New HPLC-MS method for rapid and simultaneous quantification of doxycycline, diethylcarbamazine and albendazole metabolites in rat plasma and organs after concomitant oral administration.

A sensitive and relatively fast, cost-effective high-performance liquid chromatographic method coupled with mass spectrometer (HPLC-MS) is herein reported for the first time for a simultaneous quantification of plasma and organs concentration of three therapeutic agents that are widely used in treatment of lymphatic filariasis (LF), namely, doxycycline (DOX), diethylcarbamazine (DEC) and albendazole (ABZ) metabolites. The method was developed and validated as per ICH and FDA guidelines and successfully employed to quantify DOX, DEC and ABZ metabolites (albendazole sulfoxide (ABZ-OX) and albendazole sulfone (ABZ-ON)) in the plasma and organs of Sprague Dawley rats after oral concomitant administration of the above mentioned therapeutic agents. Importantly, a simple, one-step protein precipitation and extraction method was used to extract the four compounds efficiently with a recovery in the range of 79.88 ± 5.02%-90.71 ± 5.13%, 85.72 ± 7.22%-93.17 ± 5.55%, 94.38 ± 7.35%-101.00 ± 8.88% and 94.38 ± 7.35%-99.87 ± 10.22% in plasma and organs for DOX, DEC, ABZ-OZ and ABZ-ON, respectively. Separation of all analytes was performed on a Xselect CSH™ C18 HPLC column (Waters, 3.0 x 150 mm, 3.5 µm particle size) with gradient elution employing a mobile phase consisting of 0.1% v/v formic acid in water and methanol with a run time of 20 min. Quantification was carried out employing a single, quadruple MS detector operated with single ion monitoring (SIM) mode and the ion transitions at m/z of 445.4, 200.2, 282.3 and 298.3 for DOX, DEC, ABZ-OX and ABZ-ON respectively. The MS response for plasma samples was linear across the concentration range of 2.5-2500 ng/mL for DOX, 0.5-500 ng/mL for DEC, 1-1000 ng/mL for ABZ-OX and ABZ-ON with a correlation coefficient (r2) ≥ 0.998. The method was selective, precise and accurate. This method allowed us to get an insight into the pharmacokinetics and biodistribution of the three therapeutic agents after simultaneous oral administration to Sprague Dawley rats. This bioanalytical method could provide a reliable, reproducible and excellent tool for routine therapeutic drug monitoring of the above mentioned therapeutic agents and also support other clinical pharmacokinetic-based studies.

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.

Gastrointestinal Motility Variation and Implications for Plasma Level Variation: Oral Drug Products.

The oral route of administration is still by far the most ubiquitous method of drug delivery. Development in this area still faces many challenges due to the complexity and inhomogeneity of the gastrointestinal environment. In particular, dosing unpredictably relative to motility phase means the gastrointestinal environment is a random variable within a defined range. Here, we present a mass balance analysis that captures this variation and highlights the effects of gastrointestinal motility, exploring what impacts it ultimately has on plasma levels and the relationship to bioequivalence for high solubility products with both high and low permeability (BCS I and III). Motility-dependent compartmental absorption and transit (MDCAT) mechanistic analysis is developed to describe the underlying fasted state cyclical motility and how the contents of the gastrointestinal tract are propelled.

Liquid chromatography-mass spectrometry analysis of diethylcarbamazine in human plasma for clinical pharmacokinetic studies.

A sensitive and selective liquid chromatographic method using mass spectrometric detection was developed for the determination of diethylcarbamazine (DEC) in human plasma. DEC and its stable isotope internal standard d3-DEC were extracted from 0.25mL of human plasma using solid phase extraction. Chromatography was performed using a Phenomenex Synergi 4µ Fusion-RP column (2mm×250mm) with gradient elution. The retention time was approximately 4.8min. The assay was linear from 4 to 2200ng/mL. Analysis of quality control samples at 12, 300, and 1700ng/mL (N=15) had interday coefficients of variation of 8.4%, 5.4%, and 6.2%, respectively (N=15). Interday bias results were -2.2%, 6.0%, and 0.8%, respectively. Recovery of DEC from plasma ranged from 84.2% to 90.1%. The method was successfully applied to clinical samples from patients with lymphatic filariasis from a drug-drug interaction study between DEC and albendazole and/or ivermectin.

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.

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.

Pharmacokinetics of diethylcarbamazine: prediction by concentration in saliva.

The concentration of diethylcarbamazine in saliva was used to determine pharmacokinetic parameters, in comparison to plasma and urine concentrations. Six healthy adult male volunteers were administered 150 mg diethylcarbamazine with 400 ml of water. At seven different time intervals, blood, urine and saliva samples were taken, and different pharmacokinetic parameters measured. The plasma-saliva concentration ratio was calculated as 1.53 whereas the observed ratio was 3.82. The half lives, times to reach peak plasma concentration, and elimination rate constants did not show any significant difference in the different samples. The plasma peak concentration and areas under the curve were significantly (p<0.05) increased from those of the saliva. At 24 h, when diethylcarbamazine was absent in urine, the plasma and saliva concentrations were almost zero. Diethylcarbamazine is secreted in saliva, and its concentration in saliva can be used to monitor drug therapy.

Age-related alteration of carbamazepine-serum protein binding in man.

