LC-MS/MS method for simultaneous determination of diethylcarbamazine, albendazole and albendazole metabolites in human plasma: Application to a clinical pharmacokinetic study.

Combination therapy with anti-filarial drugs is now widely used for treatment of lymphatic filariasis. A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantitation of diethylcarbamazine (DEC), albendazole (ABZ) and albendazole metabolites in human plasma. Separation and detection of analytes were achieved on a reversed phase column (Acquity UPLC®BEH C18 column (100 × 2.1 mm, 1.7 µm) with gradient elution using 0.05% formic acid in methanol and 0.05% formic acid as mobile phase. Solid phase extraction was utilized for elution of analytes from the matrix. Thereafter, analytes were monitored by using MS/MS with electrospray ionization source in positive multiple reaction monitoring mode. The MS/MS response was linear over the concentration range from 0.1-200 ng/mL for ABZ and ABZ-ON, 0.5-1000 ng/mL for ABZ-OX and 1-2000 ng/mL for DEC with a correlation coefficient (r2) of 0.998 or better. The within- and between-batch precisions (relative standard deviation, % RSD) and the accuracy (% bias) were within the acceptable limits as per FDA guideline. The validated method was successfully applied to the clinical pharmacokinetic study. Due to high sensitivity and low requirement of sample volume, the method will be applicable for therapeutic drug monitoring of this regimen.

Temporary shift of microfilariae of Brugia pahangi from the lungs to muscles in Mongolian jirds, Meriones unguiculatus, after a single injection of diethylcarbamazine.

A single-dose treatment with diethylcarbamazine (DEC) reduced microfilaria (mf) counts of Brugia pahangi by >90% at 30 min post-treatment in Mongolian jirds (Meriones unguiculatus). The reduction was followed by a rapid increase in microfilaremia, with the count reaching pretreatment level in 3 hr. The mechanisms behind this temporary reduction of mf were investigated. Without treatment, mf accumulated in the lungs. At 30 min post-treatment, they had moved from the lungs and accumulated in the muscle. At the same time, electron microscopy revealed many mf in the muscle interstitium. DEC concentrations at 30 min were much lower in the muscle (12.2 microg/g of tissue) than in the lungs, liver, and kidneys (19.8-40.7 microg/g), all of which declined to < 0.6 microg/g by 3 hr. The presence of mf in the muscle would be advantageous for avoiding high DEC concentrations, and their extravascular location could prevent attack by host effector cells.

Semi-automated quantification of ivermectin in rat and human plasma using protein precipitation and filtration with liquid chromatography/tandem mass spectrometry.

Ivermectin is a parasiticide commonly used in humans and livestock. It is currently under development for the treatment of pediculosis of humans (head lice) that does not respond to established treatments. A liquid chromatography/turbo ion spray tandem mass spectrometry (LC/TIS-MS/MS) method for the determination of ivermectin in rat and human plasma has been developed that uses emamectin [4"-epi-(methylamino)-4"-deoxyavermectin] as the internal standard. Sample preparation involved protein precipitation and filtration of fortified plasma in the 96-well format. Chromatographic separation was accomplished using fast gradient conditions on a C8 stationary phase. The analytes were detected with the mass spectrometer operated in the positive ion, multiple reaction monitoring mode. The method exhibited good intra- and interday accuracy and precision, and was linear over a dynamic range of 1-2000 ng/mL. In rat plasma, intraday accuracy ranged between 84-93% for the low quality control (QC) sample (1.5 ng/mL), and between 91-109% for the remaining QCs. Intraday precision ranged between 4.9-15% for the low QC, and 0.8-6.3% for the remaining QCs. Interday accuracy ranged between 88-107%, and precision between 4.1-11%. Similar data was obtained using human plasma. An investigation of matrix effects indicated that the ionization efficiency of ivermectin was favored by the presence of an ammonium ion in an aqueous environment. The implications of this observation toward assay sensitivity are discussed.

Safety, tolerability, efficacy and plasma concentrations of diethylcarbamazine and albendazole co-administration in a field study in an area endemic for lymphatic filariasis in India.

