Semisynthetic Analogues Research Articles

Targeting Apoptosis Pathway with Natural Terpenoids: Implications for Treatment of Breast and Prostate Cancer

Terpenoids represent a large and diverse class of naturally occurring compounds found in a variety of fruits, vegetables and medicinal plants. Structurally some of the terpenoids are similar to human hormones. A diet rich in terpenoids is inversely related with the risk of chronic diseases including cancers. Breast and prostate cancers are hormone-related diseases and the second leading cause of female and male cancer mortality. Diterpenoid paclitaxel, and its semi-synthetic analogue docetaxel, have entered clinical use against established breast and prostate cancers. Here we reviewed potential molecular targets and biological properties of natural terpenoids, including monoterpenoids, diterpenoids, triterpenoids and tetraterpenoids, and their applications in treatment of human breast and prostate cancers. These terpenoids are able to inhibit tumor cell proliferation and induce tumor cell death by inhibiting multiple cancer-specific targets including the proteasome, NF-kappaB, and antiapoptotic protein Bcl-2. The efficacy of these terpenoids against breast or prostate cancer cells, as demonstrated in pre-clinical studies support clinical application of these naturally occurring terpenoids in treatment of hormone-related human cancers.

Activity of topotecan given intravenously for 5 days every three weeks in patients with advanced non-small cell lung cancer pretreated with platinum and taxanes: a phase II study

Topotecan, a semi-synthetic camptothecin analogue with topoisomerase I interaction, has shown to be an active agent in the treatment of advanced refractory lung cancer. This paper describes the authors' experience with this drug when used as a single agent in patients (pts) with advanced non-small cell lung cancer (NSCLC) refractory to platinum- and taxane-containing chemotherapy regimens. Thirty-five patients with NSCLC refractory to previous chemotherapy and KI ≥ 60% were included in the study. Their characteristics are as follows: median age of 52 years (range 43-69) and Karnofsky PS of 70 (60-80); 27 were male and 8 were female. Twenty-one (60%) patients had adenocarcinoma; eleven (31.4%), squamous cell, and three (8.5%), undifferentiated carcinoma. There was a median of two disease sites and two prior chemotherapy regimens. Topotecan was administered at a dose of 1.25 mg/m(2) I.V. daily for 5 days, repeated every 21 days until disease progression, maximal response, or intolerable toxicity. After 73 cycles, patients received a median of 2 treatment cycles (1-9). All patients except one were considered evaluable for toxicity; eight episodes (24%) of nausea/vomiting and two episodes (6%) of grade 1-2 asthenia, respectively, were reported. Four (12%) patients developed grade 1-2 anemia and two (6%) subjects suffered grade 3 anemia. Seven (21%) patients had grade 1-2 neutropenia and one (3%) presented grade 5 neutropenia. In 33 patients evaluable for activity of the 35 subjects included in the study; one (2.8%) presented a partial response; nine (25.7%) had stable disease, and 23 (65.7%) exhibited disease progression. Median time to progression and overall survival were 54 (12-210) and 70 (12-324) days, respectively. Intravenous topotecan at that dose and administration schedule displays scant activity in terms of response rate in individuals with advanced NSCLC previously treated with platinum and taxanes. The role and usefulness of chemotherapy in this setting warrants further investigation and confirmation through comparative studies.

Semi-synthetic analogues of thiostrepton delimit the critical nature of tail region modifications in the control of protein biosynthesis and antibacterial activity

We report the successful production of selectively-modified tail analogues of the natural product antibiotic thiostrepton, which have been used to evaluate the critical nature of this section of the antibiotic to its inhibition of protein synthesis. This work highlights the tail region as a critical area for future semi-synthetic or synthetically bioengineered thiostrepton derivatives.

