Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques.

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+ T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Genetic modifications designed for xenotransplantation attenuate sialoadhesin-dependent binding of human erythrocytes to porcine macrophages.

The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec-1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild-type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near-complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.

MYD88 L265P mutations identify a prognostic gene expression signature and a pathway for targeted inhibition in CLL.

The L265P somatic mutation in the Myeloid Differentiation Primary Response 88 (MYD88) gene is a recurrent mutation in chronic lymphocytic leukaemia (CLL). This mutation has functional effects in various haematological malignancies but its role in CLL remains to be fully elucidated. Here, we report that MYD88 L265P mutations are associated with mutated immunoglobulin heavy-chain gene (IGHV-M) status and that among IGHV-M patients, the presence of MYD88 L265P is associated with younger age at diagnosis. Using microarray and RNA-Seq gene expression analysis, we further observe that the MYD88 L265P mutation is associated with a distinctive gene expression signature that predicts both failure-free survival and overall survival. This association was validated in an independent cohort of patients. To determine whether MYD88 L265P mutations can be therapeutically exploited in CLL, we treated primary cells with an inhibitor of interleukin 1 receptor-associated kinase 4 (IRAK4), a critical effector of the MYD88 pathway. IRAK4 inhibition decreased downstream nuclear factor-κB signalling and cell viability in CLL cells, indicating the potential of the MYD88 pathway as a therapeutic target in CLL.

Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats.

Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein-protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease.

Acetyl-coenzyme A carboxylase inhibition reduces de novo lipogenesis in overweight male subjects: A randomized, double-blind, crossover study.

NDI-010976, an allosteric inhibitor of acetyl-coenzyme A carboxylases (ACC) ACC1 and ACC2, reduces hepatic de novo lipogenesis (DNL) and favorably affects steatosis, inflammation, and fibrosis in animal models of fatty liver disease. This study was a randomized, double-blind, placebo-controlled, crossover trial evaluating the pharmacodynamic effects of a single oral dose of NDI-010976 on hepatic DNL in overweight and/or obese but otherwise healthy adult male subjects. Subjects were randomized to receive either NDI-010976 (20, 50, or 200 mg) or matching placebo in period 1, followed by the alternate treatment in period 2; and hepatic lipogenesis was stimulated with oral fructose administration. Fractional DNL was quantified by infusing a stable isotope tracer, [1-13 C]acetate, and monitoring 13 C incorporation into palmitate of circulating very low-density lipoprotein triglyceride. Single-dose administration of NDI-010976 was well tolerated at doses up to and including 200 mg. Fructose administration over a 10-hour period stimulated hepatic fractional DNL an average of 30.9 ± 6.7% (mean ± standard deviation) above fasting DNL values in placebo-treated subjects. Subjects administered single doses of NDI-010976 at 20, 50, or 200 mg had significant inhibition of DNL compared to placebo (mean inhibition relative to placebo was 70%, 85%, and 104%, respectively). An inverse relationship between fractional DNL and NDI-010976 exposure was observed with >90% inhibition of fractional DNL associated with plasma concentrations of NDI-010976 >4 ng/mL. CONCLUSION: ACC inhibition with a single dose of NDI-010976 is well tolerated and results in a profound dose-dependent inhibition of hepatic DNL in overweight adult male subjects. Therefore, NDI-010976 could contribute considerable value to the treatment algorithm of metabolic disorders characterized by dysregulated fatty acid metabolism, including nonalcoholic steatohepatitis. (Hepatology 2017;66:324-334).

Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans.

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro and in the collagen-induced arthritis model of autoimmune disease. Recently, CC-292 has entered human clinical trials with a trial design that has provided rapid insight into safety, pharmacokinetics, and pharmacodynamics. This first-in-human healthy volunteer trial has demonstrated that a single oral dose of 2 mg/kg CC-292 consistently engaged all circulating Btk protein and provides the basis for rational dose selection in future clinical trials. This targeted covalent drug design approach has enabled the discovery and early clinical development of CC-292 and has provided support for Btk as a valuable drug target for B-cell mediated disorders.

Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine.

