Rapid determination of amphotericin B in serum and urine by third-order derivative spectrophotometry

A derivative spectrophotometric method for rapid monitoring of amphotericin B in serum and urine down to 30 ng/mliters is described. Samples are treated with acetonitrile, and amphotericin B is directly quantified in the crude extracts on the basis of the intensity of the peak that appears at 402 nm when the normal absorption spectrum is submitted to third-order derivative processing. Accuracy data suggested recoveries in the range of 84.3-94.9% for serum and 85.6-93.4% for urine. The precision of the method was better than 11.3% for serum and 9.2% for urine when samples contained as low as 29.6 ng/mliters of amphotericin B. Ease of applicability, short analysis time, low cost, and reliability are the main advantages of the method.     

Comparison of a new triazole antifungal agent, Schering 56592, with itraconazole and amphotericin B for treatment of histoplasmosis in immunocompetent mice

A murine model of intratracheally induced histoplasmosis was used to evaluate a new triazole antifungal agent, Schering (SCH) 56592, for treatment of histoplasmosis. MICs were determined for SCH 56592, amphotericin B, and itraconazole by testing yeast-phase isolates from 20 patients by a macrobroth dilution method. The MICs at which 90% of the isolates are inhibited were for 0.019 microgram/ml for SCH 56592, 0.5 microgram/ml for amphotericin B, and < or = 0.019 microgram/ml for itraconazole. Survival studies were done on groups of 10 B6C3F1 mice with a lethal inoculum of 10(5). All mice receiving 5, 1, or 0.25 mg of SCH 56592 per kg of body weight per day, 2.5 mg of amphotericin B per kg every other day (qod), or 75 mg of itraconazole per kg per day survived to day 29. Only 44% of mice receiving 5 mg of itraconazole/kg/day survived to day 29. Fungal burden studies done in similar groups of mice with a sublethal inoculum of 10(4) showed a reduction in CFUs and Histoplasma antigen levels in lung and spleen tissue in animals treated with 2 mg of amphotericin B/kg qod, 1 mg of SCH 56592/kg/day, and 75 mg of itraconazole/kg/day, but not in those treated with lower doses of the study drugs (0.2 mg of amphotericin B/kg qod, 0.1 mg of SCH 56592/kg/day, or 10 mg of itraconazole/kg/day). Serum drug concentrations were measured 3 and 24 h after the last dose in mice (groups of five to seven mice), each treated for 7 days with SCH 56592 (10 and 1 mg/kg/day) and itraconazole (75 and 10 mg/kg/day). Mean levels measured by bioassay were as follows: SCH 56592, 10 mg/kg/day (2.15 micrograms/ml at 3 h and 0.35 microgram/ml at 24 h); SCH 56592, 1 mg/kg/day (0.54 microgram/ml at 3 h and none detected at 24 h); itraconazole, 75 mg/kg/day (22.53 micrograms/ml at 3 h and none detected at 24 h); itraconazole, 10 mg/kg/day (1.33 micrograms/ml at 3 h and none detected at 24 h). Confirmatory results were obtained by high-pressure liquid chromatography assay. These studies show SCH 56592 to be a promising candidate for studies of treatment of histoplasmosis in humans.     

A high-performance liquid chromatographic assay for the determination of amphotericin B serum concentrations after the administration of AmBisome, a liposomal amphotericin B formulation

A sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the determination of amphotericin B in human serum. After methanol deproteinization, amphotericin B and 3-nitrophenol (internal standard) are separated by reversed-phase chromatography and detected by ultraviolet absorbance. The analysis of human serum after the standard addition of amphotericin B (0.05-200.0 micrograms/mL) demonstrated excellent precision and accuracy over a five-day period. The HPLC assay uses two standard curve ranges. The high sensitivity curve range for low AmBisome dosage (1.0 mg/kg) is 0.05-20.0 micrograms/mL (curve 1), and the second curve range for the higher AmBisome dose regimens (2.5-5.0 mg/kg) is 0.5-200 micrograms/mL (curve 2). The intraday and interday coefficients of variations for standard curve 1 were 0.5-4.6% and 3.0-11.5%, respectively. The limit of quantitation was 0.05 microgram/mL. The intraday and interday coefficients of variation for standard curve 2 were 2.0-3.6 and 6.9-10.1, respectively. No interfering peak at the retention time for Amphotericin B and the internal standard were present in blank serums or serum samples spiked with fifteen potential co-administrated drugs with Amphotericin B treatment. The method was used to quantitate serum concentrations of amphotericin B in patients after the administration of AmBisome, a liposomal formulation of amphotericin B.     

