microns with a standard deviation of 11.9 microns. The cross-sectional area of the cuff was negatively correlated with the mitotic activity in the cuff and positively correlated with the diameter of the central vessel. The mitotic activity in the cuff of cells was inversely related to the distance from the central blood vessel. When the cuff was divided into three concentric rings, the inner ring contained a mean of 6.2 mitotic figures, the middle ring contained a mean of 2.9 mitotic figures, and the outer ring contained a mean of 0.6 mitotic figures. This pattern of growth is similar to that observed in other rapidly growing neoplasms in humans and experimental animals. In these tumors this pattern results from reduction in oxygen tension with increased distance from the central blood vessel.

A method is given for analyzing a slope ratio assay in which a test drug is compared with a standard drug, two or more response variates being measured on each subject at each of several successively increased drug doses. The method requires all subjects to receive the same number of doses, all subjects on the same drug to receive the same doses, the ratio of corresponding doses of the two drugs to be constant over the successive increases, and response variables to be measured only once on each subject at each dose with no missing data allowed. The technique is also applicable when doses are randomly assigned, provided there is no carry-over effect between doses. For each of the J response variates, the relative potency of the test drug with respect to the standard is defined and estimated in the usual way; a 100(1-alpha)% confidence region is then obtained for the vector of the J relative potencies. A procedure is given for testing the equality of some or all of the J relative potencies; an estimator of a common relative potency is obtained by a standard multivariate least squares method. A common relative potency is of interest because the multiple outcome variables are often different indicators of a general physiologic response. The procedures in the paper are illustrated by a simple example concerning the effects of two anesthetics on children.

A study was conducted at two beaches on Lake Erie to evaluate the water sampling design for the collection of several microbiological indicator organisms in relation to day, time, and location of collection. The concentrations of these organisms were generally found to vary significantly (P less than 0.05) by the specific time of day and day of weekend that collection took place. However, the concentrations of these organisms did not vary significantly (P greater than 0.05) at various locations in the bathing area. Future studies investigating the health effects of recreational water as related to microbiological variables should be designed to collect water samples at the specific time of day and day of weekend that an individual was exposed. In addition, sampling at various locations in the bathing area should probably be considered for those beaches having poor dispersion of fecal waste sources.

In response to the November 1972 ruling of the California Supreme Court requiring complete, detailed disclosure of risks for procedures and treatments, a survey was made of the reactions of one hundred patients to this decision. A fictional man was described with a clinical diagnosis of brain tumor, and then the procedure for cerebral angiography and all of that procedure's potential complications were described. The majority of the patients surveyed responded that this complete disclosure of risks helped them in making an intelligent decision about giving consent for the procedure. However, 50 percent of these patients would have withheld consent for the procedure on learning of the complications. Although physicians would have reacted differently, it is certainly the patients' right to make the choices they did.