[Web-based rehabilitation aftercare following inpatient psychosomatic treatment]. / Web-basierte Rehabilitationsnachsorge nach stationärer psychosomatischer Therapie (W-RENA).

BACKGROUND: High relapse rates following treatment for mental health disorders are a challenge for psychosomatic rehabilitation treatments. The goal of the present study is to evaluate the feasibility, acceptance and process-quality of a 12-week transdiagnostic Internet-based maintenance treatment (W-RENA) following psychosomatic rehabilitation treatment. Findings regarding effectiveness and moderators of treatment outcome that were already reported elsewhere are briefly summarized.In a preliminary study we first assessed whether rehab patients have the technical requirements and abilities to successfully participate in Internet-based treatments. Patients expressing interest for participation in W-RENA (N=400) were compared with non-participants (N=1789) with regard to sociodemographic and clinical characteristics. METHOD: In a 2-arm randomized controlled trial (N=400) we subsequently compared participants of W-RENA with participants of a treatment as usual group (TAU). Self-report measures were assessed at the beginning of inpatient treatment (t1), at discharge from inpatient treatment/start of W-RENA (t2), and at 3- (t3) and 12-months follow-ups (t4). RESULTS: The majority of assessed rehab-patients had the technical prerequisites (78.79%) and necessary skills (79.9%) to successfully participate in an Internet-based intervention. A third of the patients (32%) which were invited to take part in the intervention (and the study) expressed interest to participate. Study participants and non-participants differed only slightly. Most participants (80.6%) reported to have gained benefit from participating. Treatment achievements as well as quality of therapist alliance were rated high from both patients and therapists. Moreover, participants of the W-RENA group could stabilize their inpatient treatment outcomes up to 3- and 12-months follow-up better than controls could do (differences in symptom change from discharge to 3-months follow-up: d=0.38; to 12-months follow-up: d=0.55). Clinical significant symptom deterioration from discharge to 1-year follow-up could be reduced by 2/3 (29.45% vs. 11.45%). We could not identify any subgroup not profiting from study participation. Patients with low education benefited particularly. CONCLUSION: Internet-based aftercare interventions are a feasible, accepted and effective approach to successfully sustain treatment outcomes achieved in inpatient psychosomatic rehabilitation.

First Report of Colletotrichum sansevieriae Causing Anthracnose of Sansevieria trifasciata in Florida.

Sansevieria Thunberg, a member of the Agavaceae, contains around 60 species indigenous to Africa, Arabia, and India. Several species and their cultivars are commercially produced for use as interior and landscape foliage plants. During August 2010, several local nurseries submitted Sansevieria trifasciata samples to the Florida Extension Plant Diagnostic Clinic in Homestead. Leaves had round, water-soaked lesions and as the disease progressed, lesions rapidly enlarged and coalesced, resulting in severe leaf blight. Both young and mature leaves were affected. Closer examination of mature lesions revealed numerous brownish black acervuli that were produced in concentric rings, which is characteristic of anthracnose. The fungus was identified as Colletotrichum sansevieriae Nakamura based on typical cultural characteristics, conidial and appressoria morphology (1). Conidia were straight, cylindrical, obtuse at the apex, slightly acute at the base with a truncate attachment point, and 12.5 to 33 (18.4) × 4 to 8.9 (6.5) µm (n = 50). Hyphopodia were ovate, dark brown, single celled, and 6.2 to 8.7 (7.7) × 6.3 to 7.5 (7.3) µm (n = 25). Colonies on potato dextrose agar (PDA) were grayish white, felted with aerial mycelium, reverse gray to dark olivaceous gray, and partly cream in color. Sequences of the rDNA internal transcribed spacer (ITS) regions of two isolates (GenBank Accession Nos. JF911349 and JF911350) exhibited 99% nucleotide identity to an isolate of C. sansevieriae (GenBank Accession No. HQ433226) collected from diseased sansevieria in Australia. In addition, a maximum parsimony analysis (MEGA v.5.0) indicates that the two C. sansevieriae isolates from Florida are monophyletic (86% bootstrap support) with the type species from Japan (SA-1-2 AB212991; SA-1-1 AB212990) and the Australian isolate. Pathogenicity of our sequenced isolates was evaluated in greenhouse experiments. Twelve- to fourteen-week-old sansevieria plants were inoculated with conidial suspensions (1 × 106 conidia/ml) of C. sansevieriae. Inoculum or autoclaved water was sprayed over the foliage until runoff. Four plants of each of two economically important cultivars, Laurentii and Moonshine, were sprayed per treatment and the experiment was repeated twice. Inoculated plants were placed in a greenhouse at 29°C with 70 to 85% relative humidity. Plants were observed for disease development, which occurred within 10 days of inoculation for both cultivars. No symptoms developed on the control plants. Foliar lesions closely resembled those observed in the affected nurseries. C. sansevieriae was consistently reisolated from symptomatic tissue collected from greenhouse experiments. On the basis of molecular phylogenetics and distinguishing morphological characters, Nakamura et al. erected C. sansevieriae as a novel species that appears to be restricted to the host sansevieria (1). To our knowledge, this is the first report of C. sansevieriae causing anthracnose of sansevieria in Florida. Reference: (1) M. Nakamura et al. J. Gen. Plant Pathol. 72:253, 2006.