Filariasis control programmes are moving towards a strategy of repeated single-dose mass treatment of endemic populations. Using a combination, such as albendazole (ALB) to diethylcarbamazine (DEC) gives both macrofilaricidal and anti-helmintic activity. However, the safety of the combination versus DEC alone should be established in field studies in large populations prior to incorporation into national programmes. The present study compared the safety, tolerability, and efficacy of single doses of DEC 6 mg/kg + ALB placebo with DEC 6 mg/kg + ALB 400 mg in populations living in two filariasis endemic villages in the district of Wardha in western India. The study was double blind, parallel group, and randomized. Safety and tolerability study were studied in males and females older than 5 years. Safety was assessed by monitoring if adverse events (AEs) over 5 days affected daily acivities. Subjects in the 2 treatment groups experienced insignificantly different effects on daily activities and the combination was shown to be safe. Efficacy was evaluated by microfilaraemia (Mf), immunochromatographic test (ICT) and ultrasonography (USG) at 0, 3, 6, and 12 months of follow up. The efficacy study enrolled 103 male patients (aged 18-50 years) in microfilariae positive, clinical disease and asymptomatic, amicrofilaremic groups. There was no significant difference in efficacy between groups at 12 months. Within the Mf positive group, significant differences were seen in microfilaraemia (P < 0.001) with both treatments, and in USG (P < 0.001 and P < 0.004 respectively), at 12 months. The present field study has shown the combination of DEC + ALB to be as safe as the single drug DEC and thus the combination can be put in use in the national filariasis control programmes. Both drugs were adequately absorbed. The study at present does not provide evidence for the greater efficacy of the combination at 12 months follow up. While the safety of the combination has been ascertained, the incorporation or otherwise of ALB into national programmes for greater efficacy must await results of studies with longer follow up.

The pharmacokinetics, safety and tolerability of the co-administration of diethylcarbamazine and albendazole.

The pharmacokinetics, safety and tolerability of single, oral doses of diethylcarbamazine (DEC) and albendazole, given alone or in combination, were investigated in a double-blind, randomized and placebo-controlled trial involving 42 amicrofilaraemic subjects living in an area of India where lymphatic filariasis is endemic. The subjects (34 males and eight females, aged 18-52 years and weighing 46-66.5 kg) were randomly allocated to one of the three drug groups. Fourteen were given just DEC (6 mg/kg), another 14 were given just albendazole (400 mg) and the remaining 14 were given both DEC (6 mg/kg) and albendazole (400 mg). Blood samples for pharmacokinetic study were collected at specified intervals before and after drug administration. Plasma concentrations of DEC and albendazole/albendazole sulphoxide were estimated using gas chromatography and HPLC, respectively. The safety and tolerability of the treatments were evaluated through clinical and laboratory assessments. Both the DEC and albendazole were well tolerated when given alone or in combination, no adverse events being observed. In all three treatment groups, the drugs were rapidly absorbed from the gastro-intestinal tract although there was marked inter-individual#10; variation. The pharmacokinetics of DEC, albendazole and albendazole sulphoxide were similar, whether each drug was given alone or in combination. These results indicate that there is no adverse pharmacokinetic or pharmacodynamic reason why DEC and albendazole should not be co-administered to control lymphatic filariasis.

Pharmacokinetics of diethylcarbamazine after single oral dose at two different times of day in human subjects.

In most Wuchereria bancrofti and Brugia malayi infections, the microfilaria are found in the blood in greatest number between 10 p.m. and 2 a.m., indicating that chronotherapy may be beneficial in treating such infections. This study reports the influence of time of administration on the pharmacokinetics of diethylcarbamazine (DEC) in healthy volunteers. The study was conducted in 12 healthy volunteers by administering a 150 mg single oral dose of diethylcarbamazine citrate at 0600 or 1800 h in a balanced crossover design with the approval of an institutional ethics committee. The subjects fasted for about 10 hours before and 3 hours after drug treatment. Blood samples were collected at predetermined time intervals, and the drug content in the serum was estimated using HPLC with an electrochemical detector. Pharmacokinetic analysis was performed using noncompartmental methods employing WinNonlin (version 3.1), and the means of various pharmacokinetic parameters were compared for any dosing time-related changes using a paired t-test at a probability level of 95%. The mean +/- SD values of pharmacokinetic parameters of DEC for the treatments at 0600 versus 1800 h were as follows: Cmax, 500+/-227 versus 637+/-401 ng/ml; tmax, 2.3+/-0.7 versus 2.7+/-1 h; Ka, 2.23+/-0.72 versus 1.96+/-0.97 h(-1); t1/2, 14.6+/-6.7 versus 11.4+/-4.9 h; AUC0-t, 5,334+/-1,853 versus 6,901+/-4,203 ng x h/ml; AUC0-infinity, 5,840+/-1,922 versus 7,220+/-4,205 ng x h/ml; CL/F, 36,058+/-19,011 versus 32,189+/-25,293 ml/h/kg; Vd/F, 570+/-225 versus 533+/-447 L; and MRT 17.7+/-5.9 versus 15.3+/-5.2 h. None of the parameters was significantly changed (p > 0.05) as a function of time of administration.