Designing selective inhibitors of proteases has proven problematic, in part because pharmacophores that confer potency exploit the conserved catalytic apparatus. We developed a fundamentally different approach by designing irreversible inhibitors that target noncatalytic cysteines that are structurally unique to a target in a protein family. We have successfully applied this approach to the important therapeutic target HCV protease, which has broad implications for the design of other selective protease inhibitors.

Exploring amino acids derivatives as potent, selective, and direct agonists of sphingosine-1-phosphate receptor subtype-1.

In the quest to discover a potent and selective class of direct agonists to the sphingosine-1-phosphate receptor, we explored the carboxylate functional group as a replacement to previously reported lead phosphates. This has led to the discovery of potent and selective direct agonists with moderate to substantial in vivo lymphopenia. The previously reported selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) in the phenylamide and phenylimidazole scaffolds were crucial to obtaining selectivity for S1P receptor subtype 1 over 3.

Extensive germline genome engineering in pigs.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

Exploration of amino alcohol derivatives as novel, potent, and highly selective sphingosine-1-phosphate receptor subtype-1 agonists.

In pursuit of a potent and highly selective sphingosine-1-phosphate receptor agonists with an improved in vivo conversion of the precursor to the active phospho-drug, we have utilized previously reported phenylamide and phenylimidazole scaffolds to identify a selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) within both pharmacophores. SEM and SEO have allowed for over 100 to 500-fold improvement in selectivity for S1P receptor subtype 1 over subtype 3. Utility of SEM and SEO and further SAR study allowed for discovery of a potent and selective preclinical candidate PPI-4955 (21b) with an excellent in vivo potency and dose responsiveness and markedly improved overall in vivo pharmacodynamic properties upon oral administration.

Synthesis and evaluation of arylalkoxy- and biarylalkoxy-phenylamide and phenylimidazoles as potent and selective sphingosine-1-phosphate receptor subtype-1 agonists.

In pursuit of potent and selective sphingosine-1-phosphate receptor agonists, we have utilized previously reported phenylamide and phenylimidazole scaffolds to explore extensive side-chain modifications to generate new molecular entities. A number of designed molecules demonstrate good selectivity and excellent in vitro and in vivo potency in both mouse and rat models. Oral administration of the lead molecule 11c (PPI-4667) demonstrated potent and dose-responsive lymphopenia.

Synthesis and evaluation of alkoxy-phenylamides and alkoxy-phenylimidazoles as potent sphingosine-1-phosphate receptor subtype-1 agonists.

In the design of potent and selective sphingosine-1-phosphate receptor agonists, we were able to identify two series of molecules based on phenylamide and phenylimidazole analogs of FTY-720. Several designed molecules in these scaffolds have demonstrated selectivity for S1P receptor subtype 1 versus 3 and excellent in vivo activity in mouse. Two molecules PPI-4621 (4b) and PPI-4691 (10a), demonstrated dose responsive lymphopenia, when administered orally.

Inhibition of Acetyl-CoA Carboxylase by Phosphorylation or the Inhibitor ND-654 Suppresses Lipogenesis and Hepatocellular Carcinoma.

The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.

A novel methionine aminopeptidase-2 inhibitor, PPI-2458, inhibits non-Hodgkin's lymphoma cell proliferation in vitro and in vivo.