Analysis of overall survival from a phase III study of ixabepilone plus capecitabine versus capecitabine in patients with MBC resistant to anthracyclines and taxanes

Limited proven treatment options exist for patients with metastatic breast cancer (MBC) resistant to anthracycline and taxane treatment. Ixabepilone, a novel semisynthetic analog of epothilone B, has demonstrated single-agent activity in MBC resistant to anthracyclines and taxanes. In combination with capecitabine in a phase III trial (CA163-046) in this setting, ixabepilone prolonged progression-free survival and increased objective response rate relative to capecitabine (Thomas et al. J Clin Oncol 25:5210-5217, 2007). Here, we report the results of overall survival (OS), a secondary efficacy endpoint from the CA163-046 trial. Seven hundred fifty-two patients with MBC resistant to anthracyclines and taxanes were randomized to ixabepilone (40 mg/m(2) intravenously on day 1 of a 21-day cycle) plus capecitabine (2,000 mg/m(2) orally on days 1 through 14 of a 21-day cycle) or capecitabine alone (2,500 mg/m(2) on the same schedule). Patients receiving ixabepilone plus capecitabine treatment had a median survival of 12.9 months compared to 11.1 months for patients receiving capecitabine alone (HR = 0.9; 95%CI: 077-1.05; P = 0.19). This observed increase in median OS favored the combination; however, the difference was not statistically significant. Predefined subset analyses showed a clinically meaningful increase in OS in KPS 70-80 patients receiving ixabepilone plus capecitabine (HR = 0.75; 95% CI: 0.58-0.98). Ixabepilone plus capecitabine did not show a significant improvement in survival compared to capecitabine alone in patients with MBC resistant to anthracyclines and taxanes. The observed differences in survival favored the combination arm. A clinical benefit was also seen in patients in the KPS 70-80 subgroup.

Blockage of chloride channels and anion transporters with pesticidal natural products and their synthetic analogs

Ligand-gated chloride channels mediate a variety of functions in excitable membranes of nerve and muscle in insects, and have a long history as targets for neurotoxic insecticides. Recent findings from our laboratory confirm that the natural product silphinenes and their semi-synthetic analogs share a mode of action with the established ligand-gated chloride channel antagonist, picrotoxinin. The silphinenes are non-selective, being roughly equipotent on insect and mammalian receptors, but also possess lethal and neurotoxic effects on a dieldrin-resistant strain of Drosophila melanogaster. These findings suggest that silphinenes act on insect GABA receptors in a way that is different from picrotoxinin, and it is possible that resistant insect populations in the field could be controlled with insecticidal compounds derived from the silphinenes. Voltage-gated chloride channels and anion transporters provide additional classes of validated targets for insecticidal/nematicidal action. Anion transporter blockers are toxic to insects via an action on the gut, and RNAi studies implicate voltage-gated chloride channels in nematode muscle as another possible target. There was no cross resistance to DIDS in a dieldrin-resistant strain of Drosophilamelanogaster, and no evidence for neurotoxicity. The potent paralytic actions of anion transporter blockers against nematodes, and stomach poisoning activity against lepidopteran larvae suggests they are worthy of further investigation as commercial insecticidal/nematicidal agents.

Lyophilised liposome‐based formulations of α‐tocopheryl succinate: Preparation and physico‐chemical characterisation

α‐Tocopheryl succinate (α‐TOS) is a semisynthetic analogue of α‐tocopherol with selective toxicity to the cancer cells and anticancer activity in vivo. Yet, no suitable formulation of α‐TOS for medical application has been reported. Various formulations, for example, solutions in organic solvents, oil emulsions and vesicules prepared by spontaneous vesiculation, polyethylene glycol conjugates and liposomes of various compositions have been tested. We developed and characterised a stable lyophilised liposome‐based α‐TOS formulation. α‐TOS (15 mol%) was incorporated into large oligolamellar vesicles (OLVs) composed of soy phosphatidylcholine (SPC) by the method of lipid film hydration followed by extrusion through polycarbonate filters. Stabilised liposomal formulation was prepared by lyophilisation in the presence of sucrose (molar ratio lipid/sucrose, 1:5). The size distribution of the liposomes (130–140 nm, polydispersity index 0.14) as well as the stable lipid and α‐TOS contents were preserved during storage in the lyophilised form at 2–8°C for at least 6 months. The data indicate good physical and chemical stability of the lyophilised preparation of α‐TOS liposomes that can be used in clinical medicine. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2434–2443, 2010

Flavonoids from Artemisia annua L. as Antioxidants and Their Potential Synergism with Artemisinin against Malaria and Cancer

