Cutaneous pharmacokinetics of topically applied maxacalcitol ointment and lotion.

UNLABELLED: Maxacalcitol (22-oxacalcitriol), a vitamin D3 analogue, is widely used for the treatment of psoriasis in Japan. The effects of topically applied dermatologic preparations have routinely been assessed by their pharmacodynamic profiles and their concentrations in the skin correlate well with these profiles. OBJECTIVES: Recently, a maxacalcitol lotion formulation (M514102) was developed for the treatment of psoriatic lesions on the face and scalp. To predict the clinical efficacy of the lotion, we investigated the cutaneous bioavailability of topically applied lotion and compared this with that of its ointment formulation in healthy subjects. METHODS: In the first study, 12 subjects were divided into two groups of 6 each and were treated with the ointment or lotion. Six drug application sites were randomly selected on the left volar forearm. After 0, 2, 4, 6, 8 and 10 h, the formulations were gently removed and tape stripping was performed. Maxacalcitol was extracted from the tape strips and quantified by liquid chromatographic tandem mass spectrometry. In the second study, four drug application sites were randomly selected on the left volar forearm in the 12 subjects. The ointment was applied and spread over two sites and the lotion was applied in the same manner over the remaining two sites. After 8 h, the preparations were gently removed and followed by tape stripping. RESULTS: The average concentrations of maxacalcitol in the stratum corneum (SC) at 2, 4, 6, 8 and 10 h after application were 6.9 +/- 3.3, 12.8 +/- 6.2, 11.8 +/- 4.6, 13.1 +/- 5.2 and 12.3 +/- 3.1 microg/g for the ointment and 3.1 +/- 1.0, 9.1 +/- 3.1, 13.9 +/- 3.4, 13.1 +/- 4.1 and 15.5 +/- 3.1 microg/g for the lotion, respectively. A steady state was observed at approximately 4 and 6 h after application of the ointment and lotion, respectively. In the second study, there was no significant difference between the average of the SC concentrations of the ointment and lotion at 8 h. CONCLUSIONS: In conclusion, we observed that assessment of cutaneous bioavailability by using tape stripping was reproducible. Accordingly, the cutaneous bioavailability of the lotion was comparable to that of its ointment. Hence, treatment with the lotion is expected to be as effective as that with the ointment.

Imaging of single fluorescent molecules using video-rate confocal microscopy.

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.

Methylcellulose-immobilized reversed-phase precolumn for direct analysis of drugs in plasma by HPLC.

We evaluated a new restricted access media (RAM) precolumn for direct analysis of drugs in plasma using a column switching HPLC system. The new RAM material was prepared by the modification of the external surface of porous silica with hydrophilic methylcellulose (MC), followed by modification of the internal surface with octadecylsilane (ODS). The external surface of the MC-immobilized ODS silica material (MC-ODS) suppressed the adsorption of proteins, while the internal surface of MC-ODS retained various types of drugs, such as ketoprofen, propranolol, caffeine and atenolol in plasma samples. In addition, MC-ODS allowed direct analysis of drugs in a 1000-microL plasma sample to monitor trace amounts of analytes contained. Reduced efficiency and clogging of the MC-ODS precolumn and/or the analytical column were not observed even after the repetitive injection of plasma sample up to 40 mL. Our results indicated that the MC-ODS precolumn could be used in pharmacodynamic and clinical studies.

Surfactants usable for electrospray ionization mass spectrometry.

Surfactants suppress the electrospray ionization (ESI) of various compounds. Here we demonstrate that fluorinated surfactants such as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOSA) could be employed for ESI-MS without a significant decrease in sensitivity. Both PFOA and PFOSA could be applied to the analysis of ionic and nonionic compounds by micellar electrokinetic chromatography (MEKC)/ESI-MS, although the migration window was limited. Furthermore, in HPLC/ESI-MS, PFOA could function as a paired ion for enzymatically digested peptides as well as sulfonamides and significant effects of PFOA on the signal peak shape, retention times, and sensitivity of the analytes were observed compared to those of trifluoroacetic acid or sodium dodecyl sulfate (SDS). In addition, PFOA was applied to protein analysis by ESI-MS, and superior sensitivity was noted, compared to other surfactants such as octylglucoside, dodecylglucoside, and SDS. Although 21% of the original signal was observed in the presence of 1% PFOA, this surfactant could be easily removed by evaporation, which resulted in the recovery of 96% of the original signal. Because these fluorinated surfactants could also be used for solubilization, extraction, and disaggregation of proteins, they should greatly expand the applicability of the ESI-MS to the biological problems of proteins.