To determine whether biological maturation influences the kinetics of carbamazepine-serum protein binding, the carbamazepine free fraction (%) was investigated in the serum of 66 patients, ranging from 4 to 83 years, with epilepsy or trigeminal neuralgia, treated with carbamazepine alone or carbamazepine in combination with phenytoin, phenobarbital, and/or valproic acid, over a relatively long period. Biochemical parameters such as levels of albumin and non-glycated albumin showed a significant relationship with carbamazepine free fraction (r = -0.521, P < 0.001 for albumin; r = -0.700, P < 0.001 for non-glycated albumin). Non-glycated albumin was more strongly correlated with carbamazepine free fraction. The biochemical parameters showed a significant relationship with age (r =-0.243, P < 0.1 for albumin; r =0.666, P < 0.001 for glycated albumin; r = -0.459, P < 0.001 for non-glycated albumin; r = 0.640, P < 0.001 for carbamazepine free fraction). Glycated albumin (%), non-glycated albumin and carbamazepine free fraction (%) were strongly correlated with age, whereas albumin showed only a weak correlation with age. To evaluate the effects of ageing on carbamazepine-serum protein binding, the patients were divided into three groups according to age: children, 4-15 years; adults, 16-64 years; elderly, 65-83 years. Albumin and non-glycated albumin were much lower, and glycated albumin (%) and carbamazepine free fraction (%) much higher in the elderly group than in the other two groups. The results of this study showed that the major ligand of carbamazepine in the serum was non-glycated albumin, which decreased with age. These observations suggested that in elderly patients, the elevation of free carbamazepine concentrations in the serum caused by reduced non-glycated albumin levels, induces increases in the sensitivity of the pharmacological effects of carbamazepine and the risk of drug interactions.

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.

A direct competitive ELISA for the simple, sensitive and accurate determination of diethylcarbamazine concentration in serum.

Improved methods for measurement of the anti-filariasis drug diethylcarbamazine in serum would assist in the design of effective therapy. The method evaluated in the present paper is a direct competitive ELISA which is sensitive, specific and accurate. Horseradish peroxidase-labelled diethylcarbamazine conjugate was incubated with diethylcarbamazine and anti-diethylcarbamazine antiserum over a bound second antibody. The enzyme activity of the remaining diethylcarbamazine-horseradish peroxidase conjugate was measured. The intra- and inter-assay coefficient of variation was < 10% in the range of 1.0-30 ng ml-1 and the limit of detection was 0.3 ng ml-1. The cross reactivities of anti-diethylcarbamazine antibodies with diethylcarbamazine metabolites and ivermectin were < 0.09%. Using the ELISA, the serum levels of diethylcarbamazine were successfully determined in mongolian jirds (Meriones unguiculantus) up to 4h following a single dose of 50 mg kg-1 of body weight. The values of pharmacokinetic parameters of diethylcarbamazine in jirds were determined.

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.

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.

Development of a competitive enzyme-linked immunosorbent assay for diethylcarbamazine.

A sensitive and reproducible competitive enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of diethylcarbamazine (DEC) in biological fluids was developed. Since DEC has no functional group to conjugate with bovine serum albumin (BSA), N-(2-aminoethyl)-N-ethyl-4-methyl-1-piperazinecarboxamide (DEC-NH2) was first synthesized. This compound was then converted to carboxyl DEC (DEC-COOH) and conjugated to BSA and to poly-L-lysine for use as immunogen and solid-phase marker, respectively. The competitive ELISA was conducted by simultaneously incubating DEC with mouse anti-DEC antiserum over DEC-poly-L-lysine solid phase. Subsequently, the binding of anti-DEC antibody was detected by using sheep anti-mouse IgG peroxidase conjugate as a tracer. The reliability, determined by the coefficient of variation for inter and intra-assay, was satisfactory. The cross-reactivities of anti-DEC antibodies with DEC metabolites, related compounds and ivermectin were negligible. Using this assay, DEC levels were easily determined in serum of Mongolian jirds (Meriones unguiculatus) up to 4 hours following a single dose of DEC citrate base (100 mg/kg of body weight) via intraperitoneal route.

Carbamazepine. What physicians should know about its hematologic effects.

Transient leukopenia and, less commonly, neutropenia may occur with carbamazepine therapy. Discontinuation of therapy is usually not indicated unless symptoms are severe, persistent, or accompanied by infection. Patients with a low leukocyte or neutrophil count before treatment may be at increased risk for carbamazepine-induced leukopenia or neutropenia. Careful monitoring of blood counts, particularly during the first month of therapy, is essential. The frequency of monitoring can be determined on an individual basis. If a hematologic abnormality develops, the frequency of monitoring should be increased, especially if carbamazepine is not discontinued. Only when the neutrophil count falls below 500/mm3 does a severe risk of infection exist.

Successful long term treatment of a dog with psychomotor seizures using carbamazepine.

Psychomotor seizures (temporal lobe epilepsy) were diagnosed in a dog based on history, clinical findings and electroencephalography. Long-term seizure control was achieved with carbamazepine, despite serum drug concentrations which were low to unmeasurable. It is suggested that serum levels of carbamazepine are not a useful guide to clinical efficacy in the dog, that an unmeasured metabolite of carbamazepine may account for the anti-convulsant activity and that carbamazepine may be potentially useful in treating certain canine seizure disorders.