Developing semiochemical repellents for protecting Picea from Dendroctonus rufipennis (Coleoptera: Curculionidae) in Alaska and Utah, USA.

Spruce beetle, Dendroctonus rufipennis (Kirby) (Coleoptera: Curculionidae), is the most destructive pest of mature spruce (Picea) in western North America. Recent outbreaks in Alaska and other western US states highlight the need for tools to protect Picea from D. rufipennis. The primary antiaggregation pheromone of D. rufipennis (3-methylcyclohex-2-en-1-one, MCH) and various combinations of potential repellents (1-octen-3-ol, exo-brevicomin, endo-brevicomin, ipsdienol, ipsenol, limonene, and verbenone) were tested for their ability to disrupt the response of D. rufipennis to attractant-baited multiple-funnel traps. Two assays were conducted on the Kenai Peninsula, Alaska, in June and July 2021. All treatments significantly reduced the mean number of D. rufipennis caught compared to the baited control. No other significant differences were observed among treatments. Informed by these and other data, tree protection studies were established in Lutz spruce, Picea × lutzii, on the Kenai Peninsula in 2022 and in Engelmann spruce, Pi. engelmannii, in the Uinta Mountains, Utah, in 2021. All experimental trees were baited with frontalin. Repellent treatments included MCH (SPLAT MCH, ISCA Inc., Riverside, CA, USA) and at least 1 additional repellent combination. In Alaska, all treatments significantly reduced colonization (strip attacks + mass attacks) and mortality of individually treated Pi. × lutzii and all Picea within 11.3-m radius of each treated Pi. × lutzii compared to the control. In Utah, all treatments except for SPLAT MCH + octenol significantly reduced colonization compared to the control. Only SPLAT MCH + Acer kairomone blend (AKB) and SPLAT MCH + octenol reduced Pi. engelmannii mortality compared to the control. SPLAT MCH + AKB and SPLAT MCH + acetophenone and green leaf volatiles (PLUS) were the most effective across both studies. The implications of these and other results to the development of an effective semiochemical repellent for D. rufipennis are discussed.

Activation of Epstein-Barr Virus' Lytic Cycle in Nasopharyngeal Carcinoma Cells by NEO212, a Conjugate of Perillyl Alcohol and Temozolomide.

The Epstein-Barr virus (EBV) is accepted as a primary risk factor for certain nasopharyngeal carcinoma (NPC) subtypes, where the virus persists in a latent stage which is thought to contribute to tumorigenesis. Current treatments are sub-optimal, and recurrence occurs in many cases. An alternative therapeutic concept is aimed at triggering the lytic cycle of EBV selectively in tumor cells as a means to add clinical benefit. While compounds able to stimulate the lytic cascade have been identified, their clinical application so far has been limited. We are developing a novel anticancer molecule, NEO212, that was generated by covalent conjugation of the alkylating agent temozolomide (TMZ) to the naturally occurring monoterpene perillyl alcohol (POH). In the current study, we investigated its potential to trigger the lytic cycle of EBV in NPC cells in vitro and in vivo. We used the established C666.1 cell line and primary patient cells derived from the brain metastasis of a patient with NPC, both of which harbored latent EBV. Upon treatment with NEO212, there was an increase in EBV proteins Zta and Ea-D, key markers of the lytic cycle, along with increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum (ER) stress, followed by the activation of caspases. These effects could also be confirmed in tumor tissue from mice implanted with C666.1 cells. Towards a mechanistic understanding of these events, we used siRNA-mediated knockdown of CHOP and inclusion of anti-oxidant compounds. Both approaches blocked lytic cycle induction by NEO212. Therefore, we established a sequence of events, where NEO212 caused reactive oxygen species (ROS) production, which triggered ER stress and elevated the levels of CHOP, which was required to stimulate the lytic cascade of EBV. Inclusion of the antiviral agent ganciclovir synergistically enhanced the cytotoxic impact of NEO212, pointing to a potential combination treatment for EBV-positive cancers which should be explored further. Overall, our study establishes NEO212 as a novel agent able to stimulate EBV's lytic cycle in NPC tumors, with implications for other virus-associated cancers.