Electrophoretic mobility-assisted identification of proteins by nanoelectrospray capillary electrophoresis/mass spectrometry under methanolic conditions.

A novel method for electrophoretic mobility-assisted identifications of proteins, using capillary electrophoresis/mass spectrometry (CE/MS) under methanolic conditions, was developed. The number of functional groups of the enzymatic digest peptides was estimated from a single run CE/MS analysis and utilized as an additional tag for database searching in addition to the mass map of the peptides. The additional amino acid information thus obtained can improve the confidence level of the protein identification. The database searching software algorithm ProFound was modified to accept the tag, based on this new concept. In this study, optimization of the CE/MS conditions for the estimation of basic functional groups was performed as an example. An accurate value of the number of such functional groups was obtained from CE characteristics when methanolic buffer (methanol/formic acid/water = 60:20:20) was used, via an excellent correlation (r = 0.997) between the number of functional groups of the peptides and [MW((2/3))]. The mass spectrometry sensitivity was also improved when using the methanolic buffer in comparison with that obtained using aqueous 1% formic acid buffer. The identification of a protein of Saccharomyces cerevisiae, which was separated by two-dimensional electrophoresis, was performed using the methanolic buffer in combination with sheathless nanoelectrospray CE/MS. A protein spot that had not been identified by MALDI-TOFMS and LC/MS/MS was successfully identified using this new method.

Enantiomeric separation by capillary electrophoresis with an electroosmotic flow-controlled capillary.

Perfect control of electroosmotic flow (EOF) was achieved by dovetailing successive multiple ionic-polymer layer (SMIL) coated capillaries. The direction and magnitude of the EOF was perfectly controllable over the pH range 2-13. Zone diffusion was not observed, even if the inner wall of the dovetailed capillary was discontinuous, or if the sample zone passed through the connected part of the capillary because the RSDs of migration time, theoretical plates, symmetry factor and S/N of the marker were almost the same when seamless capillary and dovetailed capillary were compared. The dovetailed capillary was applied to cyclodextrin modified capillary zone electrophoresis. The control of the EOF enabled us to control both the resolution and the migration order of the enantiomers. The migration time was also controllable and, therefore, the best condition between separation and migration time could be determined by controlling the EOF. Partial filling affinity electrokinetic chromatography with a protein used as a chiral selector was also studied. The migration of the pseudostationary phase was controllable by EOF, and detection of the solute at 214 nm was possible. Therefore, the EOF-controlled dovetailed capillary has great potential to expand the application of the separation technique.

Characterization of lipophilicity scales using vectors from solvation energy descriptors.

Lipophilicity scales were characterized by an approach using vectors provided from solvation energy descriptors (SED) of solutes such as an excess molar refraction, the dipolarity/polarizability, the hydrogen-bond acidity, the basicity, and the McGowan characteristic volume. The five components of the SED vector were obtained from the coefficients of the five SED terms of the linear solvation energy relationship (LSER) equation for the lipophilicity scales. The analogy between two lipophilicity scales was expressed as the angle between the two SED vectors, while the difference in the contribution of the five independent SEDs to these two lipophilicity scales was quantified by the difference of the unit vectors of the SED vectors. These approaches were applied to several lipophilicity scales measured using microemulsions, micelles, an immobilized artificial membrane column, and an octanol-water system. As a result, the quantitative classification of these scales was successfully carried out, and the difference in the scales was well characterized. In addition, this vector approach was extended to the estimation of the contribution of each constituent of the microemulsions to the lipophilicity scale. Furthermore, some biological parameters such as skin permeability and the distribution between blood and brain could be predicted by the summation of the SED vectors obtained from the chromatographic systems. These results suggest that complex biological systems can be expressed quantitatively by simple chemical models with their SED vectors.

[Reduction in the window period of an acute infection by chemiluminescence enzyme immunoassay of HIV-1 p24 antigen].