First Report of Fruit Rot on Hylocereus undatus Caused by Bipolaris cactivora in South Florida.

Pitahaya (Hylocereus undatus (Haw.) Britton & Rose), a cactus grown for its edible fruit, is gaining popularity in South Florida as part of the specialty tropical fruit market. In July 2009, flowers and fruit were discovered with an uncharacterized rot. Small, circular, light brown, depressed lesions expanded to form large areas of rot on flowers and fruit in 7 to 10 days. The lesions produced large amounts of dark fungal spores. Single-spore isolates were identified morphologically and by aligning internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) DNA sequences from the isolates with previously published sequences of Bipolaris, Drechslera, and Cochliobolus species. Conidia from the dark, blackish brown colonies were formed at the tips of pale golden brown, straight to flexuous conidiophores, 99 (184) 313 × 3 (6) 8 µm and slightly swollen at the apex and base. Conidia were pale-to-medium golden brown, smooth and clavate with a protuberant hilum, 24 (40) 51 × 9 (10) 13 µm, and two to four distoseptate. The isolates closely match descriptions of Bipolaris cactivora (= Drechslera cactivora) (3,4), although isolates from pitahaya had smaller conidia (30 to 65 µm) than previously reported. Conidial characteristics from a B. cactivora herbarium specimen BPI 431621 (U.S. National Fungus Collections) closely matched (29 (36) 50 × 8 (9) 11 µm, two to four distoseptate) our isolates. ITS (GenBank Accession Nox. HM598677-79) sequences aligned most closely (99.7% homology) with another B. cactivora isolate from China (GU390882), and both ITS and gpd (GenBank Accession Nos. HM598680-82) sequences indicate a close relationship to Bipolaris indica. Wounded or nonwounded mature pitahaya fruit and mature stems were inoculated with either a mycelia plug or a 15-µl 0.3% agar drop containing 105 conidia ml-1. Lesion diameters were measured after 7 days at 25°C, the fungus was reisolated on potato dextrose agar (PDA) and its identity was confirmed. Mean lesion diameters on mature fruit were 6.0 to 10.8 mm, depending on the inoculation method, and sporulation began 6 days after inoculation. On mature plant stems, wound-inoculated treatments formed 1.8 to 3.4 mm lesions, but nonwounded inoculations and controls were negative. Lesions were light tan, circular, and did not sporulate. To our knowledge, this is the first report of fruit rot caused by B. cactivora on pitahaya in Florida. The same pathogen causes stem rot of the Cactaceae in Europe and the United States (2) and a fruit rot on pitahaya in Japan (4). In Florida, it has been reported as causing a leaf spot on Portulaca oleracea (1). Our results indicate that B. cactivora causes flower and fruit rot on pitahaya, but does not seriously affect mature plant stems. The flower rot does not appear to significantly increase incidence but may provide inoculum for the fruit rot. The high incidence of fruit rot affecting commercial operations in Miami-Dade County over the past 2 years requires an effective disease management strategy. References: (1) S. A. Alfieri, Jr. et al. Bull. 14. Index of Plant Diseases in Florida (Revised). Florida Dep. Agric. Consumer Serv., Div. Plant Ind., 1984. (2) R. D. Durbin et al. Phytopathology 45:509, 1955. (3) M. B. Ellis. Page 432 in: Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, England. 1971. (4) S. Taba et al. J. Gen. Plant Pathol. 73:374, 2007.