Gas chromatographic assay of diethylcarbamazine in human plasma for application to clinical pharmacokinetic studies.

A sensitive and selective gas chromatography method using flame ionization detection was developed for the determination of diethylcarbamazine (DEC) in human plasma. DEC and the internal standard, 1-diethylcarbamyl-4-ethyl piperazine HCl (E-DEC), were extracted from human plasma after loading onto a conditioned C(18) solid phase extraction cartridge, rinsed with water and eluted with methanol. After evaporation under a stream of nitrogen and reconstitution in methanol, 3 microl were injected onto the GC system. Separation was achieved on a A Heliflex(R) AT-35 capillary column (length 30 m, internal diameter 0.32 mm). Gas flow rates were: hydrogen, 35 ml/min; carrier gas (helium), 1.5 ml/min, make-up gas (helium), 25 ml/min; and air 420 ml/min. The retention times of DEC and internal standard were approximately 5.5 and 7.28 min, respectively. The GC run time was 22 min. The assay was linear in concentration range 100-2000 ng/ml for DEC in human plasma. The analysis of quality control samples for DEC (120, 1000, 2000 ng/ml) demonstrated excellent precision with coefficients of variation of 4.5,1.3, and 1.6%, respectively (n=6). The method was accurate with all intra-day (n=6) and inter-day (n=12) mean concentrations within 4.3% from nominal at all quality control sample concentrations. DEC was found to be stable after 3 freeze-thaw cycles, and with storage at -20 degrees C for 12 weeks. The method is currently being used for pharmacokinetic studies of DEC in healthy volunteers.

Acceleration of body clearance of diethylCarbamazine by oral activated charcoal.

The effect of activated charcoal (AC) on body clearance of diethylcarbamazine (DEC) was investigated in six healthy volunteers. On three occasions at weekly intervals, each subject received 150 mg of DEC with 350 ml of water. One and two weeks later, 150 mg of DEC plus 7.5 g and 15 g of AC, respectively, in 350 ml of water as a charcoal slurry. The non-renal clearance of DEC expressed as the total body clearance of DEC was increased after treatment with AC. The 45.2, 79.6 percent and 58.6, 81.6 percent reductions in maximum concentration and area under the concentration-time curve, respectively, suggest an appreciable adsorption of DEC by AC (7.5 and 15 g) in the gut. Serum eliminating half-life was decreased upon treatment with AC (7.5 and 15 g). These results indicate that AC accelerates the body clearance of DEC by increasing non-renal elimination of the drug.

Inflammatory cytokines following diethylcarbamazine (DEC) treatment of different clinical groups in lymphatic filariasis.

In an earlier study in Indonesia we reported on adverse reactions to diethylcarbamazine (DEC) in brugian filariasis patients identified as microfilaraemics (n = 26), endemic normals (n = 11) and elephantiasis patients (n = 17). To assess the link between adverse reactions and cytokines we have now analysed an array of inflammatory mediators in plasma samples collected during the same study. Pre-treatment levels of interleukin (IL)-6 and soluble tumour necrosis factor receptor 75 (sTNF-R75) were higher in elephantiasis patients compared to microfilaraemics and endemic normals, indicating the presence of an ongoing inflammation in patients with chronic disease. After initiation of treatment, the levels of IL-6 and LPS-binding protein (LBP) were consistently and significantly higher in microfilaraemics who suffered most from adverse reactions compared with endemic normals and elephantiasis patients. In microfilaraemics the levels of sTNF-R75 increased after treatment to reach levels recorded in elephantiasis patients. IL-6 increased early, concurrent with the development of adverse reactions and peaked by 24 h post treatment. The levels of LBP and sTNF-R75 in microfilaraemics also increased to peak, later than IL-6, at 32 h post DEC therapy. Although changes were recorded in IL-8 and IL-10 levels in some individuals, no significant differences were found between the 3 clinical groups. These results demonstrate that intake of DEC leads to an increase in a selected number of inflammatory mediators in the group of filarial patients who suffer most from adverse systemic reactions.

Determination of 6,4'-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]py ridine in plasma and whole blood by high-performance liquid chromatography.

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4'-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyr idine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C18 Nucleosil ODS column (250x4.6 mm, 5 microm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25-500 ng/ml were 95.5+/-2.01% and 83.3+/-1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.

Plasma levels of diethylcarbamazine and their effects on implanted microfilariae of Brugia pahangi in rats.