NEO212, a Perillyl Alcohol-Temozolomide Conjugate, Triggers Macrophage Differentiation of Acute Myeloid Leukemia Cells and Blocks Their Tumorigenicity.

Many patients with acute myeloid leukemia (AML) are still dying from this disease. In the past, the alkylating agent temozolomide (TMZ) has been investigated for AML and found to be partially effective; however, the presence of O6-methylguanine DNA methyltransferase (MGMT; a DNA repair enzyme) in tumor cells confers profound treatment resistance against TMZ. We are developing a novel anticancer compound, called NEO212, where TMZ was covalently conjugated to perillyl alcohol (a naturally occurring monoterpene). NEO212 has revealed robust therapeutic activity in a variety of preclinical cancer models, including AML. In the current study, we investigated its impact on a panel of human AML cell lines and found that it exerted cytotoxic potency even against MGMT-positive cells that were highly resistant to TMZ. Furthermore, NEO212 strongly stimulated the expression of a large number of macrophage-associated marker genes, including CD11b/ITGAM. This latter effect could not be mimicked when cells were treated with TMZ or an equimolar mix of individual agents, TMZ plus perillyl alcohol. The superior cytotoxic impact of NEO212 appeared to involve down-regulation of MGMT protein levels. In a mouse model implanted with TMZ-resistant, MGMT-positive AML cells, two 5-day cycles of 25 mg/kg NEO212 achieved an apparent cure, as mice survived >300 days without any signs of disease. In parallel toxicity studies with rats, a 5-day cycle of 200 mg/kg NEO212 was well tolerated by these animals, whereas animals that were given 200 mg/kg TMZ all died due to severe leukopenia. Together, our results show that NEO212 exerts pleiotropic effects on AML cells that include differentiation, proliferation arrest, and eventual cell death. In vivo, NEO212 was well tolerated even at dosages that far exceed the therapeutic need, indicating a large therapeutic window. These results present NEO212 as an agent that should be considered for development as a therapeutic agent for AML.

Detection of perillyl alcohol and its metabolite perillic acid in postsurgical glioblastoma tissue after intranasal administration of NEO100: illustrative case.

BACKGROUND: Intranasal delivery of NEO100, a pharmaceutical-grade version of the natural monoterpene perillyl alcohol (POH), is undergoing clinical phase IIa testing as a treatment for glioblastoma (GBM). However, so far there is no evidence that intranasal delivery of NEO100 indeed results in POH reaching intracranial malignancies in a patient. OBSERVATIONS: After surgical removal of her recurrent GBM tumor, a patient received daily intranasal NEO100 therapy for more than 3 years before a second recurrence emerged. At that time, a final dose of NEO100 was given shortly before the tumor tissue was surgically removed, and the tissue was processed for high-performance liquid chromatography analysis of POH and its primary metabolite, perillic acid (PA). Both molecules could readily be detected in the tumor tissue. LESSONS: This is the first demonstration of POH and PA in brain tumor tissue from any patient. It reveals that intranasal administration of NEO100 is a valid approach to achieve delivery of this agent to a brain tumor. In view of the noninvasive and safe nature of this method, along with tentative indications of activity, our findings add confidence to the notion that intranasal administration of NEO100 holds potential as a new treatment option for brain-localized malignancies.

Potentially Curative Therapeutic Activity of NEO212, a Perillyl Alcohol-Temozolomide Conjugate, in Preclinical Cytarabine-Resistant Models of Acute Myeloid Leukemia.