PURPOSE: Fumagillin and related compounds have potent antiproliferative activity through inhibition of methionine aminopeptidase-2 (MetAP-2). It has recently been reported that MetAP-2 is highly expressed in germinal center B cells and germinal center-derived non-Hodgkin's lymphomas (NHL), suggesting an important role for MetAP-2 in proliferating B cells. Therefore, we determined the importance of MetAP-2 in normal and transformed germinal center B cells by evaluating the effects of MetAP-2 inhibition on the form and function of germinal centers and germinal center-derived NHL cells. EXPERIMENTAL DESIGN: To examine the activity of PPI-2458 on germinal center morphology, spleen sections from cynomolgus monkeys treated with oral PPI-2458 were analyzed. Antiproliferative activity of PPI-2458 was assessed on germinal center-derived NHL lines in culture. A MetAP-2 pharmacodynamic assay was used to determine cellular MetAP-2 inhibition following PPI-2458 treatment. Finally, inhibition of MetAP-2 and proliferation by PPI-2458 was examined in the human SR NHL line in culture and in implanted xenografts. RESULTS: Oral PPI-2458 caused a reduction in germinal center size and number in lymphoid tissues from treated animals. PPI-2458 potently inhibited growth (GI(50) = 0.2-1.9 nmol/L) of several NHL lines in a manner that correlated with MetAP-2 inhibition. Moreover, orally administered PPI-2458 significantly inhibited SR tumor growth, which correlated with inhibition of tumor MetAP-2 (>85% at 100 mg/kg) in mice. CONCLUSIONS: These results show the potent antiproliferative activity of PPI-2458 on NHL lines in vitro and oral antitumor activity in vivo and suggest the therapeutic potential of PPI-2458 as a novel agent for treatment of NHL should be evaluated in the clinical setting.

Inhibition of melanoma tumor growth by a pharmacological inhibitor of MetAP-2, PPI-2458.

Over the past few decades, melanoma has shown the fastest growing incidence rate of all cancers. This malignancy is clinically defined by its potential to rapidly metastasize, and advanced metastatic melanomas are highly resistant to existing therapeutic regimens. Here, we report that PPI-2458, a novel, orally active agent of the fumagillin class of irreversible methionine aminopeptidase-2 (MetAP-2) inhibitors, potently inhibited the proliferation of B16F10 melanoma cells in vitro, with a growth inhibitory concentration 50% (GI50) of 0.2 nM. B16F10 growth inhibition was correlated with the inhibition of MetAP-2 enzyme, in a dose-dependent fashion, as determined by a pharmacodynamic assay, which measures the amount of uninhibited MetAP-2 following PPI-2458 treatment. Prolonged exposure of B16F10 cells to PPI-2458 at concentrations of up to 1 microM, 5,000-fold above the GI50, did not alter their sensitivity to PPI-2458 growth inhibition and no drug resistance was observed. Moreover, prolonged exposure to this agent induced melanogenesis, concomitant with the elevated expression of the melanocyte-specific enzymes tyrosinase and tyrosinase-related proteins (TRP) 1 and 2, a morphological feature associated with differentiated melanocytes. PPI-2458, when administered orally (p.o.), significantly inhibited B16F10 tumor growth in mice in a dose-dependent fashion, with a maximum inhibition of 62% at 100 mg/kg. This growth inhibition was directly correlated to the amount of irreversibly inhibited MetAP-2 (80% at 100 mg/kg PPI-2458) in tumor tissue. These data demonstrate that PPI-2458 has potent antiproliferative activity against B16F10 cells in vitro and in vivo, and that both activities are directly correlated with levels of MetAP-2 enzyme inhibition. This antiproliferative activity, coupled with additional observations from studies in vitro (absence of detectable resistance to PPI-2458 and induction of morphological features consistent with differentiated melanocytes), provides a rationale for assessing the therapeutic potential of PPI-2458 in the treatment of melanoma.

Alphavbeta3 integrin antagonist S247 decreases colon cancer metastasis and angiogenesis and improves survival in mice.

