Development and validation of bioanalytical method for quantification of cycloserine in human plasma by liquid chromatography-tandem mass spectrometry: Application to pharmacokinetic study.

A selective, sensitive and high-throughput liquid chromatography-tandem mass spectrometry bioanalytical method has been developed for the estimation of cycloserine in human plasma, employing cytosine as the internal standard. The extraction of the analyte was facilitated by solid-phase extraction using 100 µL of human plasma. The separation was carried out on a BDS Hypersil C18 (150 × 4.6 mm, 5 µm) column using a mixture of 0.2% formic acid in HPLC-grade water, methanol and acetonitrile (70:15:15, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was linear over the range of 0.20-20 µg/mL with r2 > 0.99. Complete validation of the method was performed as per US Food and Drug Administration guidelines and the results met acceptance criteria. Applying the present method, the clinical pharmacokinetics of cycloserine following oral administration of 250 mg cycloserine was studied under fasting conditions. Assay reproducibility was also verified by incurred sample reanalysis.

Sensitive Ultra-performance Liquid Chromatography Tandem Mass Spectrometry Method for Determination of Cycloserine in Plasma for a Pharmacokinetics Study.

A simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry method has been developed and validated for the analysis of cycloserine in patients' plasma. Using methanol, cyloserine and propranolol (internal standard (IS)) was extracted from plasma by protein precipitation procedure. The chromatographic separation was successfully achieved on Phenomenex KinetexTM PFP C18 (2.1 mm × 100 mm, 2.6 µm) reversed-phase column. Acidified with 0.1% formic acid, water and acetonitrile were used as mobile phases for gradient elution. Cycloserine and IS were detected by Xevo® TQ MS triple quadrupole tandem mass spectrometer. The transition of protonated precursor to product ion were monitored at 103 → 75 m/z and 260.2 → 183 m/z for cycloserine and IS, respectively. The lower limit of quantification was 0.01 µg/mL. The method was linear over the concentration range 0.01-50 µg/mL with average coefficient of determination of 0.9994. The within-run and between-run precision and accuracy were in the range 3.7-19.3% (RSD) and 98.7-117.3%, respectively. Processed cycloserine sample was stable for 48 hours at 8°C and after three freeze-thaw cycles. The extraction efficiency ranged between 88.7 and 91.2%. The method was successfully applied in a pharmacokinetic study for the determination of cycloserine in plasma of patients with drug-resistant tuberculosis.

Therapeutic drug monitoring of cycloserine and linezolid during anti-tuberculosis treatment in Beijing, China.

SETTING: Plasma concentrations of cycloserine (CS) and linezolid (LZD) in tuberculosis (TB) patients are largely unknown. OBJECTIVE: To measure the plasma concentrations of CS and LZD after drug ingestion in drug-resistant TB patients. DESIGN: Patients who received CS and LZD as part of their treatment between 1 July 2012 and 1 July 2016 were studied retrospectively. CS and LZD plasma levels were determined using high-pressure liquid chromatography-tandem mass spectrometry. Plasma drug concentration, age, sex, liver disease, renal disease, administered doses and diabetes mellitus status were recorded. RESULTS: Based on 390 samples, CS plasma concentrations were below the lower limit of normal (54.87%, 214/390). There was a statistically significant difference between the low concentration group (14.0 ± 3.71 µg/ml) and the target concentration group (25.2 ± 3.73 µg/ml, P < 0.01). The mean plasma concentration of LZD was 15.6 ± 4.91 µg/ml, which was within the target concentration (12-26 µg/ml) in 65 patients. Variables that correlated with CS and LZD concentrations were not found in this retrospective study. CONCLUSION: Low plasma CS concentrations were common, while 83.1% (54/65) of plasma LZD concentrations were within the target range. Therapeutic drug monitoring is essential to maintain appropriate plasma drug concentrations in the era of precision medicine.

A selective and sensitive high performance liquid chromatography assay for the determination of cycloserine in human plasma.

BACKGROUND: Cycloserine (CYC) is a second line antitubercular drug that is used for the treatment of multidrug resistant tuberculosis (MDR-TB) along with other antitubercular agents and is often used in developing countries. Monitoring CYC levels in plasma could be useful in the clinical management of patients with MDR-TB. A high performance liquid chromatography method for the determination of CYC in human plasma was developed. METHODS: The method involved extraction of the sample using solid phase extraction cartridges and analysis of the extracted sample using a reverse phase T3 column (150mm) and detection at 240nm with Photo Diode Array (PDA) detector. The chromatogram was run for 15min at a flow rate of 0.4ml/min at 30°C. RESULTS AND CONCLUSION: The assay was specific for CYC and linear from 5.0 to 50.0µg/ml. The relative standard deviations of within- and between-day assays were less than 10%. Recovery of CYC ranged from 102% to 109%. The interference of other second line anti-TB drugs in the assay of CYC was ruled out. The assay spans the concentration range of clinical interest. The specificity and sensitivity of this assay makes it highly suitable for pharmacokinetic studies.

Validation of Cycloserine Efficacy in Treatment of Multidrug-Resistant and Extensively Drug-Resistant Tuberculosis in Beijing, China.