Despite progress in the treatment of acute myeloid leukemia (AML), the clinical outcome remains suboptimal and many patients are still dying from this disease. First-line treatment consists of chemotherapy, which typically includes cytarabine (AraC), either alone or in combination with anthracyclines, but drug resistance can develop and significantly worsen prognosis. Better treatments are needed. We are developing a novel anticancer compound, NEO212, that was created by covalent conjugation of two different molecules with already established anticancer activity, the alkylating agent temozolomide (TMZ) and the natural monoterpene perillyl alcohol (POH). We investigated the anticancer activity of NEO212 in several in vitro and in vivo models of AML. Human HL60 and U937 AML cell lines, as well as different AraC-resistant AML cell lines, were treated with NEO212 and effects on cell proliferation, cell cycle, and cell death were investigated. Mice with implanted AraC-sensitive or AraC-resistant AML cells were dosed with oral NEO212, and animal survival was monitored. Our in vitro experiments show that treatment of cells with NEO212 results in growth inhibition via potent G2 arrest, which is followed by apoptotic cell death. Intriguingly, NEO212 was equally potent in highly AraC-resistant cells. In vivo, NEO212 treatment strikingly extended survival of AML mice and the majority of treated mice continued to thrive and survive without any signs of illness. At the same time, we were unable to detect toxic side effects of NEO212 treatment. All in all, the absence of side effects, combined with striking therapeutic activity even in an AraC-resistant context, suggests that NEO212 should be developed further toward clinical testing.

Pharmacokinetic properties of the temozolomide perillyl alcohol conjugate (NEO212) in mice.

BACKGROUND: NEO212 is a novel small-molecule anticancer agent that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH) to the alkylating agent temozolomide (TMZ). It is undergoing preclinical development as a therapeutic for brain-localized malignancies. The aim of this study was to characterize metabolism and pharmacokinetic (PK) properties of NEO212 in preclinical models. METHODS: We used mass spectrometry (MS) and modified high-performance liquid chromatography to identify and quantitate NEO212 and its metabolites in cultured glioblastoma cells, in mouse plasma, brain, and excreta after oral gavage. RESULTS: Our methods allowed identification and quantitation of NEO212, POH, TMZ, as well as primary metabolites 5-aminoimidazole-4-carboxamide (AIC) and perillic acid (PA). Intracellular concentrations of TMZ were greater after treatment of U251TR cells with NEO212 than after treatment with TMZ. The half-life of NEO212 in mouse plasma was 94 min. In mice harboring syngeneic GL261 brain tumors, the amount of NEO212 was greater in the tumor-bearing hemisphere than in the contralateral normal hemisphere. The brain:plasma ratio of NEO212 was greater than that of TMZ. Excretion of unaltered NEO212 was through feces, whereas its AIC metabolite was excreted via urine. CONCLUSIONS: NEO212 preferentially concentrates in brain tumor tissue over normal brain tissue, and compared to TMZ has a higher brain:plasma ratio, altogether revealing favorable features to encourage its further development as a brain-targeted therapeutic. Its breakdown into well-characterized, long-lived metabolites, in particular AIC and PA, will provide useful equivalents for PK studies during further drug development and clinical trials with NEO212.

Cytotoxic impact of a perillyl alcohol-temozolomide conjugate, NEO212, on cutaneous T-cell lymphoma in vitro.

