Osmond J. D'Cruz

Metvan: a novel oxovanadium(IV) complex with broad spectrum anticancer activity

Among the 25 bis(cyclopentadienyl)vanadium(IV) and 14 oxovanadium(IV) compounds synthesised and evaluated for anticancer activity, bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium(IV) (metvan) was identified as the most promising multitargeted anticancer vanadium complex with apoptosis-inducing activity. At nanomolar and low micromolar concentrations, metvan induces apoptosis in human leukaemia cells, multiple myeloma cells and solid tumour cells derived from breast cancer, glioblastoma, ovarian, prostate and testicular cancer patients. It is highly effective against cisplatin-resistant ovarian cancer and testicular cancer cell lines. Metvan is much more effective than the standard chemotherapeutic agents dexamethasone and vincristine in inducing apoptosis in primary leukaemia cells from patients with acute lymphoblastic leukaemia, acute myeloid leukaemia or chronic acute myeloid leukaemia. Metvan-induced apoptosis is associated with a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species and depletion of glutathione. Treatment of leukaemia cells from acute lymphoblastic leukaemia, acute myeloid leukaemia and chronic acute myeloid leukaemia patients with metvan inhibits the constitutive expression as well as the gelatinolytic activities of matrix metalloproteinase-9 and -2. Treatment of human malignant glioblastoma and breast cancer cells with metvan at concentrations > 1 μM is associated with a nearly complete loss of the adhesive, migratory and invasive properties of the treated cancer cell populations. Metvan shows favourable pharmacokinetics in mice and does not cause acute or subacute toxicity at the dose levels tested (12.5 – 50 mg/kg). Therapeutic plasma concentrations ≥ 5 μM, which are highly cytotoxic against human cancer cells, can be rapidly achieved and maintained in mice for at least 24 h after intraperitoneal bolus injection of a single 10 mg/kg non-toxic dose of metvan. Metvan exhibits significant antitumour activity, delays tumour progression and prolongs survival time in severe combined immunodeficient mouse xenograft models of human malignant glioblastoma and breast cancer. The broad spectrum anticancer activity of metvan together with favourable pharmacodynamic features and lack of toxicity warrants further development of this oxovanadium compound as a new anticancer agent. Metvan could represent the first vanadium complex as an alternative to platinum-based chemotherapy.

PHI-443: A Novel Noncontraceptive Broad-Spectrum Anti-Human Immunodeficiency Virus Microbicide

Abstract PHI-443 (N′-[2-(2-thiophene)ethyl]-N′-[2-(5-bromopyridyl)] thiourea) is a rationally designed novel thiophene thiourea nonnucleoside reverse transcriptase inhibitor (NNRTI) with potent anti-HIV activity against the wild-type and drug-resistant primary clinical human immunodeficiency virus (HIV-1) isolates. This study examined the potential utility of PHI-443 as a nonspermicidal microbicide for prevention of sexual transmission of HIV. Our goal in this study was to test the effects of PHI-443 on in vivo sperm functions under conditions shown to inactivate viruses in human cells. PHI-443 completely prevented the vaginal transmission of a genotypically and phenotypically drug-resistant HIV-1 isolate in the humanized severe combined immunodeficient (Hu-SCID) mouse model of sexually transmitted AIDS. Exposure of human sperm to PHI-443 at doses 30u200a000 times higher than those that yield effective concentrations against the AIDS virus had no adverse effect on sperm motility, kinematics, cervical mucus penetrability, or the viability of vaginal and cervical epithelial cells. Exposure of rabbit semen to PHI-443 either ex vivo or in vivo had no adverse impact on in vivo fertilizing ability in the rabbit model. Reproductive indices (i.e., pregnancy rate, embryo implantation, and preimplantation losses) were not affected by pretreatment of rabbit semen with PHI-443. Likewise, intravaginal application of 2% PHI-443 via a self-emulsifying gel at the time of artificial insemination resulted in healthy offspring with no apparent peri- or postnatal repercussions. Repeated intravaginal administration of 0.5%– 2% PHI-443 gel was found to be safe in rabbits and lacked systemic absorption. PHI-443 has clinical potential as a prophylactic broad-spectrum anti-HIV microbicide without contraceptive activity.

