Cavernosal dysfunction in a rabbit model of hyperhomocysteinaemia

OBJECTIVE: To investigate the effect of sustained hyperhomocysteinaemia (HHCy) on cavernosal smooth muscle function in a rabbit model of HHCy, developed using a methionine-enriched diet in which cavernosal responses were characterized, as elevated plasma levels of homocysteine may be a risk factor for vasculogenic erectile dysfunction. MATERIALS AND METHODS: Six New Zealand White rabbits were fed a diet supplemented with methionine (20 g/kg chow) for 4 weeks, while six control animals were fed a standard diet. Cavernosal strips were mounted in an organ bath and relaxation assessed when stimulated with carbachol, sodium nitroprusside (SNP), or noncholinergic, nonadrenergic (NANC)-mediated relaxation to electrical-field stimulation (EFS). Cavernosal tissue cGMP levels were assessed using an enzyme-linked immunosorbent assay, and superoxide (O(2) (.-)) production assessed using an assay of the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. RESULTS: The methionine-rich diet led to an early but sustained HHCy; cavernosal strips from animals after 4 weeks of HHCy had a significantly impaired relaxation response to carbachol, an index of endothelium-dependent nitric oxide (NO)-mediated relaxation. This impairment was reversed by incubating with either SOD or catalase. Relaxation with either SNP, an index of endothelium-independent NO-mediated relaxation, or NANC-mediated EFS-induced relaxation, was unaffected by HHCy. There was a corresponding significant reduction in cavernosal cGMP levels (index of NO activity) in the HHCy group, with a more than five-fold increase in cavernosal tissue O(2) (.-) production. CONCLUSION: Supplementing the diet of rabbits with methionine for 4 weeks caused an early and sustained HHCy and promoted a marked inhibitory effect on endothelium-dependent relaxation and NO formation in isolated corpus cavernosum, an effect mediated by reactive oxygen species.     

An automated flow injection-serial dynamic dialysis technique for drug-protein binding studies

The interface of an automated flow injection analyzer with a dialysis unit to study drug-protein interactions using flow injection serial dynamic dialysis (FISDD) procedure is described. The method is based on the study of the kinetics of dialysis of the ligand in the absence and presence of protein. A study of the binding of sulfamethoxazole. sulfamethizole and sulfisoxazole to bovine serum albumin by means of such an automated system was undertaken to investigate the utility of FISDD technique for protein binding studies. The determination of dialysable sulfonamides was performed automatically by the FIA analyser. The influence of ionic strength and viscosity on the rate of dialysis was investigated. It was found that both variables did not affect the kinetic profile. Binding by the cellophane membrane was not encountered as a problem with the compounds studied. Binding parameters estimated for sulfamethoxazole were found to agree well with those reported in the literature. The Scatchard plots for the binding of sulfamethizole and sulfisoxazole with bovine serum albumin, revealed two classes of binding sites for each sulfonamide. The system was also used for the calculation of the dialytic rate constants. Experimental variables can be readily controlled to yield favoured conditions to study the protein binding phenomenon.     

Drug Binding and Solubility in Milk

The binding and solubility of nitrofurantoin, piroxicam, indomethacin, prednisolone, diazepam, dicumarol, and griseofulvin in milk were determined at 15, 25, and 37°C in bovine milk samples with fat contents of 0.75 and 3.50%. Drug binding to milk components was independent of drug concentration over the drug concentration studied, and the fat content of milk strongly affected binding values of most of the listed drugs. Further, drug binding increased with decreasing temperatures for most of the drugs examined. The solubility of all drugs is greatly enhanced in milk compared to their aqueous solubility (pH 6.5 phosphate buffer). The high solubility cannot be accounted for solely on the basis of drug binding to milk components. An attempt is made to correlate the binding and solubility data with physicochemical properties of the drugs (logP, pK _a, aqueous solubility). The potential significance of these findings is discussed with regard to preparation and in vivo delivery of drugs from drug–milk formulations.     

