Small Molecule Rescue of ATXN3 Toxicity in C. elegans via TFEB/HLH-30.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is a polyglutamine expansion disease arising from a trinucleotide CAG repeat expansion in exon 10 of the gene ATXN3. There are no effective pharmacological treatments for MJD, thus the identification of new pathogenic mechanisms, and the development of novel therapeutics is urgently needed. In this study, we performed a comprehensive, blind drug screen of 3942 compounds (many FDA approved) and identified small molecules that rescued the motor-deficient phenotype in transgenic ATXN3 Caenorhabditis elegans strain. Out of this screen, five lead compounds restoring motility, protecting against neurodegeneration, and increasing the lifespan in ATXN3-CAG89 mutant worms were identified. These compounds were alfacalcidol, chenodiol, cyclophosphamide, fenbufen, and sulfaphenazole. We then investigated how these molecules might exert their neuroprotective properties. We found that three of these compounds, chenodiol, fenbufen, and sulfaphenazole, act as modulators for TFEB/HLH-30, a key transcriptional regulator of the autophagy process, and require this gene for their neuroprotective activities. These genetic-chemical approaches, using genetic C. elegans models for MJD and the screening, are promising tools to understand the mechanisms and pathways causing neurodegeneration, leading to MJD. Positively acting compounds may be promising candidates for investigation in mammalian models of MJD and preclinical applications in the treatment of this disease.

CYP epoxygenase-derived H2O2 is involved in the endothelium-derived hyperpolarization (EDH) and relaxation of intrarenal arteries.

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are involved in the in endothelium-derived hyperpolarization (EDH)-type relaxant responses of coronary and mesenteric arterioles. The role of ROS in kidney vascular function has mainly been investigated in the context of harmful ROS generation associated to kidney disease. The present study was sought to investigate whether H2O2 is involved in the endothelium-dependent relaxations of intrarenal arteries as well the possible endothelial sources of ROS generation involved in these responses. Under conditions of cyclooxygenase (COX) and nitric oxide (NO) synthase inhibition, acetylcholine (ACh) induced relaxations and stimulated H2O2 release that were reduced by catalase and by the glutathione peroxidase (GPx) mimetic ebselen in rat renal interlobar arteries, suggesting the involvement of H2O2 in the endothelium-dependent responses. ACh relaxations were also blunted by the CYP2C inhibitor sulfaphenazole and by the NADPH oxidase inhibitor apocynin. Acetylcholine stimulated both superoxide (O2•-) and H2O2 production that were reduced by sulfaphenazole and apocynin. Expression of the antioxidant enzyme CuZnSOD and of the H2O2 reducing enzymes catalase and GPx-1 was found in both intrarenal arteries and renal cortex. On the other hand, exogenous H2O2 relaxed renal arteries by decreasing vascular smooth muscle (VSM) intracellular calcium concentration [Ca2+]i and markedly enhanced endothelial KCa currents in freshly isolated renal endothelial cells. CYP2C11 and CYP2C23 epoxygenases were highly expressed in interlobar renal arteries and renal cortex, respectively, and were co-localized with eNOS in renal endothelial cells. These results demonstrate that H2O2 is involved in the EDH-type relaxant responses of renal arteries and that CYP 2C epoxygenases are physiologically relevant endothelial sources of vasodilator H2O2 in the kidney.

Suppression of myocardial ischemia-reperfusion injury by inhibitors of cytochrome P450 in rats.

