Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

PURPOSE: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. EXPERIMENTAL DESIGN: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. RESULTS: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. CONCLUSIONS: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

Tozorakimab (MEDI3506): an anti-IL-33 antibody that inhibits IL-33 signalling via ST2 and RAGE/EGFR to reduce inflammation and epithelial dysfunction.

Interleukin (IL)-33 is a broad-acting alarmin cytokine that can drive inflammatory responses following tissue damage or infection and is a promising target for treatment of inflammatory disease. Here, we describe the identification of tozorakimab (MEDI3506), a potent, human anti-IL-33 monoclonal antibody, which can inhibit reduced IL-33 (IL-33red) and oxidized IL-33 (IL-33ox) activities through distinct serum-stimulated 2 (ST2) and receptor for advanced glycation end products/epidermal growth factor receptor (RAGE/EGFR complex) signalling pathways. We hypothesized that a therapeutic antibody would require an affinity higher than that of ST2 for IL-33, with an association rate greater than 107 M-1 s-1, to effectively neutralize IL-33 following rapid release from damaged tissue. An innovative antibody generation campaign identified tozorakimab, an antibody with a femtomolar affinity for IL-33red and a fast association rate (8.5 × 107 M-1 s-1), which was comparable to soluble ST2. Tozorakimab potently inhibited ST2-dependent inflammatory responses driven by IL-33 in primary human cells and in a murine model of lung epithelial injury. Additionally, tozorakimab prevented the oxidation of IL-33 and its activity via the RAGE/EGFR signalling pathway, thus increasing in vitro epithelial cell migration and repair. Tozorakimab is a novel therapeutic agent with a dual mechanism of action that blocks IL-33red and IL-33ox signalling, offering potential to reduce inflammation and epithelial dysfunction in human disease.

Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models.

PURPOSE: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. EXPERIMENTAL DESIGN: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. RESULTS: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. CONCLUSIONS: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Interleukin-33 is activated by allergen- and necrosis-associated proteolytic activities to regulate its alarmin activity during epithelial damage.

Interleukin (IL)-33 is an IL-1 family alarmin released from damaged epithelial and endothelial barriers to elicit immune responses and allergic inflammation via its receptor ST2. Serine proteases released from neutrophils, mast cells and cytotoxic lymphocytes have been proposed to process the N-terminus of IL-33 to enhance its activity. Here we report that processing of full length IL-33 can occur in mice deficient in these immune cell protease activities. We sought alternative mechanisms for the proteolytic activation of IL-33 and discovered that exogenous allergen proteases and endogenous calpains, from damaged airway epithelial cells, can process full length IL-33 and increase its alarmin activity up to ~60-fold. Processed forms of IL-33 of apparent molecular weights ~18, 20, 22 and 23 kDa, were detected in human lungs consistent with some, but not all, proposed processing sites. Furthermore, allergen proteases degraded processed forms of IL-33 after cysteine residue oxidation. We suggest that IL-33 can sense the proteolytic and oxidative microenvironment during tissue injury that facilitate its rapid activation and inactivation to regulate the duration of its alarmin function.

Oxidation of the alarmin IL-33 regulates ST2-dependent inflammation.

In response to infections and irritants, the respiratory epithelium releases the alarmin interleukin (IL)-33 to elicit a rapid immune response. However, little is known about the regulation of IL-33 following its release. Here we report that the biological activity of IL-33 at its receptor ST2 is rapidly terminated in the extracellular environment by the formation of two disulphide bridges, resulting in an extensive conformational change that disrupts the ST2 binding site. Both reduced (active) and disulphide bonded (inactive) forms of IL-33 can be detected in lung lavage samples from mice challenged with Alternaria extract and in sputum from patients with moderate-severe asthma. We propose that this mechanism for the rapid inactivation of secreted IL-33 constitutes a 'molecular clock' that limits the range and duration of ST2-dependent immunological responses to airway stimuli. Other IL-1 family members are also susceptible to cysteine oxidation changes that could regulate their activity and systemic exposure through a similar mechanism.

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope "tags".

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.

Development of a mouse model mimicking key aspects of a viral asthma exacerbation.