First Report of Colletotrichum capsici Causing Postharvest Anthracnose on Papaya in South Florida.

Postharvest anthracnose of papaya, Carica papaya, is an important disease in most production areas worldwide (2). Colletotrichum gloeosporioides causes two types of anthracnose symptoms on papaya: (i) circular, sunken lesions with pink sporulation; and (ii) sharply defined, reddish brown and sunken lesions, described as 'chocolate spot' (2). Colletorichum spp. were isolated from lesions of the first type on papaya fruit from the University of Florida Tropical Research and Education Center, Homestead in December 2007 and from fruit imported from Belize in March 2008 (4). Single-spore isolates were identified using colony morphology and internal transcribed spacer (ITS) and mating type (MAT1-2) sequences. Two taxa were identified in both locations: (i) C. gloeosporioides (MAT1-2; GenBank Nos. GQ925065 and GQ925066) with white-to-gray, fluffy colonies with orange sporulation and straight and cylindrical conidia; and (ii) C. capsici (ITS; GenBank Nos. GU045511 to GU045514) with sparse, fluffy, white colonies with setose acervuli and falcate conidia. In addition, in Florida, a Glomerella sp. (ITS; GenBank Nos. GU045518 and GU045520 to GU045522) was recovered with darkly pigmented colonies that produced fertile perithecia after 7 to 10 days on potato dextrose agar (PDA). In each of three experiments, mature fruit (cv. Caribbean Red) were wounded with a sterile needle and inoculated with a 15-µl drop of 0.3% water agar that contained 105 conidia ml-1 of representative isolates of each taxon. The diameters of developing lesions were measured after 7 days of incubation in the dark at 25°C, and the presence of inoculated isolates was confirmed by their recovery from lesion margins on PDA. In all experiments, C. capsici and C. gloeosporioides produced lesions that were significantly larger than those that were caused by the water control and Glomerella sp. (respectively, approximately 12, 17, 0, and <1 mm in diameter). C. gloeosporioides produced sunken lesions with dark gray centers and pink/gray sporulation, which match those previously described for anthracnose on papaya (2). In contrast, C. capsici produced dark lesions due to copious setae of this pathogen; they resembled C. capsici-induced lesions on papaya that were reported previously from the Yucatan Peninsula (3). C. capsici has also been reported to cause papaya anthracnose in Asia (4), but to our knowledge, this is the first time it has been reported to cause this disease in Florida. Since it was also recovered from fruit that were imported from Belize, it probably causes anthracnose of papaya in that country as well. Another falcate-spored species, C. falcatum, was recovered from rotted papaya fruit in Texas (1). The Glomerella sp. was recovered previously from other hosts as an endophyte and causes anthracnose lesions on passionfruit (4). However, its role as a pathogen on papaya is uncertain since it was not pathogenic in the current work; the isolates that were recovered from papaya lesions may have colonized lesions that were caused by C. capsici and C. gloeosporioides. References: (1) Anonymous. Index of Plant Diseases in the United States. U.S. Dept. of Agric. Handb. No. 165. Washington, D.C., 1960. (2) D. M. Persley and R. C. Ploetz. Page 373 in: Diseases of Tropical Fruit Crops. R. C. Ploetz, ed. CABI Publishing. Wallingford, UK, 2003. (3) R. Tapia-Tussell et al. Mol Biotechnol 40:293, 2008. (4) T. L. Tarnowski. Ph.D. diss. University Florida, Gainesville, 2009.

First Report of Colletotrichum boninense, C. capsici, and a Glomerella sp. as Causes of Postharvest Anthracnose of Passion Fruit in Florida.