Plasma level of diethylcarbamazine (DEC) was measured by using gas chromatography and was compared to the changes of microfilaremia after an intraperitoneal injection with 200 mg/kg of DEC in rats. The microfilaremia was induced artificially by an intravenous implantation with 2 x 10(5) Brugia pahangi microfilariae (mf) 1 day before DEC treatment. The rats treated with DEC showed a rapid and significant decrease in mf number in the circulation within 30 min, continued for 4 hr, and then increased rapidly. DEC seemed to cause transient but significant suppression of microfilaremia of B. pahangi in rats directly.

Tissue distribution and metabolism in rat of an ethynesulphonamide with filaricidal activity.

1. The tissue distribution and metabolism of a new filaricidal agent P903 (N-[(2-phenylethynyl)sulfonyl]morpholine) were studied in rat. 2. After s.c. administration of 14C and 13C P903, the Tmax in the blood was observed on day 2. Elimination was slow and > 95% was bound to protein. Radioactivity was distributed in the whole organism but particularly in erythrocytes and the lymphatic channel. Four days later, > 60% of the radioactivity was excreted in urine and faeces at equal amounts and 15% remained at the injection point. 3. In all biological fluids tested no P903 was found but only its metabolites. 4. One principal metabolite, the N-[(2-phenyloxo-2-ethane) sulphonyl] morpholine or oxosulphonamide was identified in blood, urine and faeces as compared with the reference compound by GC/MS and NMR. This latter molecule was detected following hydrolysis by hydrochloric acid but not with beta glucuronidase/sulphatase. 5. Unconjugated and conjugated oxosulphonamide represented > 85% of the radioactivity at all times tested in blood but only 38 and 35% respectively of urinary and faecal radioactivity on day 1 after the administration of the labelled drug. 6. Thus, P903 is rapidly converted to a reactive metabolite, probably an oxirene, which is then conjugated with endogenous components to form conjugated oxosulphonamide and an unknown metabolite. The role of this reactive metabolite in antifilarial activity seems to be very important in understanding the mechanism of action of P903.

Specific gas chromatographic analysis of diethylcarbamazine in human plasma using solid-phase extraction.

Diethylcarbamazine (DEC, 1-diethylcarbamyl-4-methylpiperazine) is an antiparasitic piperazine derivative used in the treatment of lymphatic filariasis. DEC-N-oxide is a major metabolite in humans which has antifilarial activity. Gas chromatographic analysis of DEC in plasma can be complicated by the presence of the metabolite, since the thermally unstable DEC-N-oxide is converted to a material which coelutes with DEC under the conditions of the analysis. We now report a method to separate DEC-N-oxide from DEC in plasma using solid-phase extraction with subsequent gas chromatographic analysis using a nitrogen specific detector. 1-Diethylcarbamyl-4-ethylpiperazine (E-DEC) was the internal standard. The standard curve of DEC is linear in the range of 10 to 200 ng/ml. The limit of detection is 4 ng/ml. Reproducibility at 10, 100 and 200 ng/ml concentration points of the standard curve gives coefficients of variation of 6.1%, 7.8% and 1.6%, respectively. Recovery following solid-phase extraction is 99.3% for DEC and 94.8% for the internal standard. This sensitive and specific analytical method is suitable for pharmacokinetic studies of DEC.

Ivermectin distribution in the plasma and tissues of patients infected with Onchocerca volvulus.

OBJECTIVE: To determine the distribution of ivermectin in plasma and tissues of onchocerciasis patients following a single oral dose of 150 micrograms kg-1. SETTING: Medical Department at Soba University Hospital, Khartoum. PATIENTS: Twenty five patients and fourteen healthy volunteers. METHODS: Serial blood samples were obtained from both groups. Tissue samples were removed from various patients as full thickness skin punch biopsies or during nodulectomy. Ivermectin concentration was determined by radioimmunoassay. RESULTS: The plasma pharmacokinetic variables for patients were; maximum plasma concentration 52.0 ng ml-1; time to achieve maximum concentration, 5.2 h.; elimination half life, 35.0 h; and the area under the plasma concentration curve versus time, 2852 ng.h.ml-1. In healthy volunteers, the plasma ivermectin distribution was similar to that in patients, and both groups showed a tendency for a second rise in plasma concentration of the drug suggestive of enterohepatic recirculation. Ivermectin was detected in tissues obtained from patients. Fat showed the highest and most persistent levels, whilst values for skin, nodular tissues, and worms were comparable. Subcutaneous fascia contained the lowest concentrations. CONCLUSIONS: Infection with O. volvulus does not affect the pharmacokinetics of ivermectin, and filarial infected tissues and parasites themselves do take up the drug. There may be prolonged retention of ivermectin because of depot formation in fat tissue.