Randomized, double-blind clinical trial of amphotericin B colloidal dispersion vs. amphotericin B in the empirical treatment of fever and neutropenia

The possibility that there is an inhibitory component to auditory covert orienting was addressed. Each trial consisted of a cue followed by a target, and listeners were required to detect, localize, or identify the frequency of the target. At 150-msec stimulus onset asynchrony (SOA), performance was best when stimuli sounded from the same location or were of the same frequency. However, at 750-msec SOA, performance was best when stimuli differed in location or were of different frequencies. These results document the existence of both location-based and frequency-based auditory inhibition of return.     

Efficacy of NS-718, a novel lipid nanosphere-encapsulated amphotericin B, against Cryptococcus neoformans

In vitro and in vivo efficacies of NS-718, a lipid nanosphere-encapsulated amphotericin B (AMPH-B), have been studied. Of the tested AMPH-B formulations, NS-718 had the lowest MIC for Cryptococcus neoformans. In a murine model, low-dose therapy (0.8 mg/kg of body weight) with NS-718 showed higher efficacy than that with AmBisome. High-dose therapy (2.0 mg/kg) with NS-718 was much more effective than those with Fungizone and AmBisome. In mice treated with a high dose of NS-718, only a few yeast cells had grown in lung by 7 days after inoculation. A pharmacokinetic study showed higher concentrations of AMPH-B in lung following administration of NS-718 than after administration of AmBisome. Our results indicated that NS-718, a new AMPH-B formulation, is a promising antifungal agent for treatment of pulmonary cryptococcosis and could be the most effective antifungal agent against C. neoformans infections.     

Psammaplysin F, a new bromotyrosine derivative from a sponge, Aplysinella sp

A new member of the psammaplysin family, psammaplysin F (6) has been isolated from an undescribed species of Aplysinella sponge, along with the four known psammaplysins A-C (1-3) and E (5). The structure of psammaplysin F was determined by spectral analysis.     

Photodegradation products of a new antibacterial fluoroquinolone derivative, orbifloxacin, in aqueous solution

PURPOSE: The authors sought to assess the feasibility of in vivo gene transfer to the small intestine using recombinant adenovirus in neonatal and adult mice. METHODS: H5.010CMVlacZ is a replication-defective, E1-deleted human type 5 adenovirus, which contains the lacZ gene under the control of a cytomegalovirus promoter and enhancer. The lacZ gene was used as a marker because its gene product, beta-galactosidase, is readily detected by X-gal histochemistry. Sixty neonatal (3 to 5 days old) and 45 adult (6 to 8 weeks old) C57BL/6 mice were investigated. Intestinal gene transfer was attempted with H5.010CMVlacZ by intraperitoneal (i.p.), intraluminal (IL), and intramural (i.m.) injection. Based on prior studies, the optimal dose of H5.010CMVlacZ was 1 x 10(8) plaque forming units (pfu/mL). Control animals received saline injections. Gene transfer on repeat administration of adenovirus has been shown to be prevented by neutralizing antibody. To determine if neonatal inoculation induced a humoral immune response, neonates (n = 5) that received i.p. injections were rechallenged with intravenous H5.010CMV alkphos, a similar adenoviral construct containing the alkaline phosphatase marker gene. Serum samples were analyzed by Western blot to detect the presence of adenoviral-specific antibody. RESULTS: Gene transfer to neonatal small intestine was successful by IL gastric (n = 8/10), IL jejunal (n = 9/10) and i.p. (n = 10/10) routes 2 days after injection. Macroscopic staining was present in 90% of standardized 2-cm small bowel segments. Transgene expression was identified in intestinal smooth muscle, serosa, and epithelium. Gene transfer to the adult small intestine was successful by IL jejunal (n = 4/5), i.m. (n = 5/5), and i.p. (n = 1/5) injection of adenoviruslacZ with focal staining (< 5% of 2-cm segments) in epithelium including crypts, muscle, and serosa. Three weeks after i.p. H5.010CMVlacZ in neonates, intravenous injection with H5.010CMValkphos resulted in hepatic transgene expression (n = 4/5) that was indistinguishable from a primary intravenous inoculation; persistent, lacZ expression was not detectable in the liver or intestine (n = 0/5). Western blot analysis detected adenoviral-specific antibodies after adult IM but not after neonatal i.p. injection. Furthermore, 3 weeks after neonatal i.p. injection repeat administration by the i.m. route was successful (n = 4/ 4). CONCLUSION: Gene transfer to neonatal and adult small intestine is feasible using recombinant adenovirus and is more efficient in neonates as indicated by increased surface area of marker gene expression, effectiveness of intraperitoneal delivery, and the ability to readminister recombinant adenovirus.     