Anthracnose is an important foliar and fruit disease of passion fruit, Passiflora spp. (3). In 2008, postharvest anthracnose on purple and yellow passion fruits (P. edulis Sims and P. edulis f. flavicarpa O. Degner, respectively) from a commercial planting in Miami-Dade County, FL was examined. Lesions began as light brown areas that became papery, covered much of the fruit surface, and developed pink-to-dark sporulation. Single-conidium isolates from lesions were examined morphologically and with internal transcribed spacer (ITS) sequences. Four taxa were identified: Colletotrichum boninense (GenBank No. GU045516) with felted cream-to-orange colonies and cylindrical conidia; C. capsici (synonym C. truncatum [2]) (GU045515) with sparse, white mycelia, setose acervuli, and falcate conidia; C. gloeosporioides with fluffy white-to-gray colonies and straight, cylindrical conidia; and a Glomerella sp. (GU045517) with darkly pigmented perithecia. In two experiments, four mature, yellow passion fruit were wounded at a single equatorial site with a sterile needle and inoculated with a 15-µl drop of 0.3% water agar that did not contain (noninoculated control) or contained 105 conidia per ml of representative isolates from each taxon. After 21 days at 25°C without light, anthracnose incidence was recorded and the presence of the isolates was confirmed by their recovery from lesion margins on potato dextrose agar. Anthracnose did not develop on noninoculated control fruit. Mean incidences of anthracnose exceeded 50% for isolates of C. boninense (three from passion fruit), C. capsici (two from passion fruit), and a Glomerella sp. (two from passion fruit and one each from papaya and eugenia). Despite its common indictment as a causal agent of anthracnose on passion fruit (3), symptoms developed on only one fruit that was inoculated with an isolate of C. gloeosporioides from passion fruit (13%) and did not develop after inoculation with an isolate from papaya. Work is needed to determine whether host-specific populations of C. gloeosporioides exist on passion fruit that were not assessed during this study or whether the pathogen was misidentified in previous reports on this host. C. boninense was associated previously with postharvest anthracnose of passion fruit in Japan and Colombia, whereas C. capsici was associated with leaf anthracnose of passion fruit in Florida and Japan (4); both species are reported here for the first time as causes of postharvest anthracnose of passion fruit in Florida. Glomerella sp. caused darkly pigmented lesions and produced the teleomorph on symptomatic passion fruit and in single-ascospore cultures. Isolates with ITS sequences that are 99% homologous to those from passion fruit have been recovered in South Florida from eugenia, papaya, and Piper betle (4) and from other locations on several other hosts (GenBank); they are often nonpathogenic endophytes. Almeida and Coêlho (1) reported in Brazil a Glomerella sp. that formed the teleomorph in culture and caused anthracnose on passion fruit, but did not provide ITS sequences. Additional work is warranted on the identity and ecology of these fungi. References: (1) L. C. C. Almeida and R. S. B. Coêlho. Fitopatol. Bras. 32:318, 2007. (2) U. Damm et al. Fungal Divers. 39:45, 2009. (3) B. Manicom et al. Page 413 in: Diseases of Tropical Fruit Crops. R. C. Ploetz, ed. CABI Publishing, Wallingford, UK, 2003. (4) T. L. Tarnowski. Ph.D. diss. University of Florida, Gainesville, 2009.

Activity of Fungicides Against Monilinia vaccinii-corymbosi in Blueberry Flowers Treated at Different Phenological Stages.

The activity of fenbuconazole and azoxystrobin applied to blueberry flowers at different phenological stages against subsequent gynoecial infection by the mummy berry fungus Monilinia vaccinii-corymbosi was evaluated. In the greenhouse, potted blueberry plants having flower clusters at five distinct stages (from bud scale separation to anthesis) were treated with the two fungicides. One day after anthesis (between 1 and 15 days after fungicide treatment), individual flowers were detached and inoculated with conidia of M. vaccinii-corymbosi in the laboratory. Four days after inoculation, hyphal ingress into the style was determined microscopically as a measure of fungicide efficacy. Results revealed a significant flower stage effect (P < 0.0001), whereby only fungicide application at anthesis but not at the four preanthesis stages reduced subsequent fungal ingress into the style. There was no significant difference between the two fungicides (P > 0.50) nor was there a significant fungicide-flower stage interaction (P > 0.30). In the field during 2 years, mature blueberry plants were treated with the two fungicides and exposed to natural pathogen inoculum. At the time of application, flower clusters at anthesis and at three preanthesis stages were selected and tagged. Mummy berry incidence in fruit developing from the tagged clusters was assessed to determine treatment effects. Whereas fenbuconazole lowered disease incidence for all preanthesis stages, azoxystrobin was effective only at the latest preanthesis stage. The discrepancy between these results and those of the greenhouse study (where there was no preanthesis activity of either fungicide) indirectly suggests post-infection fungicidal activity in the ovary, the base of which was exposed to the fungicide spray at the time of treatment for all flower phenology stages. Thus, although there appears to be insufficient translocation of the two fungicides in flowers treated at preanthesis stages to prevent stylar ingress by the pathogen, fungicidal activity in the ovary may be sufficient to halt subsequent fungal colonization, especially for fenbuconazole. To prescribe the most effective management program for flower-infecting fungi, translocation and post-infection activity of fungicides in floral tissues must be better understood.