Artemisia annua is currently the only commercial source of the sesquiterpene lactone artemisinin. Since artemisinin was discovered as the active component of A. annua in early 1970s, hundreds of papers have focused on the anti-parasitic effects of artemisinin and its semi-synthetic analogs dihydroartemisinin, artemether, arteether, and artesunate. Artemisinin per se has not been used in mainstream clinical practice due to its poor bioavailability when compared to its analogs. In the past decade, the work with artemisinin-based compounds has expanded to their anti-cancer properties. Although artemisinin is a major bioactive component present in the traditional Chinese herbal preparations (tea), leaf flavonoids, also present in the tea, have shown a variety of biological activities and may synergize the effects of artemisinin against malaria and cancer. However, only a few studies have focused on the potential synergistic effects between flavonoids and artemisinin. The resurgent idea that multi-component drug therapy might be better than monotherapy is illustrated by the recent resolution of the World Health Organization to support artemisinin-based combination therapies (ACT), instead of the previously used monotherapy with artemisinins. In this critical review we will discuss the possibility that artemisinin and its semi-synthetic analogs might become more effective to treat parasitic diseases (such as malaria) and cancer if simultaneously delivered with flavonoids. The flavonoids present in A. annua leaves have been linked to suppression of CYP450 enzymes responsible for altering the absorption and metabolism of artemisinin in the body, but also have been linked to a beneficial immunomodulatory activity in subjects afflicted with parasitic and chronic diseases.

Abstract 3715: Potent cytotoxins and their prodrugs

Abstract Epothilones, dolastatins (auristatins), tubulysins, duocarmycins, and enediynes are among the most potent anticancer agents, which inhibit cell growth of cancer cells with a low or subnanomolar activity. Epothilones inhibit tumor growth by stabilizing the microtubules in a manner similar to Taxol. There are hundreds of natural and synthetic epothilones known, and some of them are undergoing clinical trials. One semisynthetic analog, ixabepilone, has also been approved for the treatment of metastatic breat cancer. Similarly, there are numerous dolastatins, their synthetic analogs - auristatins, and tubulysins known, of which auristatins are currently being developed by Seattle Genetics, Inc. These compounds also inhibit microtubules, however unlike epothilones and Taxol, they function by destabilizing the microtubules. The other compounds, duocarmycins and enediynes, cause toxicity by interacting with DNA; duocarmycins alkylate DNA whereas enediynes cleave. At least one enediyne antibody conjugate, Mylotarg (or Gemtuzumab ozogamycin), prepared from calicheamicin and targeted to CD33, is an approved therapy for the treatment of acute myeloid leukemia. Not surprisingly, most of these naturally occurring compounds or their synthetic analogs also cause nonselective toxicities to the rapidly dividing normal cells, including those from hair follicles, bone marrow, and gastrointestinal lining. To minimize such indiscriminate toxicity, these compounds can be conjugated to tumor-targeting antibodies, as the case is with auristatins and calicheamicin. While we are developing antibody conjugates, we have also been focusing on a prodrug approach, in that the prodrugs can be activated selectively in tumor and tumor microenvironments by the endogenous tumor-associated proteases. Alternatively, an exogenous enzyme, such as aldolase antibody, 38C2, that are directed using low molecular weight inhibitors of the cell surface receptors overexpressed in tumor cells and tumor microenvironments, can also catalyze the prodrug activation. In this presentation, we will be discussing our studies toward the synthesis and evaluation of many of these cytotoxins and/or their prodrugs, especially those belonging to the auristatins. The prodrugs are targeting to integrins and/or proteases, and activated by the aldolase antibody or the tumor-associated proteases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3715.

Abstract 751: Synthesis of irofulven analogs

Abstract Irofulven (NSC 683863), a semi-synthetic analog of the fungal product illudin S, that has been evaluated in a variety of phase I and II clinical trials. Promising results were noted in clinical trials against a variety of solid cancers including colon, sarcoma, prostate, liver, ovarian and pancreas. Irofulven in a randomized phase IIB trial increased median overall lifespan in patients with metastatic taxane-refractory prostate cancer [HR = 0.69]. As irofulven was the first member of the acylfulvene class of agents to be created, we synthesized several hundred analogs for further testing. Modifications were specifically made to 7 sites of the irofulven molecule including the cyclopropane ring, the chiral center, the carbonyl group, the tertiary hydroxyl site, and several nucleophilic sites. Modifications at several sites completely eliminated both the cytotoxicity and antitumor activity of the analog. Analogs with modifications at one site retained cytotoxic activity, as well as in vivo toxicity in rodents, but surprisingly eliminated antitumor activity in xenograft models. Analogs in which the tertiary hydroxyl group of irofulven was replaced with specific functional groups were noted to have an increase in tumor selectivity as noted by both cell culture and xenograft studies. Specific functional groups noted to be favorable for increased activity included carbamate, sulfonamide, and urea moieties. Additional derivatives of each of these specific functional groups were synthesized and tested in a variety of cell culture and xenograft studies including multidrug resistant models. These studies identify molecular sites critical for the antitumor activity of acylfulvenes, as well as identify promising new analogs for further investigation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 751.