Superior efficacy of liposomal amphotericin B with prolonged circulation in blood in the treatment of severe candidiasis in leukopenic mice

In leukopenic mice with severe systemic candidiasis, single-dose treatment (5 mg of amphotericin B [AMB]/kg of body weight) with long-circulating polyethylene glycol-coated AMB liposomes (PEG-AMB-LIP) resulted in zero mortality and a significant reduction in the number of viable Candida albicans in the kidney, whereas 70% mortality was seen in mice treated with five daily doses of AmBisome (5 mg of AMB/kg . day). When the first of five daily doses of AmBisome was combined with a single low dose of Fungizone (0.1 mg of AMB/kg), the efficacy was equal to that of PEG-AMB-LIP.     

A high-performance liquid-chromatographic assay for amphotericin B in a hydrophilic colloidal paste base

A stability-indicating high-performance liquid-chromatographic (HPLC) assay has been developed for amphotericin B (AmB) in a paste, containing AmB, tobramycin (or gentamicin) sulphate, colistin sulphate, liquid paraffin and Orabase. Extraction of AmB was performed by partitioning the antibiotic between N,N-dimethylformamide (DMF) and cyclohexane, which led to precipitation of the polymeric materials and extraction of the liquid paraffin into the cyclohexane and AmB into the DMF. Analysis by HPLC of the latter layer gave a linear relationship between concentration and peak area response for the AmB over the range 5.0 x 10(-4) to 7.5 x 10(-3)% (w/v) (r = 0.9995) with a relative standard deviation of +/- 1.46% (n = 8). The efficiency of extraction was 1006 +/- 2.4% (n = 5).     

Effect of KCA-098, a new benzofuroquinoline derivative, on bone mineral metabolism

The effect of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one designated as KCA-098) on the bone mineral metabolism of chick embryonic bone was examined. KCA-098 dose-dependently inhibited bone resorption of cultured chick embryonic femora and calvariae. It increased the length, dry weight, and calcium and phosphorus contents of 9-d-old chick embryonic femurs cultivated for 6 d, indicating that it stimulated bone formation. These results show that KCA-098 has the unique effects of inhibiting bone resorption and stimulating bone formation of chick embryo. In addition, in an in vivo experiment, oral administration of KCA-098 (3.0 mg/kg/d) for 16 weeks led to an increase in calcium and phosphorus content as well as an increase in the amount of force required to break the femur from ovariectomized rats, suggesting that it may be useful for the treatment of bone diseases.     

Safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) in neutropenic patients

The safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) administered for empirical antifungal therapy were evaluated for 36 persistently febrile neutropenic adults receiving cancer chemotherapy and bone marrow transplantation. The protocol was an open-label, sequential-dose-escalation, multidose pharmacokinetic study which enrolled a total of 8 to 12 patients in each of the four dosage cohorts. Each cohort received daily doses of either 1.0, 2.5, 5.0, or 7.5 mg of amphotericin B in the form of AmBisome/kg of body weight. The study population consisted of patients between the ages of 13 and 80 years with neutropenia (absolute neutrophil count, <500/mm3) who were eligible to receive empirical antifungal therapy. Patients were monitored for safety and tolerance by frequent laboratory examinations and the monitoring of infusion-related reactions. Efficacy was assessed by monitoring for the development of invasive fungal infection. The pharmacokinetic parameters of AmBisome were measured as those of amphotericin B by high-performance liquid chromatography. Noncompartmental methods were used to calculate pharmacokinetic parameters. AmBisome administered as a 1-h infusion in this population was well tolerated and was seldom associated with infusion-related toxicity. Infusion-related side effects occurred in 15 (5%) of all 331 infusions, and only two patients (5%) required premedication. Serum creatinine, potassium, and magnesium levels were not significantly changed from baseline in any of the dosage cohorts, and there was no net increase in serum transaminase levels. AmBisome followed a nonlinear dosage relationship that was consistent with reticuloendothelial uptake and redistribution. There were no breakthrough fungal infections during empirical therapy with AmBisome. AmBisome administered to febrile neutropenic patients in this study was well tolerated, was seldom associated with infusion-related toxicity, was characterized by nonlinear saturation kinetics, and was effective in preventing breakthrough fungal infections.     

Antitrypanosomal activity of a new triazine derivative, SIPI 1029, In vitro and in model infections

A recently developed diaminotriazine derivative [O,O'-bis(1, 2-dihydro-2,2-tetramethylene-4,6-diamino-S-triazin-1-yl)-1, 6-hexanediol dihydrochloride; T-46; SIPI 1029] was examined for activity against African trypanosomes in in vitro and in vivo model systems. In vitro, SIPI 1029 was 50% inhibitory for growth of bloodstream trypomastigotes of four strains of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense at 0.15 to 2.15 nM (50% inhibitory concentrations). In in vivo mouse laboratory models of T. b. rhodesiense clinical isolate infections, SIPI 1029 was curative for 12 of 13 isolates at /=60% curative, and in six of these, a dose of /=60% cure rates. A number of these isolates were resistant to the standard trypanocide melarsoprol (Arsobal) and/or the diamidines diminazene aceturate (Berenil) and pentamidine. SIPI 1029 was also curative in combination with DL-alpha-difluoromethylornithine (Ornidyl) in a T. b. brucei central nervous system model infection. Some evidence of toxicity was found in dosage regimens of 10 mg/kg/day for 2 or 3 days in which deaths were observed in 6 of 65 animals given this dosage regimen. The activity of SIPI 1029 in this study indicates that this class of compounds (diaminotriazines) should be explored as leads for new human and veterinary trypanocides.     

Ceftezole, a new cephalosporin C derivative II. Distribution and excretion in parenteral administration

The distribution of ceftezole in blood and tissues and its excretion after intramuscular or intravenous administration of single doses of 10 and 20 mg/kg were compared with those of cefazolin, cephaloridine and cephalothin. Blood levels of ceftezole in rats and rabbits were lower than those of cefazolin, and higher than those of cephaloridine and cephalothin. Retention time of ceftezole in the blood was somewhat shorter than that of cefazolin. However, blood levels of ceftezole in dogs were nearly the same as those of cefazolin and cephaloridine. The rate of urinary excretion of ceftezole in 24-hour urine after administration in rats and rabbits was found to be higher than those of the other antibiotics tested. In dogs, however, the rate of urinary excretion of ceftezole was nearly the same as that of cefazolin and higher than those of cephaloridine and cephalothin. The biliary excretion of ceftezole in rats and dogs was much higher than those of cephaloridine and cephalothin, but lower than that of cefazolin. Tissue distribution of ceftezole in rats was compared with that of the other antibiotics by intramuscular and intravenous administration. The initial level of ceftezole in the kidneys was found to be substantially higher than those of the other antibiotics. The initial level of ceftezole in the liver and lungs was also slightly higher than those of the other drugs when administered intramuscularly. Tissue levels of ceftezole were somewhat lower than those of cefazolin in rabbits after intravenous administration. Ceftezole attained a higher maximum level in rat lymph by intramuscular administration than the other antibiotics tested. The maximum concentration of ceftezole present in the exudate in the rat inflammatory pouch was higher than that of cefazolin. In rabbits with cerebrospinal meningitis induced by infection of Streptococcus pyogenes, the level of ceftezole in the cerebrospinal fluid was several times higher than that in normal rabbits. The serum level and urinary excretion of ceftezole was examined in 6 healthy male volunteers after intramuscular administration of a single dose of 500 mg. Ceftezole attained a mean maximum serum level of 22.9 mug/ml 30 minutes after administration and disappeared from the blood in about 6 hours. It was excreted rapidly in the urine. The concentration in 1-hour urine was the highest (mean level: 2,667 mug/ml) and the total excretion rate was 92.6%. No metabolites with antimicrobial activity were observed in the urine. No changes in the pattern of plasma level and urinary excretion and no accumulation in the tissues were observed after repeated intramuscular administration of 20 mg/kg of ceftezole in rabbits, 26 times, for 14 days.     