An HPLC method for the determination of diastereomeric prodrug RS-79070-004 in human plasma.

Ganciclovir is an antiviral nucleoside analogue approved for treatment and prevention of cytomegalovirus infections in immunocompromised subjects. RS-79070-194, a diastereomeric monovalyl ester of ganciclovir (hydrochloride salt), is under evaluation as a prodrug to increase the bioavailability of ganciclovir. An HPLC method with column switching has been developed and validated for quantification of the corresponding free base RS-79070-004 in human plasma. In the method, proteinaceous material in 0.25 ml of plasma is precipitated by trichloroacetic acid. An aliquot of the supernatant is analyzed by HPLC, with automated column switching to remove late-eluting materials that might interfere with the analyte peaks in subsequent runs. Detection of RS-79070-004 is by UV lambda = 254 nm). The peak areas for each isomer are summed to generate a value for total RS-79070-004. The method has a validated range of 0.0400-4.00 microg/ml and a lower limit of quantification of 0.0400 microg/ml. All intra- and inter-assay %CVs were < 7.5%, and all recoveries (accuracy) were within 6% of nominal values. No interference was observed by ganciclovir, caffeine, acetaminophen, or ibuprofen. Analyte stability in plasma and in the sample extracts is adequate for the specified collection, storage, and analysis conditions. The validated method has been successfully used to analyze clinical study samples.

A rapid, sensitive HPLC method for the determination of ganciclovir in human plasma and serum.

A method for ganciclovir determination in human serum and plasma has been developed and validated. The method has a lower limit of quantification (LLOQ) adequate for sensitive pharmacokinetic studies ( < or = 0.05 microg/ml), has run times of < or = 15 min, and uses aliquot volumes adequate for pediatric studies (0.25 ml). In the method, proteinaceous material in serum or plasma is precipitated by trichloroacetic acid. An aliquot of the supernatant is analyzed by HPLC; automated column switching removes late-eluting materials that might interfere with the analyte peak in subsequent runs. Detection and quantification of ganciclovir is by fluorescence (lambda(ex) = 278 nm; lambda(em) = 380 nm). The method has a validated range of 0.0400-4.00 microg/ml and an LLOQ of 0.0400 microg/ml. All intra- and inter-assay % C.V. values were < 8%; all recoveries (accuracy) were within 7% of nominal values. No interference was observed by mycophenolic acid or its glucuronide metabolite, by AZT, salicylic acid, acetaminophen, ibuprofen, naproxen prednisone, acyclovir, or cyclosporine. Ganciclovir is very stable in the samples and the extract during storage and sample processing. Both serum and plasma methods have been validated for use and have been successfully used to analyze samples from clinical studies.

Effect of high-dose oral ganciclovir on didanosine disposition in human immunodeficiency virus (HIV)-positive patients.

This study was designed to investigate the interaction between high-dose oral ganciclovir (6,000 mg/day) and didanosine at steady state in patients who were seropositive for human immunodeficiency virus (HIV) and cytomegalovirus (CMV) infection. The study was conducted as an open-label, randomized, three-period crossover study. Patients received (in random order) multiple oral doses of didanosine 200 mg every 12 hours alone, ganciclovir 2,000 mg every 8 hours alone, and ganciclovir 2,000 mg every 8 hours in combination with didanosine 200 mg every 12 hours. Blood and urine samples for determinations of drug concentrations were obtained on day 3 of each dose regimen. When ganciclovir was administered either before or 2 hours after didanosine, the mean increases in maximum concentration (Cmax), area under the concentration-time curve (AUC0-12), and percent excreted in urine of didanosine were 58.6% and 87.3%, 87.3% and 124%, and 100% and 153%, respectively. There were no statistically significant effects of didanosine on the steady-state pharmacokinetics of ganciclovir in the presence of didanosine, irrespective of sequence of administration. There were no significant changes in renal clearance of didanosine, suggesting that the mechanism for the interaction does not involve competition for active renal tubular secretion. The mechanism responsible for increased didanosine concentrations and percent excreted in urine during concurrent ganciclovir therapy may be a result of increased bioavailability of didanosine. However, the mechanism appears to be saturated at oral ganciclovir doses of 3 g/day.

