Spectrophotometric determination of sildenafil citrate in pure form and in pharmaceutical formulation using some chromotropic acid azo dyes.

Two simple and highly sensitive spectrophotometric methods were developed for the quantitative determination of the drug sildenafil citrate (SC), Viagra, in pure form and in pharmaceutical formulations, through ion-associate formation reactions (method A) with mono-chromotropic acid azo dyes, chromotrope 2B (I) and chromotrope 2R (II) and ion-pair reactions (method B) with bi-chromotropic acid azo dyes, 3-phenylazo-6-o-carboxyphenylazo-chromotropic acid (III), bis-3,6-(o-hydroxyphenylazo)-chromotropic acid (IV), bis-3,6-(p-N,N-dimethylphenylazo)-chromotropic acid (V) and 3-phenylazo-6-o-hydroxyphenylazo-chromotorpic acid (VI). The reaction products, extractable in methylene chloride, were quantitatively measured at 540, 520, 540, 570, 600 and 575 nm using reagents, I-VI, respectively. The reaction conditions were studied and optimized. Beer's plots were linear in the concentration ranges 3.3-87.0, 3.3-96.0, 5.0-115.0, 2.5-125.0, 8.3-166.7 and 0.8-15.0 microg mL(-1) with corresponding molar absorptivities 1.02 x 10(4), 8.34 x 10(3), 6.86 x 10(3), 5.42 x 10(3), 3.35 x 10(3) and 2.32 x 10(4)Lmol(-1) cm(-1) using reagents I-VI, respectively. The limits of detection and Sandell's sensitivities were calculated. The methods were successfully applied to the analysis of commercial tablets (Vigoran) and the recovery study reveals that there is no interference from the common excipients that are present in tablets. Statistical comparison of the results was performed with regard to accuracy and precision using Student's t- and F-tests at 95% confidence level. There is no significant difference between the reported and proposed methods with regard to accuracy and precision.

Analysis for loss of heterozygosity (LOH) of p53 allele in tumors derived from p53+/- and CD-1 mice following repeated subcutaneous injections of solutions containing antioxidants.

Genomic DNA was isolated from subcutaneous masses observed in CD-1 and p53+/- heterozygous mice during the course of carcinogenicity studies in the vehicle control groups. These masses resulted after daily subcutaneous injection of an antioxidant vehicle with a pH adjusted to 3-4. The vehicle was 1.0% ascorbic acid plus 0.05% sodium metabisulfite in 0.75% saline in a dosing volume of 10 ml/kg/day. These masses were first palpable after 13 and 37 weeks of dosing among p53+/- and CD-1 mice, respectively. By week 26, the incidence of these masses was 89% and 80% in male and female p53+/- mice, respectively (n = 15 mice/sex) and was 0% in both male and female CD-1 mice (n = 60 mice/sex). These masses originated from panniculus carnosus muscle. Histopathological examination of the p53+/- mouse masses indicated the tumors to be sarcomas of spindle-cell origin. The histopathological examination of the masses in the CD-1 mice revealed fibrosarcomas. Five mice/sex/strain were randomly selected from a pool of mice that developed these masses in the course of the two studies. Frozen tissues from these masses were used to examine the DNA for loss of the functional p53 allele in the p53+/- mice (i.e., loss of heterozygosity, or LOH) or for loss of one of the alleles in the wild type (p53+/+) CD-1 mice by the polymerase chain reaction (PCR) technique. Loss of the functional allele was observed only in the tumor from one p53+/- male mouse. These results support a nongenotoxic mechanism for these injection site masses.

Synthesis of di- and tripeptide analogues containing alpha-ketoamide as a new core structure for inhibition of HIV-1 protease.

Di- and tripeptide analogues containing alpha-ketoamide as a new core structure and incorporating allophenylnorstatine (Apns) as a transition state mimic, were designed and synthesized in the hope of obtaining a novel structural type of HIV-1 protease inhibitors. The immediate precursor, Apns-Thz-NHBu(t) was prepared by coupling of Boc-Apns with N-tert x butyl Thz-4-carboxamide hydrochloride. Removal of Boc group followed by coupling with the respective alpha-ketoacid residue (P2) gave the desired dipeptides (8-12) in almost quantitative yields. The alpha-keto tripeptides (18-21) were obtained by oxidation of the hydroxyl group of Apns (PI) in the appropriate tripeptide, iQOA-Val-Apns-(un)substituted Thz(Oxa)-NHBu(t) with DMSO/DCC. Preliminary evaluation of the activity of the synthesized derivatives was determined as percentage of enzyme inhibition at 5 microM and 50 nM levels of the di- and tripeptides respectively. The alpha-ketoamides displayed a significant enhanced potency relative to their parent isosteres as inhibitors of HIV-1 protease and are shown to be a promising new core structure for the development of enzyme inhibitors. A quantitative approach was attempted, using an LFE model, correlating the effect of structural modification and HIV-1 protease inhibition activity of the prepared dipeptides. The result indicates the contribution of the torsion angle by 84% to the activity of the inhibitors.

