Packed column supercritical fluid chromatography of isomeric polypeptide pairs.

The characterization and determination of peptides is of great importance in the pharmaceutical industry as is the ability to rapidly perform targeted determinations of bioactive peptides in complex matrices. The purpose of the presented work is to assess the feasibility of packed column supercritical fluid chromatography (SFC) for the separation of two-pairs of water soluble peptides of identical mass, composition and charge that differ only in amino acid sequence. Upon evaluating a variety of conditions, trifluoroacetic acid (HTFA) in conjunction with methanol as the modifier proved to be, in general, the most successful mobile phase additive for elution of the two isomeric peptide pairs from all nitrogenous stationary phases. In contrast, water and ammonium acetate gave distorted peak shapes and therefore proved to be less satisfactory as neutral additives. The basic additive, iso-propylamine (IPAm), coupled with HA-Pyridine yielded the highest resolution factor for the complete study. Aminopropyl and HA-Pyridine columns with 5 µm particle size and 60 Å pore size were found to be best for resolution of each peptide pair. Bare silica and phenyl-hexyl stationary phases did not afford any separation. The primary roles of the carbon dioxide and methanol modifier are believed to provide (a) stationary phase solvation and (b) peptide solubility and transport; while, HTFA is postulated to fully protonate each peptide and form ion pairs between its conjugate base and cationic peptide analyte. The separation process, therefore, is best viewed as ion pair supercritical fluid chromatography (IP-SFC). For the case where IPAm gave good resolution on the HA-Pyridine column, the peptides are probably in the neutral state.

4-Hydroxy-5,6-dihydropyrones as inhibitors of HIV protease: the effect of heterocyclic substituents at C-6 on antiviral potency and pharmacokinetic parameters.

Due largely to the emergence of multi-drug-resistant HIV strains, the development of new HIV protease inhibitors remains a high priority for the pharmaceutical industry. Toward this end, we previously identified a 4-hydroxy-5,6-dihydropyrone lead compound (CI-1029, 1) which possesses excellent activity against the protease enzyme, good antiviral efficacy in cellular assays, and promising bioavailability in several animal species. The search for a suitable back-up candidate centered on the replacement of the aniline moiety at C-6 with an appropriately substituted heterocyle. In general, this series of heterocyclic inhibitors displayed good activity (in both enzymatic and cellular tests) and low cellular toxicity; furthermore, several analogues exhibited improved pharmacokinetic parameters in animal models. The compound with the best combination of high potency, low toxicity, and favorable bioavailabilty was (S)-3-(2-tert-butyl-4-hydroxymethyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-6-(2-thiophen-3-yl-ethyl)-5,6-dihydro-pyran-2-one (13-(S)). This thiophene derivative also exhibited excellent antiviral efficacy against mutant HIV protease and resistant HIV strains. For these reasons, compound 13-(S) was chosen for further preclinical evaluation.

Characterization of clinafloxacin photodegradation products by LC-MS/MS and NMR.

Exposure of clinafloxacin to light results in photochemical degradation. The polar and nonpolar photodegradation products were profiled by HPLC using two sets of conditions. Clinafloxacin was subjected to severe light exposure conditions to obtain elevated levels of the photodegradation products for characterization. The structures of eight new degradation products were elucidated based on information from LC-MS/MS fragmentation and NMR spectra following isolation by preparative HPLC. Two photodegradation routes were identified: (1) dechlorination, followed by further reactions involving the quinolone ring, to yield polar photodegradation products; and (2) degradation of the pyrrolidine side-chain, yielding the nonpolar photodegradation products.

Relationship between bisphosphonate concentration and osteoclast activity and viability.