Viral respiratory tract infections are known triggers of asthma exacerbations in both adults and children. The current standard of care, inhaled CS (corticosteroids) and LABAs (long-acting ß2-adrenoceptor agonists), fails to prevent the loss of control that manifests as an exacerbation. In order to better understand the mechanisms underlying viral asthma exacerbations we established an in vivo model using the clinically relevant aeroallergen HDM (house dust mite) and the viral mimetic/TLR3 (Toll-like receptor 3) agonist poly(I:C). Poly(I:C) alone induced a similar neutrophilic inflammatory profile in the BAL (bronchoalveolar lavage) to that of HRV1b (human rhinovirus 1b) alone, accompanied by both elevated BAL KC (keratinocyte-derived chemokine) and IL-1ß (interleukin-1ß). When mice allergic to HDM were also challenged with poly(I:C) the neutrophilic inflammatory profile was exacerbated. Increased CD8(+) T-cell numbers, increased CD4(+) and CD8(+) cell activation and elevated KC and IL-1ß were observed. No increases in Th2 cytokines or the eosinophil chemoattractant CCL11 [chemokine (C-C motif) ligand 11], above those induced by HDM alone, were observed. The poly(I:C)-exacerbated neutrophilia did not translate into changes in AHR (airways hyper-responsiveness), indicating that in this model inflammation and AHR are two mechanistically independent events. To test the clinical relevance of this model CS sensitivity was assessed using prednisone, a synthetic oral CS used to manage exacerbations in asthmatic patients already on maximal doses of inhaled CS. The increased neutrophils, and accompanying cytokines/chemokines KC and IL-1ß induced by poly(I:C) challenge of HDM-sensitized and challenged mice were insensitive to oral prednisone therapy. In summary we have described a CS-resistant mouse model mimicking the key aspects of viral asthma exacerbation using the clinically relevant aeroallergen HDM and the viral mimic poly(I:C). This model may provide better understanding of disease mechanisms underlying viral exacerbations and could be used to build early confidence in novel therapeutic axes targeting viral asthma exacerbations in Th2 asthmatics.

Pharmacodynamics of voriconazole in a dynamic in vitro model of invasive pulmonary aspergillosis: implications for in vitro susceptibility breakpoints.

BACKGROUND: Voriconazole is a first-line agent for the treatment of invasive pulmonary aspergillosis (IPA). There are increasing reports of Aspergillus fumigatus isolates with reduced susceptibility to voriconazole. METHODS: An in vitro dynamic model of IPA was developed that enabled simulation of human-like voriconazole pharmacokinetics. Galactomannan was used as a biomarker. The pharmacodynamics of voriconazole against wild-type and 3 resistant strains of A. fumigatus were defined. The results were bridged to humans to provide decision support for setting breakpoints for voriconazole using Clinical Laboratory Standards Institute (CLSI) and European Committee of Antimicrobial Susceptibility Testing (EUCAST) methodologies. RESULTS: Isolates with higher minimum inhibitory concentrations (MICs) required higher area under the concentration time curves (AUCs) to achieve suppression of galactomannan. Using CLSI and EUCAST methodologies, the AUC:MIC values that achieved suppression of galactomannan were 55 and 32.1, respectively. Using CLSI and EUCAST methodologies, the trough concentration:MIC values that achieved suppression of galactomannan were 1.68 and 1, respectively. Potential CLSI breakpoints for voriconazole are ≤ 0.5 mg/L for susceptible and >1 mg/L for resistant. Potential EUCAST breakpoints for voriconazole are ≤1 mg/L for susceptible and >2 mg/L for resistant. CONCLUSIONS: This dynamic model of IPA is a useful tool to address many remaining questions related to antifungal treatment of Aspergillus spp.

Pharmacokinetics and pharmacodynamics of posaconazole for invasive pulmonary aspergillosis: clinical implications for antifungal therapy.

BACKGROUND: Posaconazole is a triazole with anti-Aspergillus activity. However, little is known about the utility of posaconazole as primary therapy for invasive pulmonary aspergillosis. METHODS: An in vitro model of the human alveolus was used to study the impact of minimum inhibitory concentrations (MIC) on exposure-response relationships. The pharmacokinetic-pharmacodynamic relationships of posaconazole were examined in an inhalational murine model of invasive pulmonary aspergillosis. A mathematical model was fitted to the entire data set. This model was then used to describe the relationship between drug exposure, quantified in terms of the area under the concentration time curve to MIC (AUC:MIC) and the observed antifungal effect. RESULTS: The posaconazole MIC was an important determinant of exposure-response relationships and accounted for a portion of the observed variance. Murine pharmacokinetics were linear for dosages 1-20 mg/kg/day. There was a dose-dependent decline in serum galactomannan concentrations, with near-maximal suppression following 20 mg/kg/day. The murine pharmacokinetic-pharmacodynamic data were well described by the mathematical model. An AUC:MIC ratio of 167 was associated with half-maximal antifungal effect. CONCLUSIONS: These results provide the experimental foundation for the selection of candidate posaconazole regimens for the primary treatment of invasive pulmonary aspergillosis in profoundly neutropenic hosts.