Tigecycline – how powerful is it in the fight against antibiotic-resistant bacteria?

Tigecycline is a semisynthetic analogue of earlier tetracyclines and represents the first member of a novel class of antimicrobials - glycylcyclines - recently approved for clinical use. It is active against a broad range of gram-negative and gram-positive bacterial species including clinically important multidrug-resistant nosocomial and community-acquired bacterial pathogens. The exact molecular basis of tigecycline action is not clear at present, although similarly to the tetracyclines, it has been shown to inhibit the translation elongation step by binding to the ribosome 30S subunit and preventing aminoacylated tRNAs to accommodate in the ribosomal A site. Importantly, tigecycline overcomes the action of ribosomal protection proteins and is not a substrate for tetracycline efflux pumps of most bacteria - well-known and prevalent cellular mechanisms of microbial tetracycline resistance. The present review summarizes current knowledge on the molecular mechanism of the tigecycline action, antibacterial activity against various bacteria, clinical application, development of resistance to glycylcyclines.

Abstract A13: Selective role of iNOS reveals its negative regulatory influence on the expression of TNFR1, NF-κB and other cell survival signaling events during a P19-induced apoptosis in HL-60 cells

Abstract P19, a semi-synthetic analog of sesquiterpene lactone ‘Parthenin’, is a promising template to develop into novel anti-cancer leads. P19 induced robust cell death in human leukemia cell lines compared to normal cells when observed after 48h treatment with IC50 of 3.5 μM in HL-60 cells to that of 35 μM in human normal gingival fibroblast cells. Mechanistic studies into HL-60 cells death after 6 h exposure to 10 μM P19 were investigated. It killed cells by apoptosis consequent to robust generation of NO through enhanced expression of iNOS. N-acetyl-L-cysteine (NAC) and s-methyl isothiourea (sMIT) suppressed apoptosis equipotently by inhibiting endogenous NO formation. P19 disrupted mitochondrial functions by altering Bax/Bcl2 ratio, cytosolic Bid loss, Drp1 over expression, Bax and cytochrome c translocation leading predominantly to caspase-9 activation. P19 also translocated AIF from mitochondria to the nucleus which may further potentiate caspases-independent cell death, because pan caspase inhibitor offered incomplete protection. On the contrary, activation of caspase-8 was augmented with concomitant selective enhanced expression of apical death receptor DR4 rather than TNFR1 and FASR. Activation of pre-executioner caspases positively regulated the activation of caspase-3 leading to PARP-1 cleavage and therefore, DNA fragmentation. Down-regulation of activator TNFR1 and transactivator PARP-1 of NF-κB with attendant decreased expression of both the subunits p50 and p65 of NF-κB argue against the plausible involvement of NF-κB as a transcription factor in the induction of iNOS. Further, P19 potently inhibited pro-survival proteins pSTAT-3 and survivin that are overtly expressed in cancer cells. The multimodal proapoptotic features of P19 raises its potential usefulness as a promising anti-cancer therapeutic. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A13

Andrographolide as a template in the synthesis of new anticancer agents with potentially novel molecular targets

Using andrographolide (AGP) as a template, a series of semisynthetic analogues was synthesised for the discovery of new anticancer agent(s) with improved antitumour selectivity and potency. SRJ09, a lead compound displayed remarkable activity in the National Cancer Institute (NCI) of USA in vitro anticancer screen, particularly against breast and colon cancers. The treatment of MCF‐7 breast and HCT‐116 colon cancer cells with SRJ09 induced a G1 phase cell cycle arrest through the down‐regulation of CDK‐4 expression with concomitant up‐regulation of p21 expression and without affecting the cyclin D1 expression. SRJ09 induced apoptotic cell death in both cells in a p53‐ and bcl‐2‐independent manner, implicating extrinsic apoptotic pathway. The compound showed impressive in vivo activity by delaying HCT‐116 tumour xenograft growth in nude mice. Recently, a derivative of SRJ09, SRS07 showed vast improvement in the NCI anticancer activity screen compared with the parent compound. Additionally, NCI's in silico COMPARE and SOM analyses revealed that the new AGP derivatives have potentially novel molecular target(s). These findings although preliminary they are highly suggestive SRS07 have a great potential to be developed into a clinical anticancer agent. This research is funded by the Ministry of Science, Technology and Innovation of Malaysia (MOSTI) under the IRPA and Science Fund grant schemes.