Structural Requirements for Potent Anti-Human Immunodeficiency Virus (HIV) and Sperm-Immobilizing Activities of Cyclohexenyl Thiourea and Urea Non-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase

Abstract The current pandemic of sexually transmitted human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) has created an urgent need for a new type of microbicide, one that is both a spermicide and a virucide. In a systematic effort to identify a non-detergent-type antiviral spermicide, we have rationally designed and synthesized a series of cyclohexenyl thiourea (CHET) nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) with sperm-immobilizing activity (SIA). To gain further insight into the structural requirements for the optimal activity of these dual-function NNIs, we compared the effects of thiazolyl, benzothiazolyl, and pyridyl ring substitutions and functionalization with electron-donating and electron-withdrawing groups as well as the importance of thiourea and urea moieties of 15 heterocyclic ring-substituted NNIs. RT activity and p24 antigen production in HIV-infected peripheral blood mononuclear cells were used as markers of viral replication. Computer-assisted sperm analysis was used for evaluating SIA of CHET compounds. The rabbit model was used for evaluation of in vivo mucosal toxicity and contraceptive activity of the lead NNIs. Three CHET-NNIs with a bromo, chloro, or methyl substitution at the 5 position of the pyridyl ring exhibited potent anti-HIV activity at nanomolar concentrations (IC50 = 3–5 nM) and SIA at micromolar concentrations (EC50 = 45–96 μM). The dual-function CHET-NNIs were potent inhibitors of drug-resistant HIV-1 strains with genotypic and phenotypic NNI resistance. Upon substitution of the sulfur atom of the thiourea moiety with an oxygen atom, the most striking difference noted was a 38-fold reduction in time required for 50% sperm immobilization (T1/2). A quantitative structure-activity relationship (QSAR) analysis was used in deriving regression equations between 20 physicochemical properties and SIA of NNIs. QSAR analysis showed that the T1/2 values positively correlated with values for molecular refractivity (r = 0.88), hydrophobicity (r = 0.72), atomic polarizability (r = 0.70), and principal moment of inertia (r = 0.63) of spermicidal NNIs. A stepwise multiple regression model to describe the relationship of T1/2 values with these four regressors provided excellent predictability (r = 0.93). Exposure of semen to thiourea/urea NNIs either alone or in combination at the time of artificial insemination led to marked or complete inhibition of pregnancy in rabbits as assessed by the number of embryo implants versus corpora lutea on Day 8 of pregnancy. Repeated intravaginal application of a gel-microemulsion with and without 0.5%, 1%, and 2% CHET-NNI or its urea analog either alone or in combination did not induce mucosal toxicity. We hypothesize that the gain of spermicidal function by CHET-NNIs is due to their metabolic oxidation to urea analogs by sperm. Three reaction pathways are discussed. The extremely rapid SIA of the urea analog as well as the broad-spectrum anti-HIV activity of spermicidal CHET-NNIs together with their lack of mucosal toxicity and the marked ability to reduce in vivo fertility is particularly useful for the clinical development of a dual-function spermicidal microbicide. The cyclohexenyl pyridyl NNIs, especially N-[2-(1-cyclohexenyl)ethyl] N′-[2-(5-bromopyridyl)]-thiourea in combination with the urea analog, show unique clinical potential as anti-HIV spermicides aimed at curbing the sexual transmission of multidrug-resistant HIV-1 while providing effective fertility control for women.

Development and Evaluation of a Thermoreversible Ovule Formulation of Stampidine, a Novel Nonspermicidal Broad-Spectrum Anti-Human Immunodeficiency Virus Microbicide

Abstract Stampidine [2′,3′-didehydro-2′,3′-dideoxythymidine 5′-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions. Stampidine was solubilized in a thermoreversible ovule formulation composed of polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80. Does were exposed intravaginally for 14 days to an ovule formulation with and without 0.5%, 1%, or 2% stampidine corresponding to 1 × 107- to 4 × 107-fold higher than its in vitro anti-HIV IC50 value. Vaginal tissues harvested on Day 15 were evaluated for mucosal toxicity and cellular inflammation. Additionally, does were exposed intravaginally to stampidine, and plasma collected at various time points was assayed by analytical HPLC for the prodrug and its bioactive metabolites. Stampidine did not cause mucosal inflammation. The vaginal irritation scores for 0.5–2% stampidine were within the acceptable range for clinical trials. The prodrug and its major metabolites were undetectable in the blood plasma. The marked stability of stampidine at acidic pH, its rapid spreadability, together with its lack of mucosal toxicity or systemic absorption of stampidine via a thermoreversible ovule may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.

Limitations of the Human-PBL-SCID Mouse Model for Vaginal Transmission of HIV-1

SCID mice reconstituted with human peripheral blood lymphocytes (PBL) are amenable to vaginal transmission of HIV‐1. We investigated the effectiveness of this model to establish systemic HIV‐1 infection.