The effects of inhibitors of cytochrome P450 on myocardial regional ischemia-reperfusion injury were examined in rats. Ischemia-reperfusion injury was evoked by ligation of the left anterior descending coronary artery for 1 h, followed by reperfusion for 24 h. Injuries were evident in causing infarction, decreases in left ventricular systolic pressure and left ventricle (dP/dt max)/P and an increase in left ventricular end-diastolic pressure. Increases in lipid peroxidation and reactive oxygen species levels in the ischemic region were observed. Intravenous injection of the potent cytochrome P450 inhibitor sulfaphenazole at 10 and 30 mg/kg at the time of reperfusion reduced infarct size by 41.7 and 73.2%, respectively; and improved cardiac function accompanied by the decrease in content of lipid peroxide and reactive oxygen species in the area at risk. Cardiac testosterone metabolism was inhibited by sulfaphenazole administration, indicating its inhibitory effects on cardiac cytochrome P450 activity. Another cytochrome P450 inhibitor, cimetidine, given intravenously, had similar effects to sulfaphenazole on reperfusion injury. Taken together, these results indicate that reactive oxygen species derived from cytochrome P450 play an important part in myocardial regional ischemia-reperfusion injury in vivo, and strongly support the hypothesis that cytochrome P450 inhibitors are promising therapeutic agents for cardiac ischemia-reperfusion injury.

Effect of sulfaphenazole on tissue plasminogen activator release in normotensive subjects and hypertensive patients.

BACKGROUND: A nitric oxide-independent response, possibly mediated by hyperpolarization, regulates vascular tone, acting as a compensatory mechanism in the presence of impaired nitric oxide availability. Cytochrome P450 2C9 (CYP 2C9) is a source of endothelium-derived hyperpolarizing factors and modulates tissue-type plasminogen activator (tPA) release from endothelial cells; however, no effect of hyperpolarization on fibrinolysis has been documented in humans. We aimed to assess the effect of sulfaphenazole, a specific CYP 2C9 inhibitor, on tPA release in normotensive subjects and patients with essential hypertension. METHODS AND RESULTS: tPA release was measured in the forearm microcirculation of 56 normotensivesubjects and 57 patients with essential hypertension after bradykinin (0.015 microg x 100 mL(-1) x min(-1)) and acetylcholine (1.5 microg x 100 mL(-1) x min(-1)) infusions, with or without sulfaphenazole (0.03 microg x 100 mL(-1) x min(-1)). Bradykinin and acetylcholine infusions were repeated with N(G)-monomethyl-l-arginine (L-NMMA; 100 microg x 100 mL(-1) x min(-1)) and/or sulfaphenazole. tPA release by bradykinin and acetylcholine was higher in normotensive subjects than in patients with essential hypertension (P<0.01). Sulfaphenazole (P<0.01) blunted bradykinin-induced but not acetylcholine-induced tPA release in both groups. In normotensive subjects, L-NMMA infusion reduced tPA release (P<0.01). When L-NMMA was coinfused with sulfaphenazole, tPA release induced by bradykinin, but not by acetylcholine, was further reduced (P<0.01). In patients with essential hypertension, tPA release by both agonists was unaffected by L-NMMA, but only bradykinin-induced tPA release was blunted by sulfaphenazole, alone or with L-NMMA (P<001). CONCLUSIONS: Sulfaphenazole inhibits bradykinin-induced tPA release, which suggests a modulatory role of CYP 2C9-derived endothelium-derived hyperpolarizing factors in tPA release in humans. In patients with essential hypertension, tPA release depends exclusively on endothelium-derived hyperpolarizing factor, which is an ineffective compensatory mechanism in the presence of impaired nitric oxide availability.

The role of nitric oxide- and prostacyclin-independent vasodilatation in the human cutaneous microcirculation: effect of cytochrome P450 2C9 inhibition.