Members of the integrin family influence several aspects of tumor progression and metastasis, including cell survival, proliferation, and angiogenesis. Specific integrins such as alpha(v)beta(3) and alpha(v)beta(5) are involved in regulating endothelial cell function, and thus angiogenesis. We evaluated the effect of the alpha(v)beta(3)/alpha(v)beta(5) integrin antagonist S247 on the growth and angiogenesis of colon cancer liver metastases in an orthotopic murine model. Murine colon cancer cells were injected into the spleens of BALB/c mice to produce liver metastases. On day 7, miniature osmotic pumps were implanted into the subcutis to continuously infuse either saline or 70 mg/kg/day S247. All mice were sacrificed when control mice became moribund. Mice that received S247 developed significantly fewer liver metastases than did controls (P < 0.05). Using the same model, a subsequent survival study was performed. Mice were sacrificed when moribund as determined by an observer blinded to the treatment given. Treatment with S247 significantly prolonged overall survival (P < 0.05). Interestingly, primary tumors in the spleen were the cause of death in the S247-treated group as S247 appeared to have little effect on these tumors. Immunohistochemical staining demonstrated a significant reduction of vessels in liver metastases of S247-treated mice (P < 0.001), a significant increase in endothelial cell apoptosis (P < 0.05), and a significant decrease in pericyte coverage (P < 0.0001). To determine the role of S247 on angiogenesis, we examined the effect of S247 in vitro on human umbilical vein endothelial cells (HUVECs) and human vascular smooth muscle cells (hVSMCs). The addition of S247 to HUVECs and hVSMCs growing on vitronectin-coated flasks and in Matrigel significantly impaired cell growth and colony formation, respectively (P < 0.05). Furthermore, S247 completely inhibited the attachment of HUVECs and hVSMCs and increased apoptosis by six- to 9fold compared with controls. In in vitro invasion assays, S247-treated cells demonstrated decreased migration (P < 0.05). In conclusion, S247 demonstrated significant antimetastatic and antiangiogenic activity and impaired both endothelial and hVSMC/pericyte function in vitro and in vivo. The use of agents such as integrin antagonists that target multiple cell types involved in angiogenesis may be a more effective method of inhibiting angiogenesis than agents targeting only the endothelial cells.

Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models.

Continuous de novo fatty acid synthesis is a common feature of cancer that is required to meet the biosynthetic demands of a growing tumor. This process is controlled by the rate-limiting enzyme acetyl-CoA carboxylase (ACC), an attractive but traditionally intractable drug target. Here we provide genetic and pharmacological evidence that in preclinical models ACC is required to maintain the de novo fatty acid synthesis needed for growth and viability of non-small-cell lung cancer (NSCLC) cells. We describe the ability of ND-646-an allosteric inhibitor of the ACC enzymes ACC1 and ACC2 that prevents ACC subunit dimerization-to suppress fatty acid synthesis in vitro and in vivo. Chronic ND-646 treatment of xenograft and genetically engineered mouse models of NSCLC inhibited tumor growth. When administered as a single agent or in combination with the standard-of-care drug carboplatin, ND-646 markedly suppressed lung tumor growth in the Kras;Trp53-/- (also known as KRAS p53) and Kras;Stk11-/- (also known as KRAS Lkb1) mouse models of NSCLC. These findings demonstrate that ACC mediates a metabolic liability of NSCLC and that ACC inhibition by ND-646 is detrimental to NSCLC growth, supporting further examination of the use of ACC inhibitors in oncology.

Selective interleukin-1 receptor-associated kinase 4 inhibitors for the treatment of autoimmune disorders and lymphoid malignancy.

Pathological activation of the Toll-like receptor signaling adaptor protein MYD88 underlies many autoimmune and inflammatory disease states. In the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), the oncogenic MYD88 L265P mutation occurs in 29% of cases, making it the most prevalent activating mutation in this malignancy. IRAK4 kinase accounts for almost all of the biological functions of MYD88, highlighting IRAK4 as a therapeutic target for diseases driven by aberrant MYD88 signaling. Using innovative structure-based drug design methodologies, we report the development of highly selective and bioavailable small molecule IRAK4 inhibitors, ND-2158 and ND-2110. These small molecules suppressed LPS-induced TNF production, alleviated collagen-induced arthritis, and blocked gout formation in mouse models. IRAK4 inhibition promoted killing of ABC DLBCL lines harboring MYD88 L265P, by down-modulating survival signals, including NF-κB and autocrine IL-6/IL-10 engagement of the JAK-STAT3 pathway. In ABC DLBCL xenograft models, IRAK4 inhibition suppressed tumor growth as a single agent, and in combination with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or the Bcl-2 inhibitor ABT-199. Our findings support pharmacological inhibition of IRAK4 as a therapeutic strategy in autoimmune disorders, in a genetically defined population of ABC DLBCL, and possibly other malignancies dependent on aberrant MYD88 signaling.

Functional overlap and cooperativity among alphav and beta1 integrin subfamilies during skin angiogenesis.

Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.

A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.