Difluoromethylidene bisphosphonate (F2MBP) is one of the many bisphosphonates known to inhibit bone resorption in vitro and in vivo. We have developed an analytical method, employing anion exchange and postcolumn indirect fluorescence detection, by which F2MBP can be quantified in bone samples. The objective of this study was to relate the concentration of F2MBP in embryonic bones treated in organ culture to the physiological effects of the compound, such as bone resorption (i.e., the amount of 45Ca released into the medium from prelabeled bones) and viability of the osteoclast population (i.e., the incidence of abnormal osteoclasts). Osteoclasts in bones treated with F2MBP exhibited morphological features of apoptosis, such as nuclear fragmentation. Both the number and percentage of these abnormal cells increased with dose of F2MBP and duration of incubation. The decrease in normal osteoclasts was correlated with the decreased amount of 45Ca released into the medium. Bones treated with F2MBP for only the first 5 min of the 48-h incubation period had similar numbers of abnormal osteoclasts and amounts of 45Ca released, as had bones incubated with F2MBP continuously for 48 h. The uptake of F2MBP into the bone was rapid. Bones treated with F2MBP for 6 h were similar to bones treated with F2MBP for the entire 48-h incubation period, both in F2MBP concentration and the 45Ca release ratios. These relationships between concentrations of F2MBP within bone and osteoclast activity and viability implicate apoptosis in the mechanism by which this bisphosphonate inhibits bone resorption.

Anion-exchange separation and determination of bisphosphonates and related analytes by post-column indirect fluorescence detection.

Bisphosphonic acids and their salts can be detected after their liquid chromatographic separation by post-column indirect fluorescence detection (IFD). After separation the analyte is combined with the highly fluorescent Al(3+)-morin (2',3,4',5,7-pentahydroxyflavone) solution and fluorescence decreases because of the formation of the nonfluorescent Al(3+)-bisphosphonate complex. The decrease in fluorescence is proportional to the amount of bisphosphonate present. Separation of the multivalent anionic bisphosphonate analytes from other anions and sample matrix is achieved on a strong base anion-exchange column with a strong, basic eluent. The post-column reaction variables, which influence IFD, are identified and optimized for the detection of the bisphosphonates after separation on the anion exchanger. The method is selective, since only a few anions will undergo a reaction with the Al(3+)-morin solution, and sensitive, detection limit for difluoromethylene bisphosphonate, F2MDP, is 4 ng for S/N = 3. The separation-IFD method can be applied to the determination of bisphosphonates, such as F2MDP, ethane-1-hydroxy-1,1-bisphosphonic acid, dichloromethylene bisphosphonic acid, 4-amino-1-hydroxybutane-1,1-bisphosphonic acid, in biological samples. The separation-IFD method is also applicable to the detection of inositol phosphate anions.

Disposition kinetics of cobalt mesoporphyrin in mouse, rat, monkey and dog.

1. Radiometric and UV analyses indicated > 95% unchanged cobalt mesoporphyrin (CoMP) in plasma after i.v. or i.m. administration. Blood clearance of CoMP is < 2% of hepatic blood flow in mouse and rat, and < 0.5% of hepatic blood flow in monkey and dog. CoMP elimination t1/2 ranged from 3.1 to 9.9 days in animals after i.v. administration. 2. CoMP is highly (> 99.5%) bound to plasma proteins, but has low affinity for blood cells (Kp < 0.15). The volume of CoMP distribution (Vss < 0.91/kg) is reflective of a distribution to total body water following i.v. administration to mouse, rat, monkey and dog. 3. [14C]CoMP reached highest levels in rat tissue between 1 and 4 days following i.m. injection. Liver, kidney cortex, lymph node, adrenal and spleen demonstrated greatest uptake of radiolabel. Concentration in tissues was readily detectable at 60 days post-dose. 4. CoMP was slowly absorbed after i.m. administration showing dose-dependent pharmacokinetics. The major route of radiolabel elimination was faecal excretion (54% of dose) in rat after an i.m. dose of [14C]CoMP. Approximately 1% of the 14C dose was recovered in the urine over 7 days post-dose. 5. As a polar metalloporphyrin, CoMP has low clearance, restricted tissue distribution and long elimination t1/2 in the laboratory animals.

Effects of hepatocyte growth factor on viability and biotransformation functions of hepatocytes in gel entrapped and monolayer culture.