The pharmacokinetics of a single oral dose of mycophenolate mofetil in patients with varying degrees of renal function.

BACKGROUND: The purpose of this study was to determine the effect of renal function on the elimination and disposition of mycophenolic acid and its glucuronide metabolite (MPAG) after oral administration of the pro-drug mycophenolate mofetil. In addition, this study sought to examine hemodialysis removal of mycophenolic acid and its MPAG. METHODS: Subjects were stratified into five groups on the basis of iohexol clearance. After an overnight fast, all subjects received a single 1 gm dose of mycophenolate mofetil. Plasma concentrations of mycophenolic acid and MPAG were measured from 0 to 96 hours after administration. Mycophenolic acid and MPAG maximum plasma concentration (Cmax) and the time to reach Cmax (tmax) for each group were determined from the mean plasma concentration-time profiles. Area under the plasma concentration-time curve values for mycophenolic acid and MPAG were calculated by the trapezoidal rule. The half-lives of mycophenolic acid and MPAG were calculated from the terminal portions of the concentration-time profiles. RESULTS: Mycophenolic acid clearance was not associated with changes in glomerular filtration rate (GFR). Cmax tended to increase as GFR declined. MPAG clearance correlated well with GFR (r2 = 0.905). Clearance of mycophenolic acid and MPAG were unaffected by hemodialysis. CONCLUSIONS: Clearance of mycophenolic acid after a single 1 gm oral dose of mycophenolate mofetil is unaffected by renal function. Clearance of mycophenolic acid is unaffected by hemodialysis. Diminished renal function should not require preemptive adjustment of 1 gm doses of mycophenolate mofetil; however dosage adjustment may be warranted on the basis of adverse effects or toxicity in individual patients. Mycophenolate mofetil can be administered irrespective of hemodialysis session without effect on mycophenolic acid exposure.

Pharmacokinetics of oral ganciclovir alone and in combination with zidovudine, didanosine, and probenecid in HIV-infected subjects.

The aim of this study was to determine whether oral ganciclovir interacted pharmacokinetically with zidovudine (AZT), didanosine (ddI), or probenecid. A multicenter, open-label, randomized, crossover pharmacokinetic study with four phases was undertaken at an outpatient private research center and at university research clinics. Twenty-six HIV-infected adults (23 men, 3 women) with cytomegalovirus (CMV) seropositivity and CD4+ T-lymphocyte count > or =100 cells/microl were studied. Patients had to be stable on antiretroviral therapy for at least 4 weeks. Patients with a history of opportunistic infection or gastrointestinal symptoms were excluded. Measurements included serial blood and urine samples during the dosing intervals at steady state. The steady-state pharmacokinetics of ganciclovir were determined after the participants had stabilized and were tolerating AZT or ddI therapy. When a 1000-mg dose of oral ganciclovir was taken every 8 hours, there was a significant mean increase in Cmax and dosing interval area under the serum concentration time curve over a dosing interval (AUC) for the two antiretroviral drugs: for AZT, 61.6% and 19.5%, respectively; for ddI when administered sequentially (2 hours before ganciclovir), 116.0% and 114.6%; and for ddI administered simultaneously with ganciclovir, 107.9% and 107.1%, respectively. There was no significant change in renal clearance for either antiretroviral drug, suggesting that the interaction did not occur through a renal mechanism. There was no significant change in mean ganciclovir Cmax and AUC(0-8) when coadministered with AZT. Mean increases in Cmax and AUC(0-8) of oral ganciclovir averaged 40.1% and 52.5%, respectively, when coadministered with probenecid, but decreased by 22.1% and 22.7%, respectively, when oral ganciclovir was administered 2 hours after ddI. There was no change in the mean ganciclovir Cmax or AUC(0-8) when administered simultaneously with ddI. The mean renal clearance of oral ganciclovir was not affected by AZT or ddI coadministration intake, but there was a mean decrease of 19% when coadministered with probenecid. We conclude the increased serum concentration and reduced renal clearance of ganciclovir suggests competition with probenecid for secretion at the renal tubule. The mechanism of the interaction of oral ganciclovir with either AZT or ddI remains to be determined. The magnitude of the effect of oral ganciclovir on ddI pharmacokinetics may result in an increase in ddI concentration-related toxicities. Similarly, the small but significant decrease in ganciclovir concentration with sequential combination ddl therapy may impair the efficacy of oral ganciclovir. For HIV-infected patients receiving ganciclovir and ddI, clinicians should recommend administering the two drugs simultaneously, and patients should be monitored closely for ddI-associated toxicities.

