Low Content of Cyclosporine A and Its Metabolites in the Colostrum of Post-Transplant Mothers.

The rate of post-transplant mothers who breastfeed while on immunosuppression is progressively increasing. Data on breastfeeding while on cyclosporine-based regimens are limited. Therefore, we assessed the amount of cyclosporine and its metabolites that might be ingested by a breastfed infant by measuring the concentration of cyclosporine and its metabolites in the colostrum of seven post-transplant mothers. The mean concentration of cyclosporine in the colostrum was 22.40 ± 9.43 mcg/L, and the estimated mean daily dose of the drug was 1049.22 ± 397.41 ng/kg/24 h. Only three metabolites (AM1, DHCsA, and THCsA) had mean colostrum amounts comparable to or higher than cyclosporine itself, with the daily doses being 468.51 ± 80.37, 2757.79 ± 1926.11, and 1044.76 ± 948.56 ng/kg/24 h, respectively. Our results indicate a low transfer of cyclosporine and its metabolites into the colostrum in the first two days postpartum and confirm the emerging change to the policy on breastfeeding among post-transplant mothers. A full assessment of the safety of immunosuppressant exposure via breastmilk will require further studies with long-term follow-ups of breastfed children.

Long-term cross-validation of everolimus therapeutic drug monitoring assays: the Zortracker study.

BACKGROUND: This ongoing academic collaboration was initiated for providing support to set up, validate, and maintain everolimus therapeutic drug monitoring assays and to study long-term interlaboratory performance. METHODS: This study was based on EDTA whole blood samples collected from transplant patients treated with everolimus in a prospective clinical trial. Samples were handled under controlled conditions during collection, storage and were shipped on dry ice to minimize freeze-thaw cycles. For more than 1.5 years, participating laboratories received a set of 3 blinded samples on a monthly basis. Among others, these samples included individual patient samples, patient sample pools to assess long-term performance, and patient samples pools enriched with isolated everolimus metabolites. RESULTS: The results between liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and the everolimus Quantitative Microsphere System (QMS, Thermo Fisher) assay were comparable. The monthly interlaboratory variability (coefficient of variation %) for cross-validation samples ranged from 6.5% to 23.2% (average of 14.8%) for LC-MS/MS and 4.2% to 26.4% (average of 11.1%) for laboratories using the QMS assay. A blinded long-term pool sample was sent to the laboratories for 13 months. The result was 5.31 ± 0.86 ng/mL (range, 2.9-7.8 ng/mL) for the LC-MS/MS and 5.20 ± 0.54 ng/mL (range, 4.0-6.8 ng/mL) for QMS laboratories. Enrichment of patient sample pools with 5-25 ng/mL of purified everolimus metabolites (46-hydroxy everolimus and 39-O-desmethyl everolimus) did not affect the results of either LC-MS/MS or QMS assays. CONCLUSIONS: Both LC-MS/MS and QMS assays gave similar results and showed similar performance, albeit with a trend toward higher interlaboratory variability among laboratories using LC-MS/MS than the QMS assay.

Determination of pK(a) values of a triptolide derivative and its impurities by pressure-assisted capillary electrophoresis.

A pressure-assisted capillary zone electrophoresis method was developed to determine the pKa values of a triptolide derivative (LLDT-246) and its impurities. The method was performed in an uncoated fused-silica capillary under the electric voltage of 18kV and 50mbar of external pressure applied simultaneously. A series of running electrolyte buffers were used with pH ranging between 2.2 and 10.0 with the constant ionic strength of 0.05M. The values of pKa of LLDT-246 and two impurities were calculated based on the pH dependence of effective mobilities (µeff). The pKa value of LLDT-246 was in good agreement with that of determined by potentiometric titration method.

Preparation of polymeric nanoparticles of cyclosporin A using infrared pulsed laser.