The component of the flow- or agonist-dependent vasodilatation, insensitive to inhibitors of nitric oxide (NO) synthases (NOS) or cyclooxygenases (COX), is suggested to reflect the production of an endothelium-dependent hyperpolarizing factor (EDHF). The identity of EDHF in humans remains controversial; in coronary arterioles, it appears to be a cytochrome P450 (CYP) 2C9-derived metabolite, whereas there are no data for human skin microcirculation. Therefore, the aim of our study was to investigate the role of the NO- and prostacyclin (PGI(2))-independent mechanism, particularly the potential involvement of CYP 2C9, in skin microcirculation. We measured skin blood flow on the volar aspect of the forearm in 12 healthy subjects by laser-Doppler fluxmetry (LDF). The inhibitors of NOS, N(omega)-monomethyl-L-arginine (L-NMMA), and cyclooxygenase (COX), diclofenac, as well as sulfaphenazole, the specific CYP 2C9 inhibitor, and saline as control, were administered to the measurement sites by an intradermal microinjection in different combinations. Afterwards, baseline LDF was assessed and iontophoresis of acetycholine (ACh) applied. Combined NOS and COX inhibition had no effect on baseline LDF, whereas it significantly reduced the ACh-induced increase in LDF (t-test, P<0.05). Sulfaphenazole did not affect baseline LDF either in the control site or in the L-NMMA- and diclofenac-pretreated site. In addition, sulfaphenazole did not attenuate the ACh-induced vasodilatation in either site. We conclude that a NO- and PGI(2)-independent vasodilator mechanism, potentially attributable to EDHF, contributes substantialy to the ACh-induced vasodilatation in human skin microcirculation and that it is probably not a CYP 2C9-derived metabolite.

Cytochrome p450 2C enzymes contribute to peritransplant ischemic injury and cardiac allograft vasculopathy.

Peritransplant ischemia and reperfusion (I/R) injury contributes to posttransplant vascular dysfunction and cardiac allograft vasculopathy (CAV). We have previously shown that cytochrome p450 (CYP) 2C inhibition significantly reduces I/R-induced myocardial infarction and postischemic vascular dysfunction. In the latter study, pretreatment with sulfaphenazole (SP), a specific inhibitor of CYP 2C, restored postischemic NO-mediated, endothelium-dependent vasodilation and reduced vascular superoxide production. Given the association between I/R injury, early vascular dysfunction and CAV, we hypothesized that CYP 2C may also contribute to the onset of CAV. Lewis-to-Fisher rat heterotopic heart transplants were performed. Donors and recipients were treated with 5 mg/kg SP or vehicle control 1 h prior to surgery. SP did not affect posttransplant morbidity, mortality or weight gain. Coronary blood vessels from rats treated with SP exhibited significantly reduced luminal narrowing and demonstrated a corresponding decrease in smooth muscle cell (SMC) proliferation compared to controls. SP did not reduce diffuse, focal, epicardial, endocardial or perivascular immune infiltration nor did it significantly alter TUNEL positivity in myocardial, endothelial or SMC populations. In conclusion, CYP 2C contributes to SMC proliferation CAV without affecting general immune infiltration.

Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice.

Vascular dysfunction is linked with increased free radical generation and is a major contributor to the high mortality rates observed in diabetes. Several probable sources of free radical generation have been suggested in diabetes, including cytochrome P450 (CYP) monooxygenase-dependent pathways. CYP-mediated superoxide production reduces nitric oxide (NO) bioavailability. In this study, we focus on the contribution of monooxygenase enzyme-generated reactive oxygen species in vascular dysfunction in an experimental model of diabetes mellitus type II. Diabetic male mice (db/db strain) and their age-matched controls received daily intraperitoneal injections of either the CYP 2C inhibitor sulfaphenazole (5.13 mg/kg) or saline (vehicle control) for 8 weeks. Although sulfaphenazole did not change endothelium-dependent vasodilation in control mice, it restored endothelium-mediated relaxation in db/db mice. We report for the first time that CYP 2C inhibition reduces oxidative stress (measured as plasma levels of 8-isoprostane), increases NO bioavailability (measured as NO(2)(-)) and restores endothelial function in db/db mice without affecting plasma glucose levels. Based on our findings, we speculate that inhibition of free radical generating CYP 450 monooxygenase enzymes restores endothelium-dependent vasodilation to acetylcholine. In addition, it reduces oxidative stress and increases NO bioavailability.

Role of vascular heme oxygenase in reduced myogenic reactivity following chronic hypoxia.