Evaluation of apomorphine HCl effects on reproductive endpoints: studies in male rats and dogs.

Apomorphine HCl is currently in phase III clinical trials for the treatment of erectile dysfunction. The potential for reproductive toxicity in males was assessed based on a 13-week rat study--a fertility study in male rats--and a 6-month study in dogs. The subcutaneous (s.c.) route was selected to simulate the sublingual route in humans. Dosages of apomorphine were 0.0 (vehicle), 0.8, 2, and 8 mg/kg/day in the 13-week study in rats (20/group), with 8 mg/kg/day used for only 9 weeks. In the fertility study, 24 males/group were cohabited with females, and doses were 0.0, 0.2, 0.8, and 2 mg/kg/day. Males were treated for 4 weeks prior to cohabitation and for 5 weeks throughout cohabitation. Organ weights, including testis and left epididymis, sperm count and morphology in the epididymis, and sperm motility in the vas deferens were evaluated. Male fertility index, and in females, the numbers of fetuses, implantation sites, and corpora lutea were counted. Male dogs (five/group) were dosed with 0.04, 0.1, or 0.4 mg/kg/day for 6 months. Epididymal and prostate weight, and testicular and epididymal histology were evaluated. Daily morbidity/mortality, weekly clinical observations, body weight, and food consumption were evaluated in all studies. No adverse effect was observed in any of the reproductive parameters in the studies. The NOEL for reproductive toxicity was approximately equal to 0.4 mg/kg/day in dogs and > or = 2 mg/kg/day in rats. These doses in rats and dogs correlated with plasma levels of approximately 240 and 130 ng/mL, and AUCs of 200 and 100 ng.h/mL, respectively. These levels suggest a safety margin for the evaluated male reproductive endpoints of at least 104 times based on the Cmax, and 44 times based on AUC of the clinical dose.

Teratogenicity of methanol following a single oral dose in Long-Evans rats.

The teratogenicity of methanol was investigated following a single oral exposure preceded by an equal volume of mineral oil to guard against local gastric irritation. Four groups of pregnant Long-Evans rats were gavaged on day 10 of gestation with the following solutions: 0.0 (n = 13), 1.3 (n = 12), 2.6 (n = 11), and 5.2 (n = 10) mL MeOH/kg. Wilson sectioning (head only), gross necropsy, and Alizarin red skeletal examinations were performed on day 20 of gestation. At 5.2 mL/kg, the dams demonstrated > 20% decrease in weight gain in comparison to the control, which was the only clinical toxic manifestation or histopathologic change noted for the dams. Methanol at all doses failed to produce any significant change in standard reproductive indices (e.g., postimplantation loss). A significant decrease in fetal body weight (11 to 19.5%), however, was associated with prenatal oral ingestion of methanol. Both internal and external examination of the fetuses demonstrated a dose-dependent increase in anomalies [0 = 0.6%, 1.3 mL/kg = 3.7%, 2.6 mL/kg = 7%, 5.2 mL/kg = 16.5% (litter percents)]. The dose-related anomalies were undescended testes, exophthalmia, and anophthalmia. Thus, acute methanol given orally produces anomalies, even when there is no apparent maternal toxic response.

Simple neurobehavioral functional observational battery and objective gait analysis validation by the use of acrylamide and methanol with a built-in recovery period.