Nanoparticle formation of poorly water-soluble drugs is a means of providing much benefit for improving solubility and bioavailability. We showed that laser irradiation of drugs can be a novel tool for dispersing drug nanoparticles in water. Using our method, we were able to produce nanoparticles containing immunosuppressant drug, cyclosporin A, which shows poor solubility toward water, with high levels of the drug using polyvinyl pyrrolidone and sodium dodecyl sulfate as stabilizing agents. The absence of degradation products was confirmed and the loss of pharmaceutical activity with an inhibitory effect on the interleukin-2 production of Jurkat T cells did not occur. Cyclosporin A nanoparticles showed a spherical shape and their particle size was distributed uniformly around 200 nm. Powder X-ray diffraction analysis suggested that cyclosporin A in the nanoparticles was in an amorphous state. In the measurement of solubility rate, the nanoparticle formulation showed a higher rate than that which had not been processed. At present, although this laser irradiation technology has low productivity, it is expected as a new technology for drug nanoparticle manufacturing together with the development of a new laser device.

Hydrogel containing dexamethasone-loaded nanocapsules for cutaneous administration: preparation, characterization, and in vitro drug release study.

CONTEXT: Our group previously reported the development of dexamethasone-loaded polymeric nanocapsules as an alternative for topical dermatological treatments. OBJECTIVE: Our study aimed to prepare and characterize a hydrogel containing this system to improve the effectiveness of the glucocorticoid for cutaneous disorders. METHODS: For the antiproliferative activity assay, a dexamethasone solution and D-NC were tested on Allium cepa root meristem model. D-NC were prepared by the interfacial deposition of preformed polymer. Hydrogels were prepared using Carbopol Ultrez 10 NF, as polymer, and characterized according to the following characteristics: pH, drug content, spreadability, viscosity, and in vitro drug release. RESULTS AND DISCUSSION: Nanocapsules showed mean particle size and zeta potential of 201 +/- 6 and -5.73 +/- 0.42 nm, respectively. They demonstrated a lower mitotic index (4.62%) compared to free dexamethasone (8.60%). Semisolid formulations presented acidic pH values and adequate drug content (between 5.4% and 6.1% and 100% and 105%, respectively). The presence of nanocapsules in hydrogels led to a decrease in their spreadability factor. Intact nanoparticles were demonstrated by TEM as well as by dynamic light scattering (mean particle size < 300 nm). In vitro studies showed a controlled dexamethasone release from hydrogels containing the drug associated to the nanocapsules following the Higuchi's squared root model (k = 20.21 +/- 2.96 mg/cm(2)/h(1/2)) compared to the hydrogels containing the free drug (k = 26.65 +/- 2.09 mg/cm(2)/h(1/2)). CONCLUSION: Taking all these results together, the hydrogel containing D-NC represent a promising approach to treat antiproliferative-related dermatological disorders.

Etanercept en la psoriasis. Experiencia clínica / Clinical experience with etanercept in the treatment of psoriasis

Introducción. Etanercept es uno de los nuevos fármacos biológicos surgidos para el tratamiento de la psoriasis. Ha demostrado ser una opción eficaz en un alto porcentaje de pacientes, provocando mejoras en el PASI (Psoriasis Assessment and Severity Index) que se mantienen en el tiempo. Además, resulta igualmente adecuado para el control de la artritis psoriásica. Por otra parte, el perfil de seguridad es excelente, con una toxicidad órgano específica mucho menor que los fármacos clásicos y la aparición de escasos efectos secundarios. Muchos de los datos publicados hasta el momento proceden de los distintos estudios clínicos que se han realizado con esta medicación, pero se necesitan trabajos que reflejen la experiencia en la práctica clínica diaria con el manejo de esta terapia en condiciones normales. Métodos. Estudio observacional, retrospectivo, en el que se recogen los 36 pacientes con psoriasis a los que administramos etanercept durante el período de tiempo comprendido entre marzo de 2004 y marzo de 2006. Exponemos la experiencia de nuestro centro en la utilización de este fármaco, con la evolución clínica y los problemas a los que nos hemos enfrentado. Resultados. El PASI se evaluó antes de comenzar el tratamiento y a los tres y seis meses de seguimiento de los pacientes. A los tres meses de tratamiento 13 de los pacientes (36,11 %) habían alcanzado el PASI 50, y 16 pacientes (44,44 %) habían alcanzado el PASI 75. Dos de los pacientes (5,56 %) experimentaron una mejoría de su psoriasis, sin alcanzar el PASI 50, y sólo 4 pacientes (11,11 %) no mostraron mejoría clínica o incluso empeoraron. A los 6 meses se observó un aumento de la eficacia, con 27 pacientes (75 %) que alcanzaron el PASI75 y 6 pacientes (16,67 %) que llegaron a obtener el PASI 50, 2 pacientes (5,56 %) no mostraron ningún beneficio tras la terapia. A los 6 meses 13 de los pacientes (36,1 %) habían alcanzado el PASI 90. En ninguno de los casos se presentaron acontecimientos adversos de importancia que obligaran a suspender el tratamiento. Once de los pacientes siguen en tratamiento con etanercept en el momento actual, ya que se ha mantenido la eficacia y no han presentado efectos adversos importantes. Conclusiones. Exponemos nuestra experiencia clínica con la utilización de etanercept para el tratamiento de la psoriasis en placas, con un perfil muy favorable de eficacia y seguridad. Proponemos la estandarización de la visita clínica al paciente con psoriasis, con recogida exhaustiva de datos en cada visita y la creación de un sistema nacional de registro de datos de pacientes con tratamientos biológicos (AU)

Background. Etanercept is one of the new biologic agents available for treating psoriasis. It has proved an effective option in a high percentage of patients, leading to sustained improvements in the psoriasis are aseverity index (PASI). Likewise, it is effective at controlling psoriatic arthritis, and its safety profile is excellent, with a much lower specific organ toxicity than traditional drugs and few side effects. Many of the data published to date are derived from clinical trials with this medication, but further studies are needed on the use of this therapy to manage patients in daily clinical practice. Methods. This was a retrospective observational study of 36 patients with psoriasis who received etanercept between March 2004 and March 2006. We describe the experience of using this agent at our hospital, with the clinical outcomes and the problems we have faced. Results. The PASI score was assessed before treatment and at 3 and 6 months of patient follow-up. After 3 months of treatment, 13 patients (36.11 %) had achieved a 50 % improvement in PASI score (PASI50), and 16 patients (44 %) had achieved a 75 % improvement (PASI75). Two of the patients (5.56 %) experienced an improvement in their disease without reaching PASI50 and only 4 patients (11.11 %) did not show clinical improvement or deteriorated. After 6 months, efficacy improved, with 27 patients (75 %) achieving PASI75, 6 patients (16.67 %) achieving PASI50, and 2 patients (5.56 %) showing no benefit from treatment. After 6 months, 13 of the patients (36.1 %) had achieved a 90% improvement in PASI score. No adverse events of sufficient significance to warrant discontinuation of treatment were reported. At present, 11 of the patients remain on etanercept treatment as efficacy has been sustained and they have not experienced any adverse events of note. Conclusions. Our clinical experience with the use of etanercept for treating plaque psoriasis shows a favorable efficacy and safety profile. We propose a standardized procedure for consultations with psoriasis patients involving extensive data collection on each visit and the creation of a national surveillance system for patients under treatment with biologic agents (AU)

Pharmacokinetic study of triptolide, a constituent of immunosuppressive chinese herb medicine, in rats.

Triptolide is a potential anti-immune agent, and has shown multi-organic toxicity, however its toxic mechanism remained undiscovered. This paper aimed at characterizing the pharmacokinetic profiles of triptolide in rats to provide the clue to approach the toxic mechanism. The absorption, distribution, metabolism and excretion of triptolide were investigated in male Sprague-Dawley rats after single doses of oral and i.v. administration. After oral administration of 0.6, 1.2 and 2.4 mg/kg, the concentration of triptolide in plasma reached the maximum within 15 min, and declined rapidly with an elimination half-life from 16.81 to 21.70 min. The triptolide kinetics was fitted into one-compartment model after i.v. administration. Oral absolute bioavailability was 72.08% at the dose of 0.6 mg/kg. Triptolide was also rapidly distributed and eliminated in all selected tissues. Less than 1% triptolide of the dose was recovered from the bile, urine or feces as parent drug within 48 h. While triptolide could not be detected in tissues and plasma at 4 h post dose, rats in the group C (oral: 1.2 mg/kg) and D (oral: 2.4 mg/kg) showed obvious toxic response to triptolide and some of rats even died out. It was indicated that triptolide was metabolized extensively, eliminated rapidly, and also showed that the toxicity produced by the triptolide was lag behind the exposure concentration.

Glucose oxidase assisted homogeneous electrochemical receptor binding assay for drug screening.

Although the idea of homogeneous electrochemical immunoassay using antibody and an electroactive modified antigen as a probe looks to be very useful for high-throughput drug screening, there have been few reports. One reason for this is the difficulty experienced making an electroactive probe, because the introduction of electroactive compounds to antigens often interferes with the antigen-antibody interaction. To apply a homogeneous electrochemical assay to drug screening, we have designed new probes referring to the information of immobilization on beads which could identify the drug receptor. FK506 (also called Tacrolimus), immunosuppressive agent is modified with ferrocene derivatives as an electron mediator between glucose oxidase and an electrode, at a non-obstructing part. One of the probes still indicated the electrochemical activity as a mediator and had the specific binding capability for FKBP12 (FK506 binding protein). The current decrease in response to the additional FKBP12, detected with constant voltage amperometry using the probe, was observed within 5 min. Then, free FK506 as a leader drug, rapamycin and cyclosporine A as unknown drugs were used as a model for drug screening. Since the order of response currents at the same concentration of each drug reflected their binding constants, it was shown that binding capacity of an unknown drug candidate could be estimated by comparison of response currents between the leader drug and the unknown drug candidate. Thus, this glucose oxidase assisted homogeneous electrochemical drug-receptor binding assay has been proved to be a useful tool for drug screening.

[Analysis of phenylethanoid glycosides of Herba cistanchis by RP-HPLC].

The Chinese drug "Rou Cong-rong" (Herba Cistanchis) is one of the commonly used drugs in Chinese traditional medicine. It is used to reinforce the vital function of kidney, especially that of the sexual organs and induce laxation, for the treatment of impotence, premature ejaculation in men, infertility, morbid leukorrhea, profuse metrorrhagia in women, and chronic constipation in the aged. This paper deals with the qualitative and quantitative analysis of phenylethanoid glycosides of four species and one variety of Genus Cistanche and 23 lots of commercial crude drugs of Herba Cistanchis by RP-HPLC. The results were as follows: the chemical constituents of Cistanche deserticola Ma, C. salsa (C. A. Mey) G. Beck, C. salsa var. albiflora P. F. Tu et Z. C. Lou and C. tubulosa were similar while those of C. sinensis were different from the others; the contents of echinacoside and acteoside of C. salsa, which were 2.13% and 1.51%, were the highest of the genus Cistanche. An ODS column (Alltima C18, 5 microns, 250 x 4.6 mm) was employed. Linear gradient elution of acetonitrile--1.5% acetic acid was used as mobile phase, and concentration of acetontrile was from 8% to 20% (0-60 min) in the qualitative analysis, and from 11.5 to 20% (0-35 min) in the quantitative analysis. The flow rate was 1.2 ml.min-1. The detection wavelength was set at 335 nm.

[A histochemical study of saikosaponins].

The root of Bupleurum chinese and B.scorzonerifolium was examined histochemically in order to clarify the distribution of saikosaponins in various tissues and parts of the root. By means of qualitative and quantitative analyses, saikosaponins, the bioactive principles in the root, were found to be abundant in the cortex outside the cambium and very little in the xylem of the root. It was also found that the thinner root hairs had the highest contents of saikosaponins, but decreased towards the thicker root head.

[Dynamic variation of saikosaponin contents].

In this paper both the cultivated and wild Bupleurum chinense and B. scorzonerifolium from northeast China of different growing periods and ages were analyzed for the contents of saikosaponins a and d by HPLC and total saikosaponins by spectrophotometry. The results indicated that the saikosaponin content of B. chinense was higher than that of B. scorzonerifolium, and that the saikosaponin contents of the cultivated species were higher than or almost equal to those of the wild ones. It was also found that both the cultivated and wild B. chinense and B. scorzonerifolium featured the highest contents of saikosaponins in the nutrition-growing period.

Purification of immunomodulatory factors in human peripheral blood leukocytes.

Procedure is described for purifying low-molecular-weight factors with antigen-aspecific properties from a dialysate of human leukocyte extract. It includes gel chromatography on Sephadex G-25 and G-15, ion-exchange chromatography, reversed-phase high-performance liquid chromatography (HPLC) on a C18 hydrophobic column and gel permeation HPLC. The immunosuppressive factor (mol.wt. 800-1000) was purified to near homogeneity. It is probably of peptidic nature, although it is pronase resistant. The enhancer factor (mol.wt. 300-600) is eluted from chromatographic columns together with a hypoxanthine-like substance. Nevertheless, the biological activity cannot be attributed to the purine derivative. Identification of this amplifier activity is still lacking.

Simultaneous determination of ginsenosides and saikosaponins by high-performance liquid chromatography.

Octadecyl porous glass was used as the packing for reversed-phase high-performance liquid chromatography. A mixture of ginsenosides and saikosaponins (saponins of ginseng and bupleurum root, respectively) were analysed with detection at 203 nm. A well resolved chromatogram of ginsenoside Rb1, Rc, Rb2 and Rd and saikosaponin a, b2 and c was obtained with acetonitrile-water (25.5:74.5) as the mobile phase. The whole separation was achieved in 20 min with a flow-rate of 1.5 ml/min. Calibration graphs for ginsenoside Rb1, Rc, Rb2 and Rd and saikosaponin a and c were linear up to 5 micrograms. Rapid and accurate simultaneous determinations of the saponins are possible by the described method.

An ethnopharmacognostic approach to the search for immunomodulators of plant origin.

The search for immunomodulating plant constituents through basic and field inquiries into the literature and practices of traditional Indian medicine is treated. The strategy of data collecting proceeds through aspects of an ethnobotanical, an ethnopharmaceutical, an ethnopharmacological, and an ethnomedical nature. In the experimental immunopharmacognostic phase, immunomodulatory compounds are isolated and purified through action-guided fractionation procedures. The results described here refer to activities found on human complement activation and on PMN leucocytes activation. The immunomodulating plant compounds included in this report were isolated from Azadirachta indica bark, Woodfordia fructicosa flowers, Picrorhiza kurroa roots, and Jatropha multifida latex.

Search for new plant constituents with potential antiphlogistic and antiallergic activity.

Structure-activity relationships obtained from in vitro screening results obviously indicate that the highest inhibition effects on cyclooxygenase and 5-lipoxygenase are found amongst the class of phenolic compounds (flavonoids, polyphenols, coumestans, phenol carboxylic acids) and arachidonic acid analogous (alkylamides, retinoids, arylheptanoids, thiosulfinates, sulfinyl disulfides). The antiinflammatory activities of some triterpenenic acids, sesquiterpene lactones, and polysaccharides may be due to their immunomodulating activities on the complement and/or T-lymphocyte populations, respectively. In the search for potential antiallergic and antiasthmatic compounds, the thiosulfinates of onion were found to be active principles of the drug. The mechanism of action of some other antiallergic plant drugs (i.e. Tylophora asthmatica, Adhatoda vasica, etc.) has not yet been clarified.

Biopharmaceutical aspects of FK-506.

FK is a potent immunosuppressive agent. FK can be analyzed in biologic fluids by EIA. The oral absorption of FK is rapid but variable in dogs. After intramuscular administration, FK is slowly and continuously absorbed. FK is primarily eliminated by metabolism. Less than 1% of the administered dose is excreted in the bile or the urine. After chronic intramuscular administration FK inhibits drug metabolism. Monitoring of FK levels in plasma is essential for the proper interpretation of efficacy and toxicity studies.