OBJECTIVE: Exposure to chronic hypoxia (CH) results in a persistent endothelium-dependent vascular smooth muscle hyperpolarization that diminishes vasoconstrictor reactivity. Experiments were performed to test the hypothesis that products of both cytochrome P450 epoxygenase (CYP) and heme oxygenase (HO) are required for the persistent diminished myogenic reactivity following CH. METHODS: The authors examined myogenic responses of mesenteric arteries isolated from control and CH (48 h; P(B) = 380 mmHg) rats in the presence of a HO inhibitor (zinc protoporphyrin IX; ZnPPIX) or combined HO and CYP epoxygenase inhibition (sulfaphenazole). Arteries were isolated and cannulated and the vascular smooth muscle was loaded with the Ca2+ indicator Fura-2. RESULTS: Control vessels maintained their internal diameter in response to step increases in intraluminal pressure, whereas arteries from CH animals passively distended. ZnPPIX augmented myogenic reactivity and [Ca2+] in arteries from CH animals. Combined administration of sulfaphenazole and ZnPPIX did not have an additional effect compared to ZnPPIX alone. Myogenic reactivity in control vessels was not altered by ZnPPIX or ZnPPIX + sulfaphenazole. CONCLUSIONS: HO appears to play a role in regulating myogenic reactivity following CH. Furthermore, these data suggest that products of HO and CYP are both required for the observed attenuation in vasoreactivity following CH.

Sulphaphenazole, streptomycin and sulphaphenazole combination, trimethoprim, and erythromycin in the treatment of chancroid.

One hundred and thirty six patients with chancroid were treated with four different treatment regimens; (A) Sulphaphenazole 1 g 12 hourly by mouth x 10 days (B) Inj streptomycin 1 g intramuscularly daily with sulphaphenazole 1 g 12 hourly orally x 10 days; (C) trimethoprim 200 mg 12 hourly by mouth x 7-10 days, and (D) erythromycin 500 mg 6 hourly orally x 7-10 days. Cure rates of 9% with sulphaphenazole alone, 48% with streptomycin and sulphaphenazole combination, 93% with trimethoprim and 100% with erythromycin were obtained. Sulphaphenazole alone or in combination with streptomycin were thus inferior in the treatment of chancroid. There is need for modification of treatment regimens recommended for chancroid in the textbooks of dermatology and venereology. Trimethoprim can be recommended as first line of treatment for chancroid in developing countries like India where resistance to trimethoprim is uncommon and erythromycin is suggested as a second line of therapy because by that time syphilis can be easily ruled out.

Urinary tract infections in children. Part I-- Young girls with non-refluxing ureters.

This paper is the study of 144 girls with histories of urinary tract infection followed at the Stanford Medical Center who were found to be free of ureterovesical reflux. The mean age at onset of infection for the entire group was about four years and ranged from the first few months of life to age 10. Ninety-two percent of the 505 infection episodes in these children presented with symptoms referable to the lower tract, and bacterial localization studies confirmed that 85 percent of the infections were limited to the bladder. Escherichia coli was the most common organism isolated and most infections were caused by a pure culture of a single bacteria. In only two of the 144 patients studied was there any evidence of upper tract damage related to infection. The possibility that these patients had reflux at an earlier age could not be discounted. In response to short-term antibacterial therapy in 66 of the patients followed closely for an average of 40 months each, 20 percent of the patients had no further infections and 80 percent went on to recurrence. With each succeeding treatment an additional 20 percent of the patients were "cured," but the remainder experienced recurrent infections during the follow-up. This reinfection pattern supports the use of long-term antibacterial prophylaxis in all girls who have more than three or four recurrences of infections. Urethral dilation appeared to have no value in reducing the reinfection rate. While it appears that in the absence of ureterovesical reflux few, if any, of these children will go on to develop upper tract damage, long-term prophylactic suppressive medication can clearly be justified on the grounds of reducing patient morbidity.