BACKGROUND: Mycosis fungoides (MF) and Sézary syndrome (SS) are subtypes of primary cutaneous lymphomas and represent complex diseases regarding their physiopathology and management. Depending on the stage of the disease, different treatment regimens are applied, but there is no consensus on an optimal approach. Prognosis for patients with early stage MF is favorable, but significantly worsens in advanced disease and in SS, where patients frequently relapse and require multiple therapies. METHODS: We investigated the potential anticancer effects of NEO212, a novel compound generated by covalently conjugating perillyl alcohol (a natural monoterpene) to temozolomide (an alkylating agent), on MF and SS cell lines in vitro. HUT-78, HUT-102, and MyLa cells were treated with NEO212 under different conditions, and drug effects on proliferation, viability, and apoptosis were characterized. RESULTS: NEO212 inhibited proliferation, diminished viability, and stimulated apoptosis in all cell lines, although with varying degrees of potency in the different cell lines. It down-regulated c-myc and cyclin D1 proteins, which are required for cell proliferation, but triggered endoplasmic reticulum stress and activation of caspases. Pretreatment of cells with antioxidants ascorbic acid and beta-mercaptoethanol prevented these NEO212-induced effects. CONCLUSIONS: NEO212 exerted promising anticancer effects on SS and MF cell lines. The generation of reactive oxygen species (ROS) appears to play a key role in the NEO212-induced cell death process, because the blockage of ROS with antioxidants prevented caspase activation. We propose that NEO212 should be investigated further toward clinical testing in these tumor types.

Preclinical evaluation of a 64Cu-labeled disintegrin for PET imaging of prostate cancer.

A novel recombinant disintegrin, vicrostatin (VCN), displays high binding affinity to a broad range of human integrins in substantial competitive biological advantage over other integrin-based antagonists. In this study, we synthesized a new 64Cu-labeled VCN probe and evaluated its imaging properties for prostate cancer in PC-3 tumor-bearing mice. Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar) was conjugated with PEG unit and followed by coupling with VCN. The precursor was then radiolabeled with positron emitter 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer to provide 64Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe (64Cu-Sar-RGD). 64Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of > 98%. The specific activity of 64Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64Cu-Sar-RGD, 64Cu-Sar-PEG-VCN produces higher tumor-to-muscle (T/M) imaging contrast ratios at 2 h (4.66 ± 0.34 vs. 2.88 ± 0.46) and 24 h (4.98 ± 0.80 vs. 3.22 ± 0.30) post-injection (pi) and similar tumor-to-liver ratios at 2 h (0.43 ± 0.09 vs. 0.37 ± 0.04) and 24 h (0.57 ± 0.13 vs. 0.52 ± 0.07) pi. The biodistribution results were consistent with the quantitative analysis of microPET imaging, demonstrating good T/M ratio (2.73 ± 0.36) of 64Cu-Sar-PEG-VCN at 48 h pi in PC-3 tumor xenografts. For both microPET and biodistribution studies at 48 h pi, the PC-3 tumor uptake of 64Cu-Sar-PEG-VCN is lower than that of 64Cu-Sar-RGD. 64Cu-Sar-PEG-VCN has the potential for in vivo imaging of prostate cancer with PET, which may provide a unique non-invasive method to quantitatively localize and characterize prostate cancer.

Efficient brain targeting and therapeutic intracranial activity of bortezomib through intranasal co-delivery with NEO100 in rodent glioblastoma models.

OBJECTIVE: Many pharmaceutical agents are highly potent but are unable to exert therapeutic activity against disorders of the central nervous system (CNS), because the blood-brain barrier (BBB) impedes their brain entry. One such agent is bortezomib (BZM), a proteasome inhibitor that is approved for the treatment of multiple myeloma. Preclinical studies established that BZM can be effective against glioblastoma (GBM), but only when the drug is delivered via catheter directly into the brain lesion, not after intravenous systemic delivery. The authors therefore explored alternative options of BZM delivery to the brain that would avoid invasive procedures and minimize systemic exposure. METHODS: Using mouse and rat GBM models, the authors applied intranasal drug delivery, where they co-administered BZM together with NEO100, a highly purified, GMP-manufactured version of perillyl alcohol that is used in clinical trials for intranasal therapy of GBM patients. RESULTS: The authors found that intranasal delivery of BZM combined with NEO100 significantly prolonged survival of tumor-bearing animals over those that received vehicle alone and also over those that received BZM alone or NEO100 alone. Moreover, BZM concentrations in the brain were higher after intranasal co-delivery with NEO100 as compared to delivery in the absence of NEO100. CONCLUSIONS: This study demonstrates that intranasal delivery with a NEO100-based formulation enables noninvasive, therapeutically effective brain delivery of a pharmaceutical agent that otherwise does not efficiently cross the BBB.