Azole resistance of Aspergillus fumigatus biofilms is partly associated with efflux pump activity.

This study investigated the phase-dependent expression and activity of efflux pumps in Aspergillus fumigatus treated with voriconazole. Fourteen strains were shown to become increasingly resistant in the 12-h (16- to 128-fold) and 24-h (>512-fold) phases compared to 8-h germlings. An Ala-Nap uptake assay demonstrated a significant increase in efflux pump activity in the 12-h and 24-h phases (P<0.0001). The efflux pump activity of the 8-h germling cells was also significantly induced by voriconazole (P<0.001) after 24 h of treatment. Inhibition of efflux pump activity with the competitive substrate MC-207,110 reduced the voriconazole MIC values for the A. fumigatus germling cells by 2- to 8-fold. Quantitative expression analysis of AfuMDR4 mRNA transcripts showed a phase-dependent increase as the mycelial complexity increased, which was coincidental with a strain-dependent increase in azole resistance. Voriconazole also significantly induced this in a time-dependent manner (P<0.001). Finally, an in vivo mouse biofilm model was used to evaluate efflux pump expression, and it was shown that AfuMDR4 was constitutively expressed and significantly induced by treatment with voriconazole after 24 h (P<0.01). Our results demonstrate that efflux pumps are expressed in complex A. fumigatus biofilm populations and that this contributes to azole resistance. Moreover, voriconazole treatment induces efflux pump expression. Collectively, these data may provide evidence for azole treatment failures in clinical cases of aspergillosis.

Pharmacokinetics and pharmacodynamics of amphotericin B deoxycholate, liposomal amphotericin B, and amphotericin B lipid complex in an in vitro model of invasive pulmonary aspergillosis.

The pharmacodynamic and pharmacokinetic (PK-PD) properties of amphotericin B (AmB) formulations against invasive pulmonary aspergillosis (IPA) are not well understood. We used an in vitro model of IPA to further elucidate the PK-PD of amphotericin B deoxycholate (DAmB), liposomal amphotericin B (LAmB) and amphotericin B lipid complex (ABLC). The pharmacokinetics of these formulations for endovascular fluid, endothelial cells, and alveolar cells were estimated. Pharmacodynamic relationships were defined by measuring concentrations of galactomannan in endovascular and alveolar compartments. Confocal microscopy was used to visualize fungal biomass. A mathematical model was used to calculate the area under the concentration-time curve (AUC) in each compartment and estimate the extent of drug penetration. The interaction of LAmB with host cells and hyphae was visualized using sulforhodamine B-labeled liposomes. The MICs for the pure compound and the three formulations were comparable (0.125 to 0.25 mg/liter). For all formulations, concentrations of AmB progressively declined in the endovascular fluid as the drug distributed into the cellular bilayer. Depending on the formulation, the AUCs for AmB were 10 to 300 times higher within the cells than within endovascular fluid. The concentrations producing a 50% maximal effect (EC50) in the endovascular compartment were 0.12, 1.03, and 4.41 mg/liter for DAmB, LAmB, and ABLC, respectively, whereas, the EC50 in the alveolar compartment were 0.17, 7.76, and 39.34 mg/liter, respectively. Confocal microscopy suggested that liposomes interacted directly with hyphae and host cells. The PK-PD relationships of the three most widely used formulations of AmB differ markedly within an in vitro lung model of IPA.

Pharmacokinetics and pharmacodynamics of a novel triazole, isavuconazole: mathematical modeling, importance of tissue concentrations, and impact of immune status on antifungal effect.

Isavuconazole is a triazole with broad-spectrum activity against medically important fungal pathogens. We investigated the pharmacokinetics and pharmacodynamics of isavuconazole in a murine model of disseminated candidiasis. We determined the pharmacokinetics in both plasma and kidney. The relationship between tissue concentrations and the resultant antifungal effect was described using a mathematical model. The pharmacodynamic parameter that optimally links drug exposure with the antifungal effect was determined using dose fractionation studies. The impact of the immune status of mice receiving isavuconazole was determined in persistently and temporarily neutropenic animals. The pharmacokinetics of 1.6 to 28 mg isavuconazole/kg of body weight were linear. Exposure-response relationships demonstrated near-maximal effect following the administration of >15 mg/kg. The mathematical model showed that exposures in the kidney were 5.77 times higher than those in plasma, and there was persistence of the drug at this site despite concentrations in plasma falling to undetectable levels. The in vitro and in vivo postantifungal effects were 2 to 5 and 8.41 h, respectively. The area under the concentration-time curve (AUC)/MIC ratio was the parameter that optimally linked drug exposure with the observed antifungal effect. The total drug AUC/MIC ratios associated with a 90% probability of survival in temporarily and persistently neutropenic mice were 270 and 670, respectively. Once corrected for protein binding, these values are similar to the magnitude of drug exposure associated with a high probability of a successful therapeutic outcome for other triazoles. This study provides the experimental foundation for the use of isavuconazole in patients with disseminated candidiasis.