Development and validation of a novel LC non-derivatization method for the determination of amikacin in pharmaceuticals based on evaporative light scattering detection

A novel method for the direct determination of the aminoglycoside antibiotic amikacin and its precursor component kanamycin was developed and validated, based on reversed phase LC with evaporative light scattering detector (ELSD). ELSD response to amikacin was found to be enhanced by: (a) use of ion-pairing acidic reagents of increased molecular mass, (b) increase of mobile phase volatility and (c) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity and/or ratio of organic solvent to water). Utilizing a Thermo Hypersil BetaBasic C₁₈ column, the selected optimized mobile phase was water–methanol (60:40, v/v), containing 3.0 ml l⁻¹ nonafluoropentanoic acid (18.2 mM) (isocratic elution with flow rate of 1.0 ml min⁻¹). ELSD experimental parameters were: nitrogen pressure 3.5 bar, evaporation temperature 50 °C, and gain 11. Amikacin was eluted at 8.6 min and kanamycin at 10.4 min with a resolution of 1.5. Logarithmic calibration curves were obtained from 7 to 77 μg ml⁻¹ (r > 0.9995) for amikacin and 8 to 105 μg ml⁻¹ (r > 0.998) for kanamycin, with a LOD equal to 2.2 and 2.5 μg ml⁻¹, respectively.

In amikacin sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (t_R = 2.3 min, LOD = 1.8 μg ml⁻¹, range 5–40 μg ml⁻¹, %R.S.D. = 1.1, r > 0.9997), kanamycin and amikacin was feasible. No significant difference was found between the results of the developed LC–ELSD method and those of reference methods, while the mean recovery of kanamycin from spiked samples (0.5%, w/w) was 97.3% (%R.S.D. ≤ 2.0, n = 6). Further, the developed method was applied for the determination of amikacin in pharmaceutical formulations (injection solutions) without any interference from the matrix (recovery from spiked samples ranged from 95.6 to 103.8%).     

Differing levels of testosterone and the prostate: a physiological interplay

The controversies surrounding testosterone replacement therapy (TRT) have been addressed in the past few years. Although the androgenic effects of TRT on normal and malignant prostate cells have been studied for over 70 years, little clinical prospective research exists on the physiological responses of prostate tissues to a wide range of serum testosterone levels. The prostate is both an androgen-dependent and an androgen-sensitive organ; active processes are triggered at a 'threshold' or 'saturation' level of testosterone. Despite decades of research, no compelling evidence exists that increasing testosterone beyond this threshold level has a causative role in prostate cancer, or indeed changes the biology of the prostate. Testosterone deficiency has marked physiological and clinical effects on men in middle age and beyond. With subnormal testosterone levels, the potential positive benefits of TRT on factors such as muscle mass, libido or erectile function are likely a dose-response phenomenon, and should be considered differently than the threshold influence on the prostate. This Review will re-examine classic androgen research and reflect on whether testosterone actually stimulates prostatic cellular growth and progression in a 'threshold' or a 'dose-response' (or both) manner, as well as discuss the influence of testosterone on prostate cells in the hypogonadal and eugonadal states.     

Novel targeted agents on the horizon for castration-resistant prostate cancer

Androgen deprivation treatment in prostate cancer patients is well established; however, resistance to such treatment manifests itself by progression to castration-resistant prostate cancer (CRPC). Despite significant advances in treatment options for patients with CRPC, their prognosis remains poor. Resistance results from multiple processes that facilitate cancer cell growth and survival. Mechanisms underlying the shift to castrate resistance have been attributed to a complex interplay of clonal selection, reactivation of the androgen receptor axis despite castrate levels of serum testosterone, stress-induced prosurvival genes and cytoprotective chaperone networks and alternative mitogenic growth factor pathways. This article discusses several pathways involved in the development of CRPC, with a particular focus on those mechanisms that have led to the development of new targeted therapies.     

Automated flow-injection mercurothiocyanate determination of chloride salts of drugs for routine assays: content uniformity and dissolution studies

An automated flow-injection determination of chloride salts of drugs, based on the colorimetric mercurothiocyanate determination of chloride counterion is described. The majority of these drugs can be determined, using chloride ion standards, with a coefficient of variation less than 1% and a measurement rate of 120 samples per hour. The method is evaluated by determining the interference caused by common inorganic ions, representative organic functional groups, and excipients, and by the analysis of pure drugs and their commercial formulations. The results compare well with those obtained by the official procedures. The benefits of the method are demonstrated in content uniformity tests and in automated monitoring of dissolution studies.