A functional observational battery (FOB) with modified objective gait analysis was validated using the neurotoxicants acrylamide and methanol. The FOB consisted of side cage observation (30 sec); open field observation (2 min); evaluation of handling, reflexes, and gait scoring; and grip strength of the fore limbs and hind limbs. Temperature and body weight were measured. Gait analysis was based on measurements of the distances and angles between sequential foot strides after walking in a corridor (90 x 10 x 10 cm). Landing foot splay was the distance between the inner surfaces of the fourth digits of each foot after the animals were dropped from a 30-cm height. Acrylamide was given at doses of 0 (saline), 20, and 40 mg/kg intraperitoneally. Days of dosing were 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. Evaluations were done before dosing on Days 0, 2, 5, 11, 17, and 27 (for recovery). Methanol was given to another group at doses of 0 (distilled water), 3.25 (escalated to 8.5 before end of evaluation), and 6.5 ml/kg. Days of dosing were 3, 5, 7, and 10; evaluations were conducted at 0, 6, 8, and 12 days. Acrylamide at 40 mg/kg demonstrated a biologically significant increase in foot splay in males on Days 3 and 17 and was not recovered by Day 27. A significant decrease in the hind limb grip strength was seen in high-dose females, but with incomplete recovery on Day 27. Methanol (3.25/8.5 ml/kg) induced a biological decrease of activity postdosing on Days 6, 8, and 12. A significant increase in gait length was seen in females only at the 6.5 ml/kg dose at Day 12. Selected doses and schedule of observation validated the battery for screening and better quantification of acute and chronic neurotoxicity.

Reproductive hazards of lead exposure among urban Egyptian men.

Fifty-five urban Egyptian males, aged 20-40, were assigned to two main groups to study the effects of their exposure to lead (Pb). Group I, infertile men (INF, n = 30), was divided into environmentally exposed (INF-E, n = 15) and environmentally and occupationally exposed (INF-EO, n = 15). A matching group (II) of fertile men (F, n = 25) was divided into fertile, environmentally exposed (F-E, n = 10), which was the control group, and fertile, environmentally and occupationally exposed (F-EO, n = 15). Semen parameters (i.e., count, morphology, motility, and volume), blood and semen Pb levels, and reproductive hormonal indices (i.e., serum testosterone, FSH, and LH) were measured in all subjects. Lead levels were always higher in blood than semen. Semen lead levels were significantly higher in all groups vs. the control (F-E) group. While no changes were observed in testosterone levels across groups, variable effects on LH and FSH levels were observed. Infertile-EO subjects showed a definite pattern of impaired semen parameters in comparison with infertile-E. No abnormalities were detected in hematologic, hepatic or renal function.

Synthesis and investigation of N4-substituted cytarabine derivatives as prodrugs.

Esters of Cytarabine-N4-carboxylates 2a-i and succinamates 3a-f were synthesized as prodrugs of cytarabine (Ara-C) with the aim of developing improved derivatives for oral or parentral administration. At pH 2 series 2 showed relative higher stability than 3, while both series of esters revealed matched stability at pH 7. All esters were susceptible to enzymatic hydrolysis by rat plasma and liver homogenate with half lives ranged from 0.14 h to 12 d, and showed improved stability against cytidine deaminase. A parabolic relation was shown between Kobs of enzymatic hydrolysis and Vw. All compounds are more lipophilic than the parent drug, Ara-C.

Neurobehavioral toxicity of methanol reflected by operant running.

Eleven Long-Evans male rats were trained to respond for food delivery by running in wheels under a Fixed Ratio = FR 20 schedule of reinforcement. Each 360 degrees rotation counted as a single response. Three food pellets were delivered for each reinforcement. The wheels, which provided transverse rods to be gripped by the rats, were specifically designed to reflect motor deficits produced by neurotoxicants. Each animal received two replicates of three different doses of methanol (50% in water): 1.0, 2.0, and 3.0 g/kg by gavage. The sequence for each animal was determined by a counterbalanced design. Gavage was followed by admitting the animal to the running wheel compartment 10 min later. Running wheel sessions lasted for 1 hour daily and were conducted 6 days/week. Statistical analyses showed insignificant differences between water and no-treatment control days, indicating no effect of the gavage procedure. However, a dose-effect relationship between methanol dose and responses per session proved statistically significant and linear (p < 0.0001) down to a dose equivalent to 10% of the LD50. In addition, detailed analyses of intervals between successive rotations (IRTs) indicated a displacement of the distribution toward longer intervals (decreased velocities) with increasing dose. The absence of a corresponding rise in the incidence of long pauses suggested that impaired coordination, reduced endurance, or their combination, rather than nonspecific variables, accounted for these results.

Colorimetric determination of C-2 unsubstituted phenothiazines, using morpholine and N-bromosuccinimide.