Inhibition of Tubulogenesis and of Carcinogen‐mediated Signaling in Brain Endothelial Cells Highlight the Antiangiogenic Properties of a Mumbaistatin Analog

A better understanding of the metabolic adaptations of the vascular endothelial cells (EC) that mediate tumor vascularization would help the development of new drugs and therapies. Novel roles in cell survival and metabolic adaptation to hypoxia have been ascribed to the microsomal glucose-6-phosphate translocase (G6PT). While antitumorigenic properties of G6PT inhibitors such as chlorogenic acid (CHL) have been documented, those of the G6PT inhibitor and semi-synthetic analog AD4-015 of the polyketide mumbaistatin are not understood. In the present study, we evaluated the in vitro antiangiogenic impact of AD4-015 on human brain microvascular endothelial cells (HBMEC), which play an essential role as structural and functional components in tumor angiogenesis. We found that in vitro HBMEC migration and tubulogenesis were reduced by AD4-015 but not by CHL. The mumbaistatin analog significantly inhibited the phorbol 12-myristate 13-acetate (PMA)-induced matrix-metalloproteinase (MMP)-9 secretion and gene expression as assessed by zymography and RT-PCR. PMA-mediated cell signaling leading to cyclooxygenase (COX)-2 expression and IkappaB downregulation was also inhibited, further confirming AD4-015 as a cell signaling inhibitor in tumor promoting conditions. G6PT functions may therefore account for the metabolic flexibility that enables EC-mediated neovascularization. This process could be specifically targeted within the vasculature of developing brain tumors by G6PT inhibitors.

Monoketocholate can decrease transcellular permeation of methotrexate across Caco-2 cell monolayers and reduce its intestinal absorption in rat

Abstract Objectives Bile salts have been shown to decrease the absorption of methotrexate in the rat intestine by an unknown mechanism. We aimed to examine this effect. Methods We assessed apical-to-basolateral (AP-BL) permeation of methotrexate (5 μM) across Caco-2 cell monolayers pretreated with various concentrations (0, 0.25, 0.5, 1, 3 and 5 mm) of sodium cholate or its semisynthetic analogue, sodium 12-monoketocholate. We also determined the effect of orally administered 12-monoketocholate on the intestinal absorption of methotrexate in rats to evaluate a possible in-vitro–in-vivo correlation. Key findings It was found that sodium cholate and sodium 12-monoketocholate decreased the AP-BL permeation of methotrexate at low concentrations (maximal inhibition at 0.25 and 1 mm, respectively) and increased it at higher concentrations. Determination of [14C] mannitol permeation and electrical resistance of monolayers during experiments showed that membrane integrity was not compromised at low concentrations of bile salts but was disrupted at higher concentrations. Subsequently, we examined the effect of the simultaneous oral administration of sodium 12-monoketocholate (4, 20, 40 and 80 mg/kg) on the intestinal absorption of methotrexate in rats after an oral dose (5 mg/kg). The pharmacokinetic study showed that 12-monoketocholate at 4 and 20 mg/kg did not change the methotrexate area under the serum concentration–time curve whereas sodium 12-monoketocholate at 40 and 80 mg/kg significantly reduced it. Conclusions Sodium 12-monoketocholate appears to decrease the intestinal absorption of methotrexate in rats by inhibition of transcellular active transport.