Mucosal Toxicity Studies of a Gel Formulation of Native Pokeweed Antiviral Protein

Pokeweed antiviral protein (PAP), a 29-kDa plant-derived protein isolated from Phytolacca americana, is a promising nonspermicidal broad-spectrum antiviral microbicide. This study evaluated the mucosal toxicity potential of native PAP in the in vivo rabbit vaginal irritation model as well as the in vitro reconstituted human vaginal epithelial tissue model. Twenty-two New Zealand white rabbits in 4 subgroups were exposed intravaginally to a gel with and without 0.01, 0.1, or 1.0% native PAP for 10 consecutive days. The dose of PAP used represented nearly 200- to 20,000 times its in vitro anti-HIV IC50 value. Animals were euthanized on day 11 and vaginal tissues were evaluated for histologic and immunohistochemical evidence of mucosal toxicity, cellular inflammation, and hyperplasia. Blood was analyzed for changes in hematology and clinical chemistry profiles. Reconstituted human vaginal epithelial tissue grown on membrane filters was exposed to 0.1, 0.1, or 1.0% native PAP in medium or topically via a gel for 24 hours and tissue damage was evaluated by histological assessment. In the in vivo rabbit vaginal irritation model, half of all PAP-treated rabbits (8/16) exhibited an acceptable range of vaginal mucosal irritation (total score <8 out of a possible 16), whereas nearly a third of PAP-treated rabbits (5/16) developed moderate to marked vaginal mucosal irritation (total score >11). However, no treatment-related adverse effects were seen in hematological or clinical chemistry measurements. Furthermore, in vitro exposure of a 3-dimensional human vaginal tissue grown on polycarbonate membrane filters to identical concentrations of PAP either added to culture medium or applied topically via gel formulation did not result in direct toxicity as determined by histologic evaluation. These findings indicate careful monitoring of vaginal irritation will be required in the clinical development of PAP as a nonspermicidal microbicide.

A 13-week subchronic intravaginal toxicity study of pokeweed antiviral protein in mice.

Pokeweed antiviral protein (PAP), a 29-kDa plant-derived protein isolated from Phytolacca americana, is a broad-spectrum antiviral agent. PAP shows unique clinical potential to become the active ingredient of a non-spermicidal microbicide because of its potent in vivo anti-HIV activity, non-interference with in vivo sperm functions, and lack of cytotoxicity to genital tract epithelial cells. Over 13 weeks the subchronic and reproductive toxicity potential of an intravaginally administered gel formulation of PAP was studied in mice to support its further development as a vaginal microbicide. Female B6C3F1 and CD-1 mice in subgroups of 20, were exposed intravaginally to a gel formulation containing 0, 0.025, 0.05, or 0.1% PAP, 5 days/week for 13 consecutive weeks. On a molar basis, these concentrations are 500- to 2000-times higher than the in vitro anti-HIV IC50 value. After 13 weeks of intravaginal treatment, B6C3F1 mice were evaluated for survival, body weight gain, and absolute and relative organ weights. Blood was analyzed for hematology and clinical chemistry profiles. Microscopic examination was performed on hematoxylin and eosin-stained tissue sections from each study animal. Placebo-control and PAP-dosed female CD-1 mice were mated with untreated males in order to evaluate if PAP has any deleterious effects on reproductive performance. There were no treatment-related mortalities. Mean body weight gain was not reduced by PAP treatment during the dosing period. The hemogram and blood chemistry profiles revealed lack of systemic toxicity following daily intravaginal instillation of PAP for 13 weeks. No clinically significant changes in absolute and relative organ weights were noted in the PAP dose groups. Extensive histopathological examination of tissues showed no increase in treatment-related microscopic lesions in any of the three PAP dose groups. Repeated intravaginal exposure of CD-1 mice to increasing concentrations of PAP for 13 weeks showed no adverse effect on their subsequent reproductive capability (100% fertile), neonatal survival (>90%) or pup development. Collectively, these findings demonstrate that repetitive intravaginal administration of PAP at concentrations as high as 2000 times its in vitro anti-HIV IC50 value was not associated with local or systemic toxicity and did not adversely affect the reproductive performance of mice. PAP may be useful as an active ingredient of a safe vaginal microbicide for prevention of the sexual transmission of viruses, particularly of HIV-1.

