Michael F. Skinner

Analysis of macrolide antibiotics

The following macrolide antibiotics have been covered in this review: erythromycin and its related substances, azithromycin, clarithromycin, dirithromycin, roxithromycin, flurithromycin, josamycin, rokitamycin, kitasamycin, mycinamycin, mirosamycin, oleandomycin, rosaramicin, spiramycin and tylosin. The application of various thin-layer chromatography, paper chromatography, gas chromatography, high-performance liquid chromatography and capillary zone electrophoresis procedures for their analysis are described. These techniques have been applied to the separation and quantitative analysis of the macrolides in fermentation media, purity assessment of raw materials, assay of pharmaceutical dosage forms and the measurement of clinically useful macrolide antibiotics in biological samples such as blood, plasma, serum, urine and tissues. Data relating to the chromatographic behaviour of some macrolide antibiotics as well as the various detection methods used, such as bioautography, UV spectrophotometry, fluorometry, electrochemical detection, chemiluminescence and mass spectrometry techniques are also included.

Application of dermal microdialysis for the evaluation of bioequivalence of a ketoprofen topical gel

The purpose was to investigate dermal microdialysis (DMD) for the assessment of the bioavailability of a ketoprofen topical gel formulation and to evaluate this technique as a tool for the determination of bioequivalence. Four microdialysis probes were inserted into the dermis on the volar aspect of the forearms of 18 human subjects and the probes were perfused with normal saline for 60 min. A ketoprofen (2.5%, m/m) gel formulation (50mg) was applied to the skin directly overlying the probes and samples were collected at 30 min intervals for 5h. With the probes still in place in the dermis each site was scanned by ultrasound to determine the implantation depth of these probes. Ketoprofen concentration in dialysates was determined by LC-MS/MS. The area under the curve obtained from the concentration-time profiles from pairs of application sites in each subject was evaluated in order to assess bioequivalence. Ninety percent confidence intervals were calculated using the two one-sided test procedure and limits of 80-125% based on log-transformed data were used as acceptance criteria to declare bioequivalence. The intra-subject variability was 10% between probes whereas inter-subject variability was 68% (n=18). Bioequivalence was confirmed with a power greater than 90%.

Comparison of tape stripping with the human skin blanching assay for the bioequivalence assessment of topical clobetasol propionate formulations.

PURPOSE A draft guidance on tape stripping for assessing the bioavailability/bioequivalence of topical formulations was issued by the United States Food and Drug Administration in 1998 but has since been withdrawn. This was due to problems associated with the method and also inconsistencies and variability in the resulting data. The purpose of this study was to re-visit the tape stripping technique, incorporate refinements to reduce variability and validate the method using bioequivalence data obtained from the assessment of a topical corticosteroid cream containing 0.05% clobetasol propionate using the human skin-blanching assay. METHODS A pilot tape stripping study was conducted to establish the variability of the formulations.The bioequivalence of two different commercially available clobetasol propionate cream formulations and a clobetasol propionate ointment formulation were subsequently investigated using the tape stripping method. RESULTS The data from the pilot tape stripping study correlated well with data from the human skin-blanching assay. A subsequent pivotal tape stripping study confirmed bioequivalence between the two cream formulations whereas bio-inequivalence was demonstrated between the cream and ointment formulations. CONCLUSIONS These studies show that the results from tape stripping concur with data from the human skin blanching assay and demonstrate the potential of a well-controlled tape stripping study as an option for the assessment of bioequivalence of topical corticosteroid formulations.

Application of dermal microdialysis for the determination of bioavailability of clobetasol propionate applied to the skin of human subjects.

Dermal microdialysis was used to assess the bioavailability of a topical corticosteroid, clobetasol propionate, following application onto the skin of human subjects. The penetration of clobetasol propionate from a 4% m/v ethanolic solution applied onto 4 sites on one forearm of healthy human volunteers was studied. A lipid emulsion, Intralipid®, was used as the perfusate and linear microdialysis probes with a 2-kDa cutoff were inserted intradermally at the designated sites. The results indicated that Intralipid could be used as a suitable perfusate for in vivo microdialysis of this lipophilic drug of interest. Furthermore, the study clearly demonstrated the application of dermal microdialysis as a valuable tool to assess the bioavailability/bioequivalence of clobetasol propionate penetration into the skin following topical application.

High-performance liquid chromatographic analysis of josamycin in serum and urine

A high-performance liquid chromatographic assay for the analysis of josamycin in human serum and urine is presented. The assay involves a simple solid-phase extraction procedure coupled with a phase separation step, separation on a reversed-phase C18 column with UV detection by a multi-wavelength programmable detector. The mobile phase was acetonitrile-0.015 M phosphate buffer, pH 6.0 (5:2) at a flow-rate of 1.2 ml/min. The column temperature was maintained at 35 degrees C. Linear calibration curves over the concentration ranges 0.1-2.0 mg/l (serum) and 0.5-5 mg/l (urine) were obtained with correlation coefficients of 0.9983 and 1.0000, respectively. The relative standard deviations of five replicate samples at the upper and lower limits of each calibration curve were below 7%. The recoveries at the upper and lower ends of the calibration range for serum were 77% and 70%, respectively, and those for urine were 76% and 80%, respectively.