To evaluate the detectability of HIV-1 p24 antigen in the window period of an acute infection, 172 HIV-1-infected and 78 HIV-1-uninfected adult serum (plasma) samples were assayed by chemiluminescence enzyme immunoassay (CLEIA, 2 step sandwich method) and EIA. All 78 HIV-uninfected samples (100%) were p24 antigen negative by both methods. Thirty five of the 172 HIV-1-infected samples (32.1%) were p24 antigen negative by EIA, and p24 antigen positive by CLEIA. The HIV-1 RNA levels of the 35 discrepant samples were 8.1 x 10(3)-7.9 x 10(5) copies/ml, TH/I lymphocyte levels of them were less than 200 cells/microliter except 9 cases, and phases of them were AIDSs of CDC 1993 classification except 7 cases. The antigen detection limit of CLEIA was 3.1-6.3 pg/ml, and that of EIA was 12.5-25.0 pg/ml. The coefficient of variations of CLEIA were 3.0-6.3% determined after the assay on 5 samples on 5 different days, 6 times per day. On the basis of these results, it was inferred that the reason for the discrepancy between the two methods was due to the fact that the detection limit of CLEIA was lower than that of EIA. On an antibody-negative RNA-positive (5.0 x 10(4) copies/ml) serum sample in the window period of an acute infection, p24 antigen was detected by CLEIA 7 days earlier than by EIA. CLEIA was judged to be a useful assay for reducing the window period, and a cost-effective (effect/cost > 1.0) method.

Stable cationic capillary coating with successive multiple ionic polymer layers for capillary electrophoresis.

A coated capillary modified with a cationic polymer was developed by using a novel coating procedure, successive multiple ionic-polymer (SMIL) coating. The SMIL coating was achieved by first attaching the cationic polymer to the capillary inner wall, and then the anionic polymer to the cationic polymer layer, and finally the cationic polymer to the anionic polymer layer. The stability of Polybrene (PB)-modified capillary made by SMIL coating was remarkably improved in comparison with a conventional PB-modified capillary. It endured during 600 replicate analyses and also showed strong stability against 1 M NaOH and 0.1 M HCl. The relative standard deviation of the run-to-run, day-to-day, and capillary-to-capillary coating was all below 1%, and good reproducibilities were obtained. The PB-modified capillary made by SMIL coating was applied to the basic protein analyses. It gave good performances for the protein analyses even when the pH of the electrolyte was near the isoelectric point (pI) of the protein. In addition, 0.1 M NaOH rinse prior to the sample injection allowed the reproducible analysis of a highly adsorptive sample such as plasma because the adsorbed sample could be flushed out of the capillary. Besides protein analyses, an efficient analysis of the cationic drugs by capillary electrophoresis/mass spectrometry (CE/MS) was also possible.

Stable capillary coating with successive multiple ionic polymer layers.

A stable modification of the inner wall of a fused silica capillary was established by a simple coating procedure, successive multiple ionic-polymer layer (SMIL) coating. An anionic polymer was tightly fixed to the capillary wall by the SMIL coating, in which a cationic polymer was sandwiched between the anionic polymer and the uncoated fused silica capillary by noncovalent bonding. The SMIL-coated capillary showed a long lifetime. The endurance of the SMIL-coated capillary was more than 100 runs, and it was also tolerant to organic solvents, 1 M NaOH, and a surfactant. The coating efficiency did not depend on capillary sources, and the relative standard deviation of capillary-to-capillary reproducibility was less than 1%. In this study, dextran sulfate (DS) was used as the anionic polymer, and Polybrene was used as the cationic polymer for SMIL modification. The DS-modified capillary (SMIL-DS capillary) exhibited a pH-independent electroosmotic flow (EOF) from anode to cathode in the pH range of 2-11. The SMIL-DS capillary showed good performance for acidic protein analyses under physiological conditions (pH 7.4). Also, the presence of EOF under acidic conditions permitted new applications. Simultaneous separations of cationic, anionic, and neutral amino acids were achieved by capillary zone electrophoresis, and separations of cresol isomers were achieved by micellar electrokinetic chromatography under the acidic conditions. The SMIL-DS capillary was also useful for fast and precise determination of the pKa of acidic functional groups.

Cyanocysteine-mediated molecular dissection of dihydrofolate reductase: occurrence of intra- and inter-molecular reactions forming a peptide bond.