A colorimetric method for quantitative determination of 6 C-2 unsubstituted phenothiazine derivatives is described. The method is based on reaction of phenothiazine derivative with morpholine and N-bromosuccinimide reagents in aqueous methanol at 60 degrees C to produce a blue color. Beer's law is obeyed in a wide range of concentrations for all 6 compounds at 660 nm. Optimum analytical conditions were determined and the produced color is stable for at least 24 h. Analytical data for determination of the pure compounds are presented together with the application of the proposed method to analysis of some pharmaceutical preparations. The results compare favorably with those of official methods. Furthermore, the method is specific for intact drug in the presence of oxidative decomposition products. The reaction product has been isolated and identified and a possible reaction mechanism is suggested.

Colorimetric determination of certain phenothiazine drugs by using morpholine and iodine-potassium iodide reagents.

A colorimetric method was developed for the quantitative estimation of 11 phenothiazine drugs. The method is based on the interaction of unsulfoxidized drug with morpholine and iodine-potassium iodide reagents. The interaction for all studied phenothiazine drugs yields a blue product with 2 absorption maxima: one in the range of 620-640 nm with lower molar absorptivity and the other in the range of 662-690 nm with higher molar absorptivity. The color was stable for at least 10 h. The reproducibility and recovery of the method were excellent. The method was applied successfully to the analysis of various commercially available phenothiazines in different dosage forms. The results were comparable to those obtained by official procedures. The suitability of the method for detection and estimation of promethazine excreted in urine has been suggested by preliminary experiments. Reaction products have been isolated and identified.

Dihydralazine sulfate analysis using 2-methyl-o-nitropyridine-6-carboxaldehyde.

A sensitive, selective colorimetric assay was developed for the quantitative analysis of dihydralazine sulfate. The method is based on the interaction of buffered (pH 4) dihydralazine sulfate with a methanolic solution of 2-methyl-3-nitropyridine-6-carboxaldehyde upon heating to give an orange color. This color can be quantified spectrophotometrically at 450 nm,with a lower limit of detection of 1 mug/ml. The color is stable for at least 24 hr. There is no interference from other drugs likely to be present along with dihydralazine sulfate and common excipients. The method was used successfully for the determination of dihydralazine sulfate in combination with other drugs in different commercial tablets. The developed method was applicable as a stability-indicating assay.

Effects of contraceptive pills and intrauterine devices on urinary bladder.

The incidence of bacteriuria and cystoscopic changes in women on oral contraceptives or users of IUD (intrauterine devices) were evaluated compared with a control group. Subjects with bacteriuria accounted for 40.5 per cent of pill users, 20 per cent of women fitted with IUD, and 16 per cent of the control group. Bladder trabeculations were found in 50.5 per cent of the pill group, 8.7 per cent of the IUD group, and 8 per cent of the controls. Congested bladder trigone was observed in 24.6 per cent of women fitted with an IUD. The mode of action of ovarian hormones and their possible side effects on the bladder are discussed. The relation of IUD to pelvic inflammatory disease and its effect on the urinary bladder are evaluated.

PIP: The incidence of bacteriuria and cystoscopic changes in 200 women using oral contraceptives from 1 month to 2 years and 150 users of IUD were compared with 50 women using neither method. Bacteriuria in midstrean urine samples of all subjects were identified qualitatively and quantitatively by the method of Stamey et al. Cystoscopy was performed on all subjects. Positive bacteriuria were found in 40.5% of patients on oral contraceptives, 20% of IUD users, and 16% of the control group. Positive pathogenic bacterial cultures in women using oral contraceptives were proportional to the length of time the pill had been used: 27% for 1 year users, 35% for 2 year users, and 50% for longer users. The potency and dosage is correlated with the pathogenic effects observed. Bladder trabeculations were found in 50.5% of oral contraceptive users, 8.7% of IUD users, and 8% of control subjects. The highest incidence was in subjects using pills for 2 years or more, and is correlated with the dosage used. Bladder trigone congestion was observed in 24.6% of IUD users; 56% of users from 1 to 3 months, 35% of users more than 2 years, and no users between these time extremes. The high incidence of bladder trabeculations might be explained by the effect of progestogens and estrogens on bladder tone; progestogens producing hypotonia and estrogens hypertonia. The IUD should not be used more than 2-3 years continuously to avoid urinary bladder effects and inflammatory pelvic disease.