Effect of African Potato (Hypoxis Hemerocallidea) on the Pharmacokinetics of Efavirenz

PURPOSE The purpose of this study was to evaluate the effect of the African potato (AP) on the pharmacokinetics of efavirenz. METHODS; A single-dose, two-phase sequential study was conducted over 31 days in 10 healthy volunteers. On day 1 of the study, volunteers were administered a 600 mg efavirenz tablet, and blood samples were collected before dosing and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 18, 24, 36 and 48 hours after dosing. From day 16, a traditionally prepared AP decoction was administered daily until day 30. On day 29, volunteers were administered a single 600 mg dose of efavirenz, as was done on day 1. Plasma samples were harvested immediately after blood sample collection and frozen at -80 degrees C until assayed. Plasma concentrations of efavirenz were determined by a validated high performance liquid chromatography (HPLC) method with UV detection, and pharmacokinetic parameters were calculated. Geometric mean ratios of C(max) and AUC(0-48) of efavirenz before and after co-administration of 14 successive daily doses of AP were compared. RESULTS All subjects completed the study. The geometric mean ratios of C(max) and AUC(0-48) were 97.30 and 102.82 with corresponding 90% confidence intervals (CIs) of 78.81 - 120.14 and 89.04 - 118.80, respectively. CONCLUSION Pharmacokinetic data generated during this study indicated that AP did not significantly alter the pharmacokinetics of efavirenz. Hence, co-administration of AP is unlikely to affect the clinical usage of efavirenz.

The pH-stability and acid degradation of the macrolide antibiotic, josamycin

Abstract The influence of pH, ionic strength and buffer concentration on the stability of josamycin has been investigated. The pH-rate profile for josamycin over the pH range 1–12 was characterised and the equation describing the profile determined. Results indicated that josamycin is subject to specific acid catalysis whilst catalysis in alkali media appeared to be more complex. The rate constant for catalysis by hydronium ion ( k H ) was 54.11 M −1 · h −1 and the rate constant for catalysis by hydroxide ion ( k OH ) was 60.35 M −1 · h −1 . Catalysis due to water was insignificant and the water catalysed rate constant was found to be 3.37 × 10 −5 h −1 . The pH of maximum stability was determined as pH 6.5 whilst degradation at pH 1.0 and 12.0 is about five orders of magnitude greater than at pH 6.5. The degradation of josamycin in acid is subject to a significant primary salt effect; however, no secondary salt effect was evident. Concentration vs. time profiles for josamycin in acidic media were biphasic which indicated that the degradation reaction did not follow a simple pathway whereby josamycin degrades directly to products. Further investigations suggest that josamycin undergoes a reversible isomerisation step, with subsequent degradation of josamycin and possibly its isomer, by cleavage of the mycarose moiety to desmycarose compounds. Studies to determine the stability of josamycin in simulated gastric fluids demonstrated that acid degradation could be appreciable after oral administration. However, extensive degradation in vivo will only occur at the most acidic gastric pHs of about pH 1.0 to 2.0.

Effect of the African Traditional Medicine, Sutherlandia frutescens, on the Bioavailability of the Antiretroviral Protease Inhibitor, Atazanavir

The objective of this study was to investigate the effect of Sutherlandia frutescens (SF) on the bioavailability of atazanavir (ATV) in twelve healthy male subjects. During Phase I (Day 1), subjects ingested a single dose of ATV and blood samples were drawn before dose and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 9.0, 12, 18, and 24 hours after dose. From Day 3 to Day 14, a single dose of milled SF was administered twice daily to each subject. During Phase II, Day 15, subjects ingested single doses of ATV and SF. Blood samples were drawn as previously described. Plasma was harvested from blood samples and the concentration of ATV therein was determined. For each phase, the mean ATV plasma concentration-time profile was plotted and the means of AUC0–24 and C max for ATV were computed. The geometric mean ratios and confidence intervals (CIs) for C max and AUC0–24 hr were 0.783 (0.609–1.00) and 0.801 (0.634–1.01), respectively. The CIs for both PK parameters fell below the limits of the “no-effect” boundary, set at 0.8–1.25, indicating that SF significantly reduced the bioavailability of ATV. This may potentially result in subtherapeutic plasma concentrations and thus reduced anti-HIV efficacy of ATV.

Bioequivalence Assessment of Generic Products: An Innovative South African Approach

Concurrent with the implementation of new legislation mandating Generic Substitution in South Africa, a new set of guidelines for bioavailability and bioequivalence have been published. Since one of the main objectives of the new legislation in South Africa relating to Generic Substitution is to ensure that medicines of high quality, safety, and efficacy are made more accessible and more affordable to the wider public, the need to speed up approval of such multi-source products has become a regulatory priority. In order to facilitate this process, various bioequivalence issues have been addressed including important issues such as the acceptance criteria and associated bioequivalence intervals, use of a foreign reference product and the issue of assessing highly variable drugs (HVDs). In addition, dispensations have been made with respect to food effect assessment and variability relating to genetic polymorphism in drug metabolism (genotyping/phenotyping). Furthermore, the use of “old” biostudies submitted in support of an application is subject to expiry date. Acceptance of appropriate data requires that specific criteria such as Cmax and AUC, in addition to the usual considerations, also meet the limits specified by the particular registration authority of the country where such products are intended to be marketed. Generally, these limits require that the 90% confidence interval (CI) for AUC and Cmax test/reference ratios lies within the acceptance interval of 0.80–1.25 calculated using log-transformed data. While such acceptance criteria are, in general, ubiquitous, some differences in acceptance criteria do exist between various countries. The new guidelines for bioavailability/bioequivalence studies developed by the South African regulatory authority, the Medicines Control Council (MCC), makes provision for highly variable drugs and the use of a non-South African reference product. The MCC requires that the acceptance criterion for Cmax ratios be set at 0.75–1.33 while maintaining AUC ratios at 0.80–1.25 using a 90% CI. Furthermore, provision is made to apply scaling based on average bioequivalence assessment and, as an interim measure, consideration has also been given to the use of a foreign reference product provided that equivalence between that product and the innovator product currently available on the South African market can be shown using in vitro testing.