Determination of the antifilarial drug UMF-078 and its metabolites UMF-060 and flubendazole in whole blood using high-performance liquid chromatography.

A rapid and selective high-performance liquid chromatographic (HPLC) method for the simultaneous determination of the antifilarial drug UMF-078 (I) and its metabolites UMF-060 (II) and flubendazole (III) is described. After a simple extraction from whole blood, the compounds were determined by HPLC using a C18 Inertsil ODS-2 reversed-phase column with methanol-0.05M ammonium acetate (pH 4.0) as the mobile phase and ultraviolet detection at 291 nm. The average recoveries of I, II and III over the concentration range 20-500 ng ml-1 were 69.9 +/- 4.7, 85.6 +/- 4.4 and 85.1 +/- 6.0%, respectively. The minimum detectable concentrations in whole blood for I, II and III were 10, 7 and 7 ng ml-1, respectively. This method was found to be suitable for pharmacokinetic studies.

Determination of a new antifilarial drug, UMF-058, and mebendazole in whole blood by high-performance liquid chromatography.

A rapid and selective high-performance liquid chromatographic assay for simultaneous quantitative determination of a new antifilarial drug (UMF-058, I) and mebendazole (MBZ) is described. After a simple extraction from whole blood, both compounds were analysed using a C18 Nova Pak reversed-phase column and a mobile phase of methanol-0.05 M ammonium dihydrogenphosphate (50:50, v/v) adjusted to pH 4.0, with ultraviolet detection at 291 nm. The average recoveries of I and MBZ over a concentration range of 25-250 ng/ml were 92.0 +/- 7.7 and 84.4 +/- 4.4%, respectively. The minimum detectable concentrations in whole blood for I and MBZ were 7 and 6 ng/ml, respectively. This method was found to be suitable for pharmacokinetic studies.

Concentrations of amocarzine in plasma of 71 Ecuadorian patients of two different races receiving 3 mg/kg b.i.d. and 5 mg/kg o.d. oral postprandial doses for 3 days.

The possible influence of sex, race and of postprandial administration conditions (either immediately after the end of meal or one hour later) on the plasma concentrations of amocarzine and its N-oxide metabolite, CGP 13 231, was investigated. 71 Ecuadorian patients (48 males and 23 females) of two different races (Indio and Negro) infected with Onchocerca volvulus participated in the study. The concentrations were measured on day 3 at times 3 and 6 h after postprandial administration in the morning of a treatment with either a dose of 5 mg/kg of amocarzine once daily (12 patients) or 3 mg/kg twice daily (59 patients) for 3 days. The concentrations of unchanged drug and of CGP 13 231 measured after the 5 mg/kg treatment were in the low range of those expected from dose proportionality by the comparison with the 3 mg/kg. After the 3 mg/kg dose, no significant difference in concentration of both compounds were detected between the male and female patients between Indio and Negro patients, between the administration immediately after food intake and one hour later. The only detected difference (P = 0.05) was that between Indio and Negro patients for the concentrations of CGP 13 231 at time 3 h. This difference was not confirmed at time 6 h. Therefore, the administration of amocarzine either immediately or one hour after food intake appeared to produce reproducible absorption conditions which were not influenced by sex and race.

Pharmacokinetics of CGP 6140 (amocarzine) after oral administration of single 100-1600 mg doses to patients with onchocerciasis.

The concentrations of CGP 6140 [4-nitro-4'-(N-methyl-piperazinylthiocarbonylamido)-diphenylamine] and of its N-oxide metabolite, CGP 13,231, were measured in plasma and urine after single oral dose of 100-1600 mg of CGP 6140 to 41 fasted Ghanaian patients with Onchocerca volvulus infections. The absorption of CGP 6140 was rapid and its terminal elimination half-life was about 3 h. The plasma concentrations of CGP 6140 were essentially proportional to the dose. A greater variability in plasma concentrations was apparent after the 800 and 1600 mg doses indicating a poor bioavailability of the drug administered in fasting conditions to several patients. In plasma, the concentrations of CGP 13,231 were similar to those of CGP 6140. The amount of CGP 13,231 excreted in urine was 25-40% of the dose of CGP 6140 whereas only 1.5% was excreted as unchanged drug. If a single dose of drug is used for the treatment, the plasma concentration would be maintained for 3-4 h at a high level. At 8 h, the concentration falls to about 10% of the Cmax. If sustained plasma concentrations of the drug are needed for efficacy, twice daily administration would maintain the minimum concentration at about 10% of the Cmax.