Cycloserine (Cs) is recommended by the World Health Organization as a second-line drug to treat multidrug-resistant tuberculosis (MDR-TB); however, its efficacy has never been sufficiently evaluated. To gain some insights into the value of cycloserine for MDR-TB treatment, in vitro bacteriostatic effect was determined and patient validations were performed prospectively. The in vitro activity of Cs against 104 wild-type Mycobacterium tuberculosis strains was determined, and serum Cs concentrations were measured for 73 MDR TB patients 2 h after administration. The treatment outcomes for 27 MDR-TB patients who had baseline isolates and were treated with Cs-containing regimens were followed up. The MICs for 90% of the recruited 104 wild-type strains were below 32 µg/ml. Eighteen out of 52 patients had peak serum concentrations (Cmax) below 20 µg/ml at the dosage of 500 mg daily, while 13 out of 21 patients had peak serum concentrations higher than 35 µg/ml at the dosage of 750 mg daily. The percentage of favorable treatment outcomes among patients with a Cmax/MIC ratio of ≥1 was statistically significantly higher than that among the group with a Cmax/MIC ratio of <1 (P = 0.022). The epidemiological cutoff value for Cs susceptibility testing was 32 µg/ml. A high percentage of patients receiving the recommended dosage of 10 mg/kg for Cs administration could not acquire desirable blood concentrations; therefore, adjusting the dosage according to drug concentration monitoring is necessary. The Cmax/MIC ratio might be a good indicator for predicting the treatment outcome for patients with MDR-TB or extensively drug-resistant TB (XDR-TB) who are being administered Cs-containing regimens.

Determination of cycloserine in microdialysis samples using liquid chromatography-tandem mass spectrometry with benzoyl chloride derivatization.

A new method for the analysis of cycloserine (4-amino-3-isoxazolidinone, CYC) in rat microdialysis samples has been developed. This method consists of derivatizing the CYC with benzoyl chloride, which transforms primary amines into highly stable derivatives. An attractive feature of this method was that the derivatization reaction is straightforward and can be completed within 10 min. The formed derivative, in contrast to the non-derivatized analyte, exhibited increased chromatographic retention and decreased matrix effects resulting from the co-elution of other components using reversed-phase liquid chromatography and on-line switching. Detection on a quadrupole-linear ion trap mass spectrometer (AB3200 Q-Trap) was performed using electrospray tandem mass spectrometry in multiple reaction monitoring mode. Various derivatization parameters were optimized in order to improve chromatographic separation and minimize ion suppression. In particular, the benzoylation reaction was improved to enhance the reproducibility and sensitivity of the chromatographic method. The transition m/z 207.1 → 105.1 was acquired to monitor the CYC derivatization products. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. A good linearity over the selected range (r > 0.99, range = 22-2200 mg/L), as well as accuracy and precision within ±7% of the target values, was obtained. The assay described herein was successfully applied to quantitatively measure CYC in the lung and blood of anesthetized rats.

A simplified LC-MS/MS method for rapid determination of cycloserine in small-volume human plasma using protein precipitation coupled with dilution techniques to overcome matrix effects and its application to a pharmacokinetic study.

Matrix effects have been a major concern when developing LC-MS/MS methods for quantitative bioanalysis of cycloserine. Sample handling procedures including solid phase extraction or derivatization have been reported previously by researchers to overcome matrix effects of cycloserine. In the present study, the possibility of reducing matrix effects of cycloserine using protein precipitation coupled with dilution techniques was investigated. Plasma samples were pretreated by protein precipitation with methanol followed by a 40-fold dilution with methanol-water (50:50, v/v). The analyte and the internal standard (mildronate) were chromatographed on a Shim-pack XR-ODS (100 mm × 2.0 mm, 2.2 µm) column using methanol-0.01% formic acid (70:30, v/v) as mobile phase and detected by multiple reaction monitoring mode via positive electrospray ionization. The total run time was only 2 min per sample. The suppression of cycloserine response was reduced with the matrix effects ranging between 80.5 and 87.9%. A lower limit of quantification (LLOQ) of 0.300 µg/mL was achieved using only 10 µL of plasma. The intra- and inter-day precisions were less than 4.8% and the accuracy ranged from -2.6 to 6.6%. The method was successfully applied to a pharmacokinetic study of cycloserine in 30 healthy Chinese male subjects after oral administration of a single dose of cycloserine at 250, 500 and 750 mg under fasting conditions. The newly developed method is simpler, faster, cost-effective, and more robust than previously reported LC-MS/MS methods.

Low Serum Concentrations of Moxifloxacin, Prothionamide, and Cycloserine on Sputum Conversion in Multi-Drug Resistant TB.

PURPOSE: Low serum concentrations of drugs used to treat multi-drug resistant tuberculosis (MDR-TB) have occasionally been associated with treatment failure. We determined the frequencies of low serum concentrations of anti-MDR-TB drugs, and assessed the effects of these concentrations on 2-month sputum conversion. MATERIALS AND METHODS: The serum levels of moxifloxacin (MF), prothionamide (PTH), and cycloserine (CS) were determined for 89 serum samples by high-pressure liquid chromatography-tandem mass spectrometry. RESULTS: Low serum concentrations of MF, PTH, and CS below the minimal levels of the normal ranges were 83.3% (20/24), 59.2% (29/49), and 71.2% (47/66), respectively. There were no significant differences between the 2-month sputum conversion group (n=25) and the 2-month sputum non-conversion group (n=4) in median drug concentrations (µg/mL) of MF (1.46 vs. 1.60), PTH (0.91 vs. 0.70), and CS (14.90 vs. 14.90). However, a poor compliance rate was significantly greater in the 2-month sputum non-conversion group (75.0%, 3/4) than in the 2-month sputum conversion group (0%, 0/25) (p=0.001). CONCLUSION: The frequency of low serum concentrations of anti-MDR-TB drugs was substantial and might not affect the 2-month sputum conversion rate. Larger prospective studies with timely sampling are needed to investigate the role of therapeutic drug monitoring in MDR-TB.

Pharmacokinetic Properties and Tolerability of Cycloserine Following Oral Administration in Healthy Chinese Volunteers: A Randomized, Open-Label, Single- and Multiple-Dose 3-Way Crossover Study.

PURPOSE: A new generic formulation of cycloserine has been developed in China but the