A Novel Venom-Derived Peptide for Brachytherapy of Glioblastoma: Preclinical Studies in Mice.

We developed a bacterial expression system to produce a recombinant disintegrin, vicrostatin (VCN), whose structure is based on a natural disintegrin isolated from southern copperhead snake venom. Our goal is to develop VCN for potential clinical translation as an anti-cancer agent. VCN is a peptide of 69 amino acids with a single tyrosine residue. We have employed VCN as integrin-targeted radionuclide therapy (brachytherapy) for treatment of glioblastoma (GBM, glioma). GBM is a deadly brain cancer that doesn't discriminate between sexes and knows no age limit. We established that the tyrosine residue in VCN can be radioiodinated with full retention of bioactivity. 131I-VCN was utilized for integrin-targeted radionuclide therapy using mouse models of glioma. The combination of radioiodinated VCN plus temozolomide (a DNA alkylating agent) significantly prolonged survival of glioma-bearing mice. We also obtained similar results using an immunocompetent mouse model and a murine glioma cell line. In summary, as demonstrated in studies reported here we have shown that VCN as targeted radionuclide therapy for GBM has significant translational potential for therapy of this deadly disease.

NEO412: A temozolomide analog with transdermal activity in melanoma in vitro and in vivo.

Despite new treatments introduced over the past several years, metastatic melanoma remains difficult to cure. Although melanoma in situ (MIS) has better prognosis, it relies heavily on thorough surgical excision, where ill-defined margins can pose a challenge to successful removal, potentially leading to invasive melanoma. As well, MIS in the head and neck area can create serious aesthetic concerns with regard to the surgical defect and substantial scar formation. Toward improved treatment of localized melanoma, including the targeting of unrecognized invasive components, we have been studying a novel agent, NEO412, designed for transdermal application. NEO412 is a tripartite agent that was created by covalent conjugation of three bioactive agents: temozolomide (TMZ, an alkylating agent), perillyl alcohol (POH, a naturally occurring monoterpene with anticancer properties), and linoleic acid (LA, an omega-6 essential fatty acid). We investigated the anti-melanoma potency of NEO412 in vitro and in mouse models in vivo. The in vitro results showed that NEO412 effectively killed melanoma cells, including TMZ-resistant and BRAF mutant ones, through DNA alkylation and subsequent apoptosis. in vivo, NEO412 inhibited tumor growth when applied topically to the skin of tumor-bearing animals, and this effect involved a combination of increased tumor cell death with decreased blood vessel development. At the same time, drug-treated mice continued to thrive, and there was no apparent damage to normal skin in response to daily drug applications. Combined, our results present NEO412 as a potentially promising new treatment for cutaneous melanoma, in particular MIS, deserving of further study.

NPPB is a novel candidate biomarker expressed by cancer-associated fibroblasts in epithelial ovarian cancer.

Most solid tumors contain cancer-associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF-like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF-specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n = 145) but not in the stroma of normal ovaries (n = 4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n = 8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC.

Reversal of thienopyridine-induced platelet dysfunction following desmopressin administration.