Impact of targeting transforming growth factor β-2 with antisense OT-101 on the cytokine and chemokine profile in patients with advanced pancreatic cancer

Background Overexpression of the cytokine – transforming growth factor-beta 2 (TGF-β2) – has been implicated in the malignant progression of pancreatic cancer (PAC). OT-101 (trabedersen) is an antisense oligodeoxynucleotide designed to target the human TGF-β2 mRNA. In a Phase I/II study, OT-101 treatment with subsequent chemotherapy was characterized by outstanding overall survival (OS) in patients with PAC. Objective This study sought to identify 1) co-regulated sets of cyto-/chemokines; 2) potential mechanisms that link TGF-β receptor type 2 receptor inhibition that may result in the induction of a cytokine storm; and 3) predictive biomarkers for OS outcome in OT-101-treated patients with PAC. Materials and methods Plasma levels of 31 cyto-/chemokines were tracked over three cycles of OT-101 therapy (140 mg/m2/day) in 12 PAC patients. Samples were acquired before onset of OT-101 therapy and at eight selected time points during therapy. A mixed ANCOVA model was developed for 19 cyto-/chemokines with median expression >1 following OT-101 therapy. Regression and hierarchical clustering analyses were performed to identify correlated expressions in each patient across cyto-/chemokines or in each cyto-/chemokine across patients. Plasma cyto-/chemokine levels were compared with OS with and without subsequent chemotherapy. Results Three highly correlated subsets of cyto-/chemokines (Cluster 1: EGF, MIP-1α, MIP-1β; Cluster 2: FGF-2, MIG, IP-10, IL-15, IFN-α, IL-12; and Cluster 3: HGF, IL-6, IL-8) were identified following OT-101 therapy. Suppression of TGF-β signaling by OT-101 led to upregulation of IL-8, IL-15, IP-10, and HGF. Protein–protein interaction networks constructed using STRING10 algorithm identified a relationship between IL-8, IL-15, and TGF-β receptor type 2 inhibition. The mixed analysis of covariance model that examined the levels of 19 cyto-/chemokines with OS as the covariate at each of the time points resulted in IL-8 and IL-15 exhibiting a significant association with OS during Cycle 1 of therapy. In the whole-blood culture model, the cytokines with the most pronounced increase after OT-101 treatment were IL-1β, IL-8, and MCP-1. Conclusion No consistent responses in cyto-/chemokine levels were observed due to OT-101 treatment. Levels of IL-8 and IL-15 during Cycle 1 were positively associated with OS across 12 patients with PAC and served as potential biomarkers for treatment outcome following OT-101 therapy.

RNAi-mediated beta-catenin knockdown in the gastrointestinal mucosa (GI) of patients with familial adenomatous polyposis (FAP): Results of START-FAP trial.

e15065Background: FAP is an inherited GI disorder that predisposes patients to early-onset of colorectal cancer due to mutations in the APC gene resulting in accumulation of β-catenin contributing ...

Abstract 2800: Transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) OT-101 synergizes with chemotherapy in preclinical tumor models

Background: Overexpression of TGF-β2 has been implicated in the malignant progression of tumors by inducing immunosuppression, proliferation, angiogenesis and metastasis. Clinical failures of TGF-β inhibitors in targeting the tumor promoting arm of TGF-β signaling is attributed to multiple isoforms and receptor functions. OT-101 (Trabedersen) is a phosphorothioate ASO designed to specifically target human TGF-β2 mRNA with superior clinical activity in patients with TGF-β2-overexpressing tumors. Herein, we report the synergizing effect of OT-101 with chemotherapy in multiple human tumor xenograft models for further exploration of clinical combinations. Methods: The in vivo efficacy studies of intraperitoneal (IP, 16 or 50 mg/kg) or subcutanous (SC, 1-64 mg/kg) repeated administration (qdx3/wk or qdx21) of OT-101 as single agent and in combination with Gemcitabine (15 mg/kg, qdx2/wk, IP), Dacarbazine (1-10 mg/kg, qdx4/wk, IP) or Paclitaxel (10 mg/kg, qdx5, IV) were evaluated in nude mice (10/subgroup) bearing either (i) orthotopic human L3.6pl pancreatic cancer (PAC), (ii) human metastatic C8161 melanoma, (iii) subcutaneous glioblastoma (U87-MG) or (iv) subcutaneous ovarian (SK-OV-3) tumors. Mice were monitored thrice a week for adverse effects, body weight loss, tumor size and survival outcome. The incidence of lymph node and liver surface and micro-metastases as well as size and weight of the pancreatic tumors were determined. Tumor sections were stained with anti-5-bromo-29-deoxy-uridine antibody to determine tumor cell proliferation and with anti-CD31/PECAM-1 antibody to determine vascularization. Results: OT-101 significantly reduced tumor growth (p = 0.0084), lymph node metastasis (p = 0.023), and tumor angiogenesis (p Conclusions: The preclinical data lay the groundwork for further exploration of combination therapy in the clinic as well as demonstrating that TGF-β2 is a druggable target. Of interest is the preferential synergy between OT-101 and Paclitaxel or Dacarbazine, but not with Gemcitabine. Citation Format: Osmond D9Cruz, Cynthia Lee, Vuong Trieu, Larn Hwang. Transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) OT-101 synergizes with chemotherapy in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2800. doi:10.1158/1538-7445.AM2017-2800