In vitro and in vivo antibacterial activities of T-3761, a new quinolone derivative

T-3761, a new quinolone derivative, showed broad and potent antibacterial activity. Its MICs for 90% of the strains tested were 0.20 to 100 micrograms/ml against gram-positive bacteria, including members of the genera Staphylococcus, Streptococcus, and Enterococcus; 0.025 to 3.13 micrograms/ml against gram-negative bacteria, including members of the family Enterobacteriaceae and the genus Haemophilus; 0.05 to 50 micrograms/ml against glucose nonfermenters, including members of the genera Pseudomonas, Xanthomonas, Acinetobacter, Alcaligenes, and Moraxella; 0.025 micrograms/ml against Legionella spp.; and 6.25 to 25 micrograms/ml against anaerobes, including Bacteroides fragilis, Clostridium difficile, and Peptostreptococcus spp. The in vitro activity of T-3761 against these clinical isolates was comparable to or 2- to 32-fold greater than those of ofloxacin and norfloxacin and 2- to 16-fold less and 1- to 8-fold greater than those of ciprofloxacin and tosulfoxacin, respectively. When administered orally, T-3761 showed good efficacy in mice against systemic, pulmonary, and urinary tract infections with gram-positive and gram-negative bacteria, including quinolone-resistant Serratia marcescens and Pseudomonas aeruginosa. The in vivo activity of T-3761 was comparable to or greater than those of ofloxacin, ciprofloxacin, norfloxacin, and tosufloxacin against most infection models in mice. The activities of T-3761 were lower than those of tosufloxacin against gram-positive bacterial systemic and pulmonary infections in mice but not against infections with methicillin-resistant Staphylococcus aureus. The activities of T-3761 against systemic quinolone-resistant Serratia marcescens and Pseudomonas aeruginosa infections in mice were 2- to 14-fold greater than those of the reference agents.     

Beditine, a new benzodioxane derivative, as a suppressor of human polymorphonuclear leukocyte and platelet activation

Beditine, 2-(2-amino-4-thiazolyl)-1,4-benzodioxane hydrochloride is a new substance which reduced platelet activation and degranulation, prevented aggregation and superoxide generation by activated human polymorphonuclear leukocytes (PMNL) and inhibited the activation of arachidonate 5-lipoxygenase. Beditine may, therefore, be a useful agent in the treatment of cardiovascular disease.     

A phase I and pharmacokinetic study of a new camptothecin derivative, 9-aminocamptothecin