OBJECTIVES: An extracorporeal bioartificial liver device must maintain viability and differentiated function of hepatocytes cultivated at high cell density. Growth factors, such as hepatocyte growth factor, found in high concentrations in the plasma of patients with fulminant hepatic failure, have the potential to promote hepatocyte dedifferentiation and thus, decrease function. We tested the hypothesis that hepatocyte growth factor would improve viable cell density and decrease biotransformation functions of liver cells in monolayer culture and in hepatocytes entrapped in collagen cylindrical gel "noodles" as found in the extracorporeal bioartificial liver. DESIGN: In vitro, controlled study. SETTING: University research laboratory. SUBJECTS: Adult Sprague Dawley Rats. INTERVENTIONS: Hepatocytes were harvested by a two-step collagenase technique. Harvested hepatocytes were plated onto type 1 collagen coated plates or entrapped in type 1 collagen cylindrical gels and cultured in different concentrations of hepatocyte growth factor. Interval measurements of 3H-thymidine incorporation, albumin synthesis, biotransformation functions, and viability were made. MEASUREMENTS AND MAIN RESULTS: In monolayer culture, the addition of hepatocyte growth factor caused a dramatic increase in 3H-thymidine incorporation. This increase was accompanied by a decrease in the appearance of the lidocaine metabolite, monoethyglycinexylidide. Albumin production was unchanged. In cylindrical gel entrapment cultures, hepatocyte growth factor caused a significant increase in 2-day viability but had no effect on the metabolite appearance of lidocaine or 4-methyl umbelliferone or albumin production. CONCLUSIONS: Hepatocyte growth factor induces dedifferentiation of hepatocytes in monolayer culture. Collagen matrix entrapment appears to abrogate this effect and improve liver cell viability. There may be reciprocal regulation of hepatocyte reproductive and differentiated functions, such as biotransformation, which can be influenced by the entrapment of hepatocytes in an extracellular type 1 collagen matrix.

Effect of Streptococcus pneumoniae and influenza A virus on middle ear antimicrobial pharmacokinetics in experimental otitis media.

Antimicrobial treatment failures in children with acute otitis media and concomitant viral respiratory tract infection prompted us to study the effects of influenza A virus infection on middle ear antimicrobial drug penetration. Using a chinchilla model of Streptococcus pneumoniae we compared middle ear elimination rates in 4 groups of chinchillas: (1) control, (2) influenza A virus inoculation alone intranasally, (3) both influenza A and S. pneumoniae inoculation directly into the middle ear, and (4) S. pneumoniae inoculation alone into the middle ear. After infection was established, a solution containing amoxicillin, sulfamethoxazole, and trimethoprim was instilled into the middle ear and removed 4 hours later. The rate constant of elimination and half-life were calculated from measured drug concentrations initially and at 4 hours. S. pneumoniae infection alone significantly shortened the middle ear elimination half-life compared with the control group: amoxicillin, 2.65 +/- 0.73 vs. 6.63 +/- 2.55 hr; sulfamethoxazole, 1.75 +/- 0.28 vs. 2.74 +/- 0.6 hr; and trimethoprim, 1.06 +/- 0.14 vs. 1.56 +/- 0.34 hr (n = 16 ears, p values all < 0.01). The combined influenza virus and S. pneumoniae infection significantly lengthened the half-life compared with the S. pneumoniae infection alone: amoxicillin, 5.65 +/- 6.44 vs. 2.65 +/- 0.73 hr; sulfamethoxazole, 2.5 +/- 0.85 vs. 1.75 +/- 0.28 hr; and trimethoprim, 1.26 +/- 0.42 vs. 1.06 +/- 0.14 hr (n = 16 ears, p values all < 0.01). Influenza virus produced the longest half-lives for all 3 antimicrobials: amoxicillin 25.52 +/- 14.96 hr; sulfamethoxazole, 5.46 +/- 0.87 hr; and trimethoprim, 2.57 +/- 0.75 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of two otitis media models for the study of middle ear antimicrobial pharmacokinetics.