Spectaline, cassine and semi-synthetic analogues as potential candidate drugs for the treatment of Alzheimer disease

Este artigo descreve o estudo fitoquimico de Senna spectabilis que conduziu ao isolamento de espectalina, cassina e outros alcaloides piperidinicos, a obtencao de analogos semi-sinteticos da espectalina e cassina, e a avaliacao de suas propriedades farmacologicas e toxicologicas que resultaram no estabelecimento de dois candidatos a farmacos para o tratamento da doenca de Alzheimer (DA). DOI: 10.5935/1984-6835.20100005

Larvicidal and Structure–Activity Studies of Natural Phenylpropanoids and Their Semisynthetic Derivatives against the Tobacco Armyworm Spodoptera litura (Fab.) (Lepidoptera: Noctuidae)

The larvicidal activity of 18 phenylpropanoids, 1-18, including phenylpropenoate, phenylpropenal, phenylpropene, and their semisynthetic analogues, were evaluated against the tobacco armyworm, Spodoptera litura (Fab.), to identify promising structures with insecticidal activity. Amongst various phenylpropanoids, isosafrole, a phenylpropene, showed the best activity, with an LC(50) value of 0.6 microg/leaf cm(2), followed by its hydrogenated derivative dihydrosafrole (LC(50)=2.7 microg/leaf cm(2)). The overall larvicidal activity of various phenylpropene derivatives was observed in the following order: isosafrole (6)>dihydrosafrole (16)>safrole (12)>anethole (4)>methyl eugenol (11)>eugenol (13)>beta-asarone (8)>dihydroasarone (18)>dihydroanethole (15). Dihydrosafrole might be a promising compound, although presenting a lower larvicidal activity than isosafrole, because of its better stability and resistance to oxidative degradation (due to the removal of the extremely reactive olefinic bond) in comparison to isosafrole. Such structure-activity relationship studies promote the identification of lead structures from natural sources for the development of larvicidal products against S. litura and related insect pests.

Natural Compounds as Antagonists of Canonical Wnt/β-Catenin Signaling

Overwhelming evidence that implicates aberrant activations of the canonical Wnt/β-catenin signaling pathway in oncogenesis and cancer progression has emerged in recent times. Accordingly, disruption of this signaling pathway offers an opportunity for effective cancer therapy. Despite various approaches to target components of the Wnt/β-catenin signaling pathway, safe and efficacious therapeutic agents have yet to be identified. As the search for novel small molecule inhibitors remains pressing and continues, natural products, which are traditionally excellent sources of lead compounds in the drug discovery and development process, are gaining prominence as effective antagonists of the signaling pathway. In this review, we will provide a comprehensive summary on the current use of natural compounds ranging from plant-derived polyphenols, anti-malarial artemisinins to several antimicrobial products as Wnt therapeutics and the development of more efficacious semi-synthetic analogues using these compounds as lead structures. We will also discuss pertinent issues that are limiting the rate of drug development based on these compounds and the chemical-biological significance across the diverse classes of natural compounds. The findings reviewed here should inspire and motivate more efforts to harness rich and diverse plant and other natural sources in the search for new leads as effective antagonists of the Wnt/β-catenin pathway. Keywords: Wnt/β-catenin, inhibitors, antagonists, natural compounds, drug development

Novel antimicrobial lipopeptides with long in vivo half-lives

The pharmacokinetic (PK) properties of novel lipopeptides (semi-synthetic amphomycin analogues) with potent activity against Gram-positive organisms were evaluated in mice and rats following single intravenous (i.v.) and oral administration. Following oral administration at 50 mg/kg, plasma concentrations of amphomycin analogues were <0.3–0.9 μg/mL, suggesting that oral availability was low. Following i.v. administration (5–10 mg/kg), the majority of lipopeptides demonstrated a long half-life (5.2–8.0 h in mice and 4.6–7.1 h in rats), low clearance (0.005–0.016 mL/min in mice and 0.050–0.084 mL/min in rats) and a volume of distribution indicative of extracellular penetration (0.118–0.339 L/kg in mice and 0.121–0.133 L/kg in rats). The area under the plasma concentration–time curve extrapolated to infinity (AUC 0−∞) for a 10 mg/kg i.v. dose was determined to be 601.7–791.7 μg h/mL in mice and 511.1–850.2 μg h/mL in rats. The long half-life and low clearance observed with these novel lipopeptides indicate that drug serum concentrations will remain above the target minimal inhibitory concentration (MIC) levels for significant periods of time. When combined with the potent efficacy of these agents against Gram-positive organisms, the results of the present study support further development of these lipopeptide analogues towards clinical evaluation.