Camptothecins are the only available antitumor agents which target the nuclear enzyme topoisomerase I. 9-Aminocamptothecin (9-AC) is a water-insoluble derivative of camptothecin which has demonstrated impressive antitumor activity in preclinical models. While two other water-soluble derivatives, CPT-11 and topotecan, have successfully completed Phase I and Phase II testing, biochemical and tissue culture studies suggest that camptothecin analogues differ in characteristics which may be important in determining antitumor activity. We performed a Phase I trial of 9-AC to determine the pharmacokinetics, dose-limiting toxicity, and maximum tolerated dose of this agent when administered as a 72-h continuous i.v. infusion. Thirty-one patients with resistant solid cancers received 5-60 microgram/m2/h 9-AC for 72 h, repeated at 3-week intervals. The drug was administered in a vehicle containing dimethylacetamide, polyethylene glycol, and phosphoric acid. Blood samples were collected and the lactone (closed ring) form of 9-AC was quantitated. The maximum tolerated dose of 9-AC was determined to be 45 microgram/m2/h. Dose-limiting toxicity consisted of neutropenia. Thrombocytopenia was also prominent. There were no significant nonhematological toxicities. Minimal responses were seen in patients with gastric, colon, and non-small cell lung cancer. Although significant interpatient variation in plasma 9-AC lactone levels was observed, pooled data were fit to a two-compartment model, with a terminal half-life of 36 h. Analyses of topoisomerase protein levels in peripheral blood cells indicated decreases in topoisomerase I accompanied by increases in topoisomerase II in two of three patients. 9-AC is an active antitumor agent and may be administered safely as a 72-h infusion in patients with cancer. Although Phase II trials with a 72-h infusion of 9-AC are warranted, alternate schedules should be evaluated given the dramatic preclinical activity seen with more prolonged administrations.     

Effect of a new rifamycin derivative, rifalazil, on liver microsomal enzyme induction in rat and dog

1. The effect of a new rifamycin derivative, rifalazil (KRM-1648), on liver microsomal enzyme induction was studied in rat and dog with repeated oral administration of the compound. Relative liver weight, cytochrome b5 and P450 contents, enzyme activities of NADPH-cytochrome c reductase, aniline hydroxylase, p-nitroanisole O-demethylase, aminopyrine N-demethylase, and erythromycin N-demethylase were measured. 2. In rat, rifalazil treatment at 300 mg/kg/day for 10 days increased cytochrome b5 content but it did not affect liver weight, P450 content or enzyme activities. In contrast, rifampicin and rifabutin increased relative liver weights, cytochrome contents and enzyme activities under similar conditions. 3. In dog, rifalazil did not affect any parameters at 30 or 300 mg/kg/day for 13 weeks. 4. These findings indicate that rifalazil is not an enzyme inducer in rat and dog. This property differs from other rifamycin derivatives such as rifampicin and rifabutin.     

Ceftezole, a new cephalosporin C derivative I. In vitro and in vivo antimicrobial activity

Ceftezole, a new cephalosporin antibiotic similar to cefazolin, has the following chemical structure: (6R,7R)-8-oxo-7[2-(1H-tetrazol-1-yl)acetamido]-3-[(1,3,4-thiadiazol-2-ylthio)methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-carboxylic acid. Ceftezole was found to be a broad-spectrum antibiotic, active in vitro against many species of gram-positive and gram-negative bacteria except Pseudomonas aeruginosa, Serratia marcescens and Proteus vulgaris. The activity of ceftezole against clinical isolates of Escherichia coli and Klebsiella spp. appeared to be nearly equal to that of cefazolin and higher than those of cephaloridine and cephalothin. Cross-resistance was observed between ampicillin and cephaloridine, but not between ampicillin and ceftezole, in susceptibility tests on clinical isolates of P. mirabilis. The in vitro activity was little affected by the inoculum size, the presence of human serum or the test medium. Ceftezole exhibited apparent bactericidal activity at the concentrations above the minimum inhibitory concentration (MIC) against both S. aureus and E. coli. The development in vitro of resistance by S. aureus 209p and E. coli NIHJ to ceftezole after 16 transfers was similar to or somewhat slower than that to other drugs tested. Ceftezole was relatively stable in nutrient broth and minimally degraded in the serum or tissue homogenates of rats. Ceftezole, in a single subcutaneous administration, exhibited somewhat less efficacy in mice against intraperitoneal infections with Streptococcus pyogenes, S. pneumoniae, E. coli, K. pneumoniae or P. mirabilis than either cephaloridine or cefazolin. However, ceftezole exhibited efficacy similar to that of cephaloridine or cefazolin when administered in three doses. Furthermore, ceftezole was as effective as cefazolin in the treatment of experimental abscesses in mice caused by subcutaneous inoculation with S. aureus.