Adenosine diphosphate (ADP)-receptor antagonists are widely used for thrombus prevention, although reversing their platelet dysfunction is difficult. This study evaluated the ability of desmopressin to reverse clopidogrel-induced platelet dysfunction. Sprague-Dawley rats received either clopidogrel (30 mg/kg) or placebo, followed 4 h later by saline or desmopressin (0.15, 0.3, or 0.6 µg/kg). Bleeding times and platelet aggregation studies were subsequently performed. A bleeding time >25 min was considered "prolonged." The median bleeding time for clopidogrel-exposed rats was 21 min, vs. 6 min for controls (p < 0.01). Progressively higher doses of 1-deamino-8-D-arginine vasopressin (DDAVP) were associated with a reduced number of rats with prolonged bleeding time (p = 0.001). Higher doses of DDAVP were also associated with a reduction in the median (IQR) bleeding time; 29 (13.5-30) min in rats receiving clopidogrel without DDAVP vs. 19 (12-28) min in rats receiving clopidogrel and 0.6 µg/kg DDAVP. The step-wise dosing of DDAVP resulted in a 54 % reduction in meeting the endpoint of prolonged bleeding time (OR 0.46; p = 0.025; 95 % CI 0.23-0.91). Platelet aggregation was observed in all control rats, but only some of those clopidogrel-treated rats who received 0.6 µg/kg DDAVP. In this model of an ADP-receptor antagonist, DDAVP results in partial reversal of clopidogrel-induced platelet dysfunction.

Chemical surface modification of parylene C for enhanced protein immobilization and cell proliferation.

To introduce the adhesion site of proteins and/or cells on parylene C (PC)-coated medical devices that can be used as implantable biosensors or drug delivery capsules, the PC surfaces were initially modified by the Friedel-Crafts acylation reaction to generate active chlorines. These chlorines were then employed to initiate the atom transfer radical polymerization of tert-butyl acrylate (TBA) and form a polymer brush layer of polyTBA on PC; the acrylate groups in the polymer brushes were hydrolyzed to carboxylic acid groups and further activated into succinimidyl ester groups via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide coupling reaction. The PC surface grafted with polymer brushes and activated by succinimide showed efficient attachment of proteins, including gelatin, contortrostatin (CN) and bovine serum albumin (BSA), all at high density on the PC surface. The CN density on the surface was evaluated for both monolayer and polymer brush-based coatings. Based on fluorescence measurements, the polymer brush gives a 60-fold higher surface protein density than the monolayer-based system. Gelatin was used as a model protein and covalently coated onto the modified PC surface for cell culture study. Substrates with gelatin coating showed a significantly higher cell attachment and proliferation in 7 days cultures as compared to the uncoated substrates. In addition, a conventional photolithography technique was coupled with the surface chemistry to successfully pattern the BSA labeled with fluorescein isothiocyanate on the modified PC surfaces.

Fibrolase: trials and tribulations.

Fibrolase is the fibrinolytic enzyme isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria including reverse phase HPLC, molecular sieve chromatography and SDS-PAGE. The purified enzyme is a zinc metalloproteinase containing one mole of zinc. It is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. Fibrolase shares a significant degree of homology with enzymes of the reprolysin sub-family of metalloproteinases including an active site homology of close to 100%; it is rapidly inhibited by chelating agents such as EDTA, and by alpha2-macroglobulin (α2Μ). The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(ß)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin, cleaving the α-chain more rapidly than the ß-chain. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phaase II stroke trial, and Nuvelo dropped further development in 2008.

Phase II clinical trial of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer: a trial of the California, Chicago, and Princess Margaret Hospital phase II consortia.