Effect of atorvastatin treatment on isoproterenol-induced myocardial infarction in rats.

In the present study, chronic treatment of atorvastatin was evaluated on isoproterenol-induced myocardial infarction. Male Sprague-Dawley rats (200 +/- 25 g) were randomized into the following four groups: (1) control group, (2) isoproterenol-treated group, (3) atorvastatin-treated group, and (4) isoproterenol- and atorvastatin-treated group. Various serum and tissue parameters as well as histopathological studies were carried out in all groups. Isoproterenol administration produced severe myocardial damage and oxidative stress in rats. Atorvastatin treatment reduced myocardial infarction which has been reflected by improvement in serum parameters, ATPase activities and histopathological lesions. However, it could not reduce oxidative stress and hypertrophy induced by isoproterenol. Hence, it can be concluded that atorvastatin may protect myocardial infarction induced by isoproterenol independent of its antioxidant properties.

Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocin-induced diabetic rats.

Effect of metformin treatment on blood pressure, endothelial function and oxidative stress in streptozotocin (STZ)-induced diabetes in rats was studied. In vitro effect of metformin on vascular reactivity to various agonist in the presence of metformin in untreated nondiabetic and STZ-diabetic rats were also studied. Sprague-Dawley rats were randomized into nondiabetic and STZ-diabetic groups. Rats were further randomized to receive metformin (150 mg/kg) or vehicle for 4 weeks. Metformin treatment reduced blood pressure without having any significant effect on blood glucose level in STZ-diabetic rats. Enhanced phenylephrine (PE)-induced contraction and impaired acetylcholine (Ach)-induced relaxation in STZ-diabetic rats were restored to normal by metformin treatment. Enhanced Ach-induced relaxation in metformin-treated STZ-diabetic rats was blocked due to pretreatment with 100 microM of Nomega-nitro-L-arginine-methyl ester (L-NAME) or 10 microM of methylene blue but not 10 microM of indomethacin. Metformin treatment significantly increased antioxidant enzymes and reduced lipid peroxidation in STZ-diabetic rats. In vitro studies in aortic rings of untreated nondiabetic and STZ-diabetic rats showed that the presence of higher concentration of metformin (1 mM and 10 mM) significantly reduced PE-induced contraction and increased Ach-induced relaxation. Metformin per se relaxed precontracted aortic rings of untreated nondiabetic and STZ-diabetic rats in a dose-dependent manner. Pretreatment with L-NAME or removal of endothelium blocked metformin-induced relaxation at lower concentration (up to 30 microM) but not at higher concentration (above 30 microM). Metformin-induced relaxation was blocked in the presence of 1 mM of 4-aminopyridine, or 1 mM of tetraethylammonium but not in the presence of 100 microM of barium ion or 10 microM of glybenclamide. The restored endothelial function along with direct effect of metformin on aortic rings and reduced oxidative stress contributes to reduced blood pressure in STZ-diabetic rats. From the present study, it can be concluded that metformin administration to STZ-diabetic rats lowers blood pressure, and restores endothelial function.

Time-dependent changes in antioxidant enzymes and vascular reactivity of aorta in streptozotocin-induced diabetic rats treated with curcumin.