We compared two models of acute otitis media that estimate middle ear antimicrobial pharmacokinetics. Using a crossover study design, we compared a systemic drug administration model with a diffusion model we devised that measures the disappearance of antimicrobials from the middle ear. We induced acute otitis media in 14 chinchillas by inoculating S. pneumoniae into the middle ear, then administered 3 antimicrobials: amoxicillin, trimethoprim, and sulfamethoxazole. Next we collected middle ear fluid samples to analyze drug concentrations and compare rate constants for the systemic and diffusion models by analysis of variance. We found that amoxicillin K values were not affected by model testing sequence (p = 0.827) or model type (systemic versus diffusion, p = 0.310), nor were sulfamethoxazole K values: model testing sequence (p = 0.917), model type (p = 0.963). Trimethoprim K values were also not affected by model testing sequence (p = 0/760), but were by model type (p = 0.0001). Trimethoprim elimination from the diffusion model was faster (K = 0.33 +/- 0.17 versus 0.57 +/- 0.09 hr-1) than from the systemic model, although it appears this was caused by sampling before drug distribution into the middle ear was complete. In conclusion, it appears K values derived from either systemic antimicrobial administration or direct middle ear instillation are similar for assessing middle ear antimicrobial pharmacokinetics, and these models can be used interchangeably to study factors affecting otitis media treatment response.

Determination of cefpodoxime levels in chinchilla middle ear fluid and plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed to determine cefpodoxime levels in chinchilla plasma and middle ear fluid (MEF) to be used in studying otitis media. Cefpodoxime and the internal standard, cefuroxime, were separated on an ODS column (250 x 2.1 mm I.D., 5 microns Hypersil), using a mobile phase of 25 mM acetate buffer (pH 4.3)/15 mM triethylamine-acetonitrile (92.5:7.5, v/v). Following elution of cefpodoxime and the internal standard, at 3.5 and 5.9 min respectively, the acetonitrile concentration was increased to 1:1 (v/v) in a step function to elute endogenous compounds retained on the column. Sample preparation involved protein precipitation with acetonitrile. This fast, efficient protein precipitation procedure together with UV detection allows a quantitation limit of 50 ng/ml with a 50-microliters sample size. Recoveries (mean +/- S.D., n = 3) at 0.1 microgram/ml in MEF were 90.3 +/- 2.9% and 88.6 +/- 1.2% for cefpodoxime and cefuroxime respectively. Recoveries (mean +/- S.D., n = 3) at 0.1 microgram/ml in plasma were 72.1 +/- 7.3% and 81.1 +/- 1.1% for cefpodoxime and cefuroxime respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering cefpodoxime proxetil.

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection.

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 x 2.1 mm I.D., 5 microns Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate-acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 microliters sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean +/- S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 +/- 1.2% and 91.4 +/- 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.

Liquid chromatography and postcolumn indirect detection of glyphosate.

Glyphosate [N-(phosphonomethyl)glycine] and its metabolite aminomethylphosphonic acid (AMPA) were separated and detected by a postcolumn indirect detection strategy. Separation can be done on a cation-exchange column, where glyphosate elutes before AMPA, or on an anion-exchange column, where the elution order is reversed. Detection was achieved by using a fluorescent Al(3+)-morin postcolumn reagent. When the postcolumn reagent combines with the column effluent in a mixing tee, the fluorescence decreases in the presence of both analytes. Variables affecting the postcolumn indirect fluorescence detection were established and optimized; the major factors were postcolumn pH and volume and temperature of the postcolumn reaction coil. Detection limits, defined as three times the background noise, for glyphosate and AMPA separated on an anion-exchange column were 14 and 40 ng, respectively.

The assay of clozapine and N-desmethylclozapine in human plasma by high-performance liquid chromatography.

A high performance liquid chromatographic method for the simultaneous quantitation of clozapine--a member of the dibenzodiazepine class of antipsychotic drugs--and its reduced metabolite in human plasma has been developed. Clozapine and N-desmethylclozapine are concentrated from blood samples by liquid-liquid extraction, followed by reverse-phase liquid chromatography. Accuracy of the determination is ensured by application of an internal standard, protriptyline, which is closely related to clozapine. A detection limit of 15 ng/mL for clozapine and 30 ng/ml for N-desmethylclozapine was achieved.