During a cyanocysteine-mediated dissection study of dihydrofolate reductase, a peptide fragment with a molecular mass of 18 Da less than expected was found as a major reaction product when the dissection reaction was applied to a Lys-cyanocysteine linkage. Detailed characterization of the dissection products by protease digestion, peptide sequencing, liquid chromatography/electrospray ionization mass spectrometry, and capillary electrophoresis suggested that the by-product was generated via a lactam ring formation through the intramolecular nucleophilic attack of the epsilon-amino group on the carbonyl carbon of the Lys-cyanocysteine linkage. We have also demonstrated the occurrence of intermolecular attack of an alpha-amino group of glycine on the carbonyl carbon of the X-cyanocysteine linkage to form a new X-Gly linkage, which should be a useful reaction for specific modification of proteins at the C-terminal.

[Clinical study on penicillin insusceptible/resistant Streptococcus pneumoniae in the elderly patients].

Clinical features of respiratory infection in the elderly with penicillin insusceptible (31 cases) and resistant (7 cases) Streptococcus pneumonia (PSSP/PRSP) are compared to those with penicillin sensitive S. pneumoniae (PSSP) (29 cases). Incidence of bacteremia and pneumonia was higher in the PSSP group. PISP/PRSP tend to be isolated from patients with bronchitis underlaid with chronic pulmonary disease without statistic significance. Efficacy of the penicillins and 1st and 2nd generation cephem was satisfactory except in only one case of pneumonia with PISP which needed an alternative choice to the 3rd generation cephem. Now a day the degree of resistance is not so high and the available antibiotics are sufficient for the treatment of pneumococcal infection in the elderly patients. However, the wide use of oral cephems and certain new quinolones which do not have enough activity against Streptococcus pneumoniae may increase resistance. In which case, continuous surveillance and clinical caution against this resistant strain is necessary.

Hydrophobicity of cationic solutes measured by electrokinetic chromatography with cationic microemulsions.

The effect of the structure of employed surfactants on the selectivity of microemulsion electrokinetic chromatography (MEEKC) was investigated. Both their ionic groups and their hydrocarbon chain lengths did not affect the selectivity for neutral solutes. However, for cationic solutes, ionic interactions between catonic solutes and anionic surfactants both in the aqueous phase and in the microemulsion phase were observed. Based on these results, determination of the hydrophobicity of cationic solutes was done using MEEKC with cationic microemulsions. The obtained migration index (MI), which had been proposed as a novel hydrophobicity scale measured by MEEKC (Ishihama, Y.; et al. Anal. Chem. 1995, 67, 1588), correlated with conventional hydrophobicity scales for a water/1-octanol system, a liposome system, and an HPLC system using a phospholipid-immobilized artificial membrane (IAM) column. Quantitative structure-activity relationship studies on a protein binding affinity using MI as well as the hydrophobicity scales using the liposome and the IAM column provided better results than those using the water/1-octanol system.

Microscale determination of dissociation constants of multivalent pharmaceuticals by capillary electrophoresis.

Dissociation constants (pKaS) of acidic, basic, and amphoteric pharmaceuticals were exactly determined by capillary electrophoresis. A general equation for use in calculation of pKaS from solute mobilities observed at different pHs was derived to be suitable for a multivalent compound with close pKaS such as angiotensins, which are bioactive octa- or decapeptides. The obtained pKa values were highly consistent with the values determined by conventional methods. For verapamil, the detection limit was 9.8 microM (49 fmol), the total analysis time was 12 min, and the relative standard deviation of the obtained pKa value was 0.11%. This method was also applicable to a mixture of two components, e.g., a bioactive compound and the prodrug, because they have different mobilities and are separated from each other.

Correlation of octanol-water partition coefficients with capacity factors measured by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) was used to measure hydrophobicity of 18 aromatic compounds. The capacity factors, which are proportional to the micelle-water distribution coefficients, were closely correlated with the octanol-water partition coefficients. The addition of Brij 35 to the charged micelle was effective in improving the correlation because the surface charges of the micelle were shielded.

Simple and sensitive quantitation method for mevalonic acid in plasma using gas chromatography/mass spectrometry.

The combination of gas chromatography and mass spectrometry using a polar capillary column and isobutane chemical ionization made it possible to determine mevalonic acid (MVA) as the lactone at subnanogram levels using 1 mL of plasma. The pretreatment procedure consisted of only three steps, namely lactonization, washing with chloroform and liquid/liquid extraction. This simple, rapid and sensitive method, having good precision and accuracy, is useful for evaluating the change of plasma MVA, which is well correlated with whole-body cholesterol biosynthesis.