In the present study changes in oxidative stress and vascular reactivity in aortic rings of chronic streptozotocin-diabetic (STZ-CON) and nondiabetic (ND-CON) rats is studied at 4 weekly intervals up to 24 weeks. The effect of chronic curcumin (200 mg/kg) treatment was also studied. Blood glucose and blood pressure levels were significantly higher in the STZ-CON group and curcumin administration had no significant effect on it. Superoxide dismutase and catalase activity were either unchanged or significantly increased during the early stage of diabetes whereas during the medium and late stage were significantly reduced. Reduced glutathione and lipid peroxidation levels significantly decreased as time after STZ administration increased. Phenylephrine (PE)-induced contraction was significantly (P < 0.05) increased during the early stage of diabetes, whereas it was significantly (P < 0.05) reduced at the medium and late stage of diabetes. Acetylcholine (Ach)-induced relaxation significantly decreased with respect to time after STZ administration. Sodium nitroprusside (SNP)-induced relaxation was unaltered up to initial stage but after medium stage there was a rightward shift and the pD2 value significantly decreased. Though curcumin treatment had no significant effect on superoxide dismutase, catalase, and reduced glutathione levels, it significantly reduced lipid peroxidation compared with diabetic control. Curcumin treatment attenuated the phenylephrine-induced increase in contraction during the early stage. However, curcumin treatment had no significant effect at the medium and late stage. Though curcumin administration improved Ach-induced relaxation it did not restore it to normal. Inability of curcumin to prevent oxidative stress during the late stage may be due to the fact that chronic diabetes (hyperglycemia) leads to excessive production of free radicals. Hence the present study shows that variations reported in antioxidant enzymes and vascular reactivity are due to the duration of diabetes or time after diabetes induction in STZ model and this can not be completely reversed by chronic treatment with curcumin.

Effect of pioglitazone on L-NAME induced hypertension in diabetic rats.

The present study investigates the effect of pioglitazone treatment on blood pressure, vascular reactivity and antioxidant enzymes in L-NAME induced hypertension in normal and STZ-diabetic rats. Diabetes was induced in male Sprague Dawley rats (200+/-15 g) by single intravenous injection of 55 mg/kg of streptozotocin (STZ). Rats were randomized into diabetic and nondiabetic groups, Nomega-nitro-L-arginine-methyl ester (L-NAME, 50 mg/kg) was administered in drinking water for 4 weeks. They were treated with pioglitazone (10 mg/kg/day, p.o.) for 4 weeks and following protocol was carried out. Blood pressure, blood glucose levels and body weight were measured. Thoracic aorta was isolated and dose response curve of phenylephrine (PE) with intact and denuded endothelium was recorded. Dose response curve of acetylcholine (Ach) and sodium nitroprusside (SNP) was recorded in precontracted rings. Lipid peroxidation, superoxide dismutase, catalase, and reduced glutathione were estimated in liver, kidney, and aorta. Pioglitazone produced no significant effect on blood glucose levels, body weight and blood pressure of L-NAME administered nondiabetic and diabetic rats. Pioglitazone treatment had no significant effect on PE induced contraction and Ach induced relaxation in L-NAME diabetic and nondiabetic rats. SNP completely relaxed aortic rings of all the groups. Higher oxidative stress in case of diabetic rats was significantly (p<0.05) reduced by pioglitazone treatment. Although pioglitazone reduced oxidative stress in diabetic rats, there was no significant effect on blood pressure as there was complete absence of nitric oxide due to administration of L-NAME. Hence from the present study it can be concluded that reduction in blood pressure in case of STZ-diabetic rats is nitric oxide mediated.

Effect of bis[curcumino]oxovanadium complex on non-diabetic and streptozotocin-induced diabetic rats.

The effect of the vanadium complex bis[curcumino]oxovanadium (BCOV) on blood glucose level, serum lipid levels, blood pressure and vascular reactivity were studied in non-diabetic and streptozotocin-induced diabetic (STZ-diabetic) rats and compared to that of vanadyl sulfate. Blood glucose level, serum lipid levels, and blood pressure were significantly increased in STZ-diabetic rats. Vascular reactivity to various agonists such as noradrenaline and acetylcholine were significantly increased in STZ-diabetic rats. Blood glucose and serum lipid levels were restored to normal in STZ-diabetic animals treated with vanadyl sulfate at a concentration of 0.5 mmol/kg/day (p.o.). However, vanadyl sulfate at a concentration of 0.2 mmol/kg/day (p.o.) did not produce any significant change in blood glucose and lipid levels. There was no significant effect of vanadyl sulfate (0.2 or 0.5 mmol/kg/day) treatment on blood pressure and vascular reactivity in STZ-diabetic rats. Vanadyl sulfate significantly reduced the body weight of non-diabetic and STZ-diabetic rats. Moreover, it also caused severe diarrhea in both groups of animals. Treatment with BCOV (0.05, 0.1 and 0.2mmol/kg/day, p.o.) significantly decreased blood glucose level and serum lipids in STZ-diabetic rats. Furthermore, administration of BCOV to STZ-diabetic rats restored the blood pressure and vascular reactivity to agonists to normal. There was no significant change in the body weight of BCOV treated non-diabetic and STZ-diabetic rats. Diarrhea was not observed in both BCOV treated groups. In conclusion, the present study shows that the vanadium complex BCOV has antidiabetic and hypolipedimic effects. In addition, it improves the cardiovascular complications associated with diabetes.