Abstract A1: The physiological form of MetAP2 can be inhibited through binding to either of the two active-site metals

Abstract Methionine aminopeptidases (MetAP) are metalloenzymes that remove the N-terminal initiator methionine from newly synthesized polypeptides allowing essential post-translational modifications such as acetylation and myristoylation to take place. MetAP2, one of the two eukaryotic forms of the enzyme, was identified as the target of fumagillin, a natural product with anti-angiogenic properties that inhibits the proliferation of endothelial cells. Clinical activity has been seen for a semi-synthetic analogue of fumagillin, TNP470, suggesting MetAP2 is a good target for inhibiting angiogenesis. In vitro, MetAP2 appears to have sites for two divalent metal ions within its active site but there has been much discussion around the identity and number of metal ions actually present in the physiological states of the various MetAPs. An understanding of the physiologically relevant metalloform of the enzyme is essential for designing inhibitors that are active in cells. We have used tool compounds that bind the active site metals in diverse ways to investigate the relevance of the two potential metal binding sites in MetAP2. Using our fragment-based screening approach, Pyramid™, we screened the manganese-form of the MetAP2 enzyme. We identified multiple low-molecular weight fragment hits and confirmed their modes of binding to the two metals in the active site of MetAP2 by X-ray crystallography. Three hit series, which bound metal 1 only, metal 2 only or both metals 1 and 2 were chosen for further optimization using structure-based drug design. Optimized lead compounds had potent inhibitory activity against the in vitro MetAP2 enzyme (∼100 nM) and in HUVEC proliferation assays, whilst also showing greater than 1000-fold selectivity for MetAP2 over MetAP1. Examples from each series, representing different active site metal binding modes, were used as tool compounds to investigate the mechanism of action in cells. The levels of the MetAP2 substrate, 14-3-3, were monitored by western blot in HUVECs treated with these compounds. Levels of methionylated 14-3-3 increased upon treatment with compounds from each of our series indicating the substrate was not being processed and that in each case the compound was inhibiting MetAP2 in these cells. These data indicate that the physiological form of MetAP2 can be inhibited by compounds which bind solely to either of the two active-site metals, suggesting that both metals must be present in the intra-cellular form of MetAP2 and allowing multiple approaches to inhibiting this key angiogenic target. The lead series identified here provide chemically diverse scaffolds for further optimization of drug like properties. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A1.

Abstract IA-11: Discovery and preclinical activity of IPI-926, a Hedgehog pathway inhibitor

Abstract Malignant activation of the Hedgehog (Hh) pathway plays a key role in multiple types of human tumors, supporting the development of Hedgehog pathway antagonists as therapeutics for cancer. This pathway involves two cell-membrane proteins, Patched (Ptc) and Smoothened (Smo), and is regulated by the absence or presence of Hh ligand. Smo can promote growth of certain cancers via three modes of malignant activation of the Hh pathway: ligand-dependent signaling either to the tumor cell itself or from the tumor to its microenvironment, and ligand-independent signaling activated by genetic mutation. Smo has a critical role in the malignant activation of the Hh pathway because it initiates a signal transduction cascade that can result in cell differentiation, migration, and proliferation. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the Hh pathway. Efforts to improve the pharmaceutical properties and potency of cyclopamine led to the discovery of IPI-926, a novel semi-synthetic analogue that inhibits the Hh pathway by binding to Smo. IPI-926 demonstrates excellent oral bioavailability, a long plasma half-life and duration of action, and is highly distributed to tumor tissue. Daily oral administration of IPI-926 results in dose-dependent inhibition of tumor growth in multiple preclinical models of cancer. Following tumor debulking in a primary human xenograft SCLC model with a chemotherapy treatment regimen similar to that used clinically in patients with SCLC, once-daily oral administration of IPI-926 leads to a statistically significant delay in tumor regrowth. In certain cancers including pancreatic and ovarian cancer, the tumor cells produce Hh ligand that diffuses out to the surrounding stroma and binds Ptc, resulting in Smo-mediated Hh pathway activation. This paracrine mode of Hh activation leads to recruitment, growth, and maintenance of the stromal tissue in the tumor microenvironment that supports tumor growth. In a genetically engineered murine model of pancreatic cancer, IPI-926 treatment results in depletion of tumor-associated stroma and prolonged survival when given in combination with gemcitabine. Because Smo plays a critical role in malignant activation of the Hh pathway in both the tumor and the stromal microenvironment, Smo may be a target for the management of a broad range of cancers. IPI-926 is currently being investigated in a Phase 1 clinical trial. Citation Information: Cancer Res 2009;69(23 Suppl):IA-11.