PURPOSE: Vascular endothelial growth factor (VEGF) plays an important role in the biology of ovarian cancer (OC). Inhibitors of VEGF suppress tumor growth in OC models. Metronomic chemotherapy, defined as frequent administration of low doses of cytotoxic chemotherapy, suppresses tumor growth, possibly by inhibiting angiogenesis. A phase II trial was conducted to evaluate the antitumor activity and adverse effects of bevacizumab and metronomic oral cyclophosphamide in women with recurrent OC. PATIENTS AND METHODS: Patients with measurable disease and prior treatment with a platinum-containing regimen were eligible. Up to two different regimens for recurrent disease were allowed. Treatment consisted of bevacizumab 10 mg/kg intravenously every 2 weeks and oral cyclophosphamide 50 mg/d. The primary end point was progression-free survival at 6 months. Plasma levels of VEGF, E-selectin, and thrombospondin-1 were obtained serially. RESULTS: Seventy patients were enrolled. The probability of being alive and progression free at 6 months was 56% (+/- 6% SE). A partial response was achieved in 17 patients (24%). Median time to progression and survival were 7.2 and 16.9 months, respectively. The most common serious toxicities were hypertension, fatigue, and pain. Bevacizumab-related toxicities included four episodes of gastrointestinal perforation or fistula, two episodes each of CNS ischemia and pulmonary hypertension, and one episode each of gastrointestinal bleeding and wound healing complication. There were three treatment-related deaths. Levels of VEGF, E-selectin, and thrombospondin-1 were not associated with clinical outcome. CONCLUSION: The combination of bevacizumab and metronomic cyclophosphamide is active in recurrent OC. Further study of this combination is warranted.

Retargeting of adenoviral vector using basic fibroblast growth factor ligand for malignant glioma gene therapy.

OBJECT: Adenovirus vector (AdV)-mediated gene delivery has been recently demonstrated in clinical trials as a novel potential treatment for malignant gliomas. Combined coxsackievirus B and adenovirus receptor (CAR) has been shown to function as an attachment receptor for multiple adenovirus serotypes, whereas the vitronectin integrins (alphavbeta3 and alphavbeta5) are involved in AdV internalization. In resected glioma specimens, the authors demonstrated that malignant gliomas have varying levels of CAR, alphavbeta3, and alphavbeta5 expression. METHODS: A correlation between CAR expression and the transduction efficiency of AdV carrying the green fluorescent protein in various human glioblastoma multiforme (GBM) cell lines and GBM primary cell lines was observed. To increase transgene activity in in vitro glioma cells with low or deficient levels of CAR, the authors used basic fibroblast growth factor (FGF2) as a targeting ligand to redirect adenoviral infection through its cognate receptor, FGF receptor 1 (FGFR1), which was expressed at high levels by all glioma cells. These findings were confirmed by in vivo study data demonstrating enhanced transduction efficiency of FGF2-retargeted AdV in CAR-negative intracranial gliomas compared with AdV alone, without evidence of increased angiogenesis. CONCLUSIONS: Altogether, the results demonstrated that AdV-mediated gene transfer using the FGF2/FGFR system is effective in gliomas with low or deficient levels of CAR and suggested that FGF2-retargeting of AdV may be a promising approach in glioma gene therapy.

The role of contortrostatin, a snake venom disintegrin, in the inhibition of tumor progression and prolongation of survival in a rodent glioma model.

OBJECT: Malignant gliomas are not curable because of diffuse brain invasion. The tumor cells invade the surrounding brain tissue without a clear tumor-brain demarcation line, making complete resection impossible. Therapy aimed at inhibition of invasion is crucial not only for prevention of tumor spread, but also for selectively blocking migrating cells that may be more resistant to chemotherapy and radiation. Recently, investigations have shown that the snake venom disintegrin contortrostatin specifically binds to certain integrins on the surface of glioma cells and thereby inhibits their interaction with the extracellular matrix (ECM), resulting in a blockage of cell motility and invasiveness. To translate these in vitro findings into clinical settings, the authors examined the effect of contortrostatin on glioma progression in a rodent model. METHODS: Athymic mice were intracranially or subcutaneously injected with U87 glioma cells, and the effect of intratumorally administered contortrostatin on tumor progression and animal survival was then studied. In addition, the authors evaluated the pharmacological safety of contortrostatin use in the brains of tumor-free animals. CONCLUSIONS: The results demonstrate that contortrostatin is able to inhibit tumor growth and angiogenesis and to prolong survival in a rodent glioma model. Moreover, contortrostatin appears to be well tolerated by the animal and lacks obvious neurotoxic side effects. Thus, contortrostatin may have potential as a novel therapeutic agent for the treatment of malignant gliomas.