Simultaneous determination of mycophenolic acid and its glucuronide conjugate in human plasma by a single-run ion-paring method.

A quick and robust HPLC method for simultaneous determination of plasma concentrations of mycophenolic acid (MPA) and its glucuronide conjugate (MPAG) is described. Using tetrabutylammonium dihydrogen phosphate as ion-paring reagent in the mobile phase, concentrations for both MPA and MPAG can be determined in a single HPLC run on a reversed-phase C18 column at 254 nm. The method is reproducible and accurate, with lower limits of quantification of 0.100 microg/ml for MPA and 0.800 microg/ml for MPAG. The quantification ranges of the method are 0.100-40.0 microg/ml for MPA and 0.800-400 microg/ml for MPAG using a 0.5-ml aliquot in the analysis.

An indirect (derivatization) and a direct HPLC method for the determination of the enantiomers of ketorolac in plasma.

An indirect and a direct HPLC method for the quantification of the (R) and (S) enantiomers of ketorolac are described here. The indirect method employs the chiral amine (+)-R-1-(1-naphthyl)ethylamine to form disastereomeric amides; separation of the disastereomeric derivates is achieved by normal-phase HPLC with a mobile phase of ethyl acetate-hexane. The direct method uses a C18 solid-phase extraction column to extract ketorolac enantiomers from plasma; the reconstituted extract is then injected onto an alpha 1-acid glycoprotein chiral column using a mobile phase of isopropanol-phosphate buffer (0.05 M; pH 5.5). Both methods are reproducible, accurate, and stereospecific, and both have equivalent quantification limits (0.02 microgram ml-1 of plasma for each enantiomer), ranges (0.02-2.0 micrograms per aliquot of plasma), precision (% relative standard deviations of < or = 10.5% and < or = 10.8% for (R)- and (S)-ketorolac respectively), and accuracy (mean recoveries of 88.4-110% and 90.1-110% for (R)- and (S)-ketorolac respectively). Results of analyses of clinical samples by the two methods showed excellent agreement (slope near 1.0 and coefficients of correlation between 0.9740 and 0.9864 for both enantiomers).

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma.

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C18 solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-microliters aliquot of the eluate is injected onto a C18 column (5 microns particle size, 150 x 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 microgram/ml and the range is 0.400-20 micrograms/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at -80 degrees C and analyzed within four months of collection.

Chiral kinetics and dynamics of ketorolac.

It has been shown that the analgesic and cyclooxygenase inhibitor activity of ketorolac tromethamine (KT), which is marketed as the racemic mixture of (-)S and (+)R enantiomers, resides primarily with (-)S ketorolac and that the ulcerogenic activity of this agent also resides in (-)S ketorolac. Resolution of individual enantiomers for analysis in plasma samples has been accomplished by two methods: derivatization to form diastereomers that are separated by HPLC, or direct HPLC using a chiral phase column. When mice and rats were given oral solutions of (-)S and (+) KT, it was found that the kinetics and interconversion of the enantiomers were species and dose dependent. Interconversion was higher in mice than in rats; when (-)S KT was administered, 71% of the area under the concentration-time curve (AUC) was due to (+)R ketorolac in mice, compared with 12% in rats. More interconversion was observed at higher doses; the percent of AUC due to (-)S ketorolac when (+)R KT was administered increased from 12% to 25% in mice and from 2% to 8% in rats. In general, more interconversion occurred from (-)S to (+)R ketorolac in the animal studies. Human subjects were given single oral solution doses of racemic KT (30 mg), (-)S KT (15 mg), and (+)R KT (15 mg). The plasma concentrations of (-)S ketorolac were lower than (+)R ketorolac at all sample times after racemic KT (22% of the AUC was due to (-)S ketorolac). When (+)R KT was administered, (-)S ketorolac was not detectable and interconversion was essentially 0%. When (-)S KT was administered, significant levels of (+)R ketorolac were detectable and interconversion was 6.5%. After all doses, plasma half-life was shorter and clearance greater for (-)S ketorolac than for (+)R ketorolac. Thus, in humans very little or no interconversion of (+)R to (-)S was observed, and interconversion of (-)S to (+)R was minimal (6.5%). These data demonstrate that the kinetics and interconversion of the enantiomers of ketorolac is different in animals and humans as well as from most other NSAIDs. This may be due to more rapid excretion or metabolism of (-)S ketorolac and a different mechanism of interconversion.

Manual and automated (robotic) high-performance liquid chromatography methods for the determination of mycophenolic acid and its glucuronide conjugate in human plasma.

A manual and an automated (Zymark PyTechnology robot) HPLC method for simultaneous determination of plasma mycophenolic acid (MPA) and its glucuronide conjugate (MPAG) are described here. Both methods are reproducible and accurate, and both are equivalent in all respects, including quantification limits (MPA, 0.100 microgram/ml; MPAG, 4.00 micrograms/ml), range (using 0.05-0.5 ml of plasma: MPA, 0.0500-20.0 micrograms/aliquot; MPAG, 2.00-200 micrograms/aliquot), precision, and accuracy. MPA and MPAG were stable under the conditions used with both methods. Results from aliquots of paired control samples, analyzed by the manual method over three years at six analytical laboratories, showed excellent agreement in precision and accuracy.

Simple reversed-phase high-performance liquid chromatography quantitation of ganciclovir in human serum and urine.

A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 micrograms/ml. Recoveries varied from 91 to 107% with coefficients of variation ranging from 0.387 to 7.95%.

Radioimmunoassay of ganirelix in plasma or serum.

A procedure for the radioimmunoassay (RIA) of ganirelix in plasma or serum at concentrations as low as 0.050 ng/ml is described. Antiserum was produced by coupling the N-terminus glycyl analog of ganirelix to BSA by a carbodiimide reaction and immunizing rabbits with this conjugate. The antiserum did not crossreact with LHRH or with various ganirelix peptide fragments. For RIA, 125I labeled ganirelix was used as the tracer and a double antibody procedure was used to separate the free and bound fractions. No purification of the analyte was required prior to RIA. Accuracy of the method was assessed by adding known quantities of ganirelix to ganirelix-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.050-50.0 ng/ml yielded a regression equation of y = 0.97x + 0.18, r = 0.999, where x is the amount added and y is the amount measured. Additional validation was obtained from an in vivo study in which [3H]-ganirelix was administered to monkeys and plasma clearance profiles were determined by RIA and an HPLC-radiochemical method. The results were in agreement within experimental error of the two methods. Linear regression analysis of the comparative data gave the equation y = 0.92x + 33.7, r = 0.980, where x is the amount measured by RIA and y is the amount measured by HPLC-radiochemical analysis.

Use of liposome encapsulation in a combined single-liquid reagent for homogeneous enzyme immunoassay.

A technique has been developed to permit mutually reactive macromolecular reagents used in immunoassays to be combined without premature reaction. A conjugate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and theophylline has been encapsulated in 0.2-micron-diameter bi-lamellar liposomes. Suspensions of these liposomes had excellent stability. Whereas the enzyme activity of the free conjugate is rapidly inhibited by anti-theophylline antibody, a suspension of the encapsulated conjugate in a solution of the antibody and NAD+ (6.0 mmol/L) retained greater than 92% of the initial enzyme activity after standing for one year at 4 degrees C. At higher NAD+ concentrations the liposomes aggregated, and enzyme activity was inhibited by leakage of the NAD+ hydrolysis product, adenosine diphosphoryl 5-ribose (ADP-ribose), into the liposomes. Inhibition by ADP-ribose could be blocked and partly reversed by adding semicarbazide. The liposomes were efficiently lysed by Triton X-100, deoxycholate, or octyl glucoside, the kinetics and extent of lysis being affected by liposome size and correlating with the acid strength of various cholate derivatives. Addition of a serum sample and a solution of buffer, substrate, and detergent to a single reagent containing the liposomes and anti-theophylline antibody provided assay results equivalent to those obtained by conventional two-reagent EMIT homogeneous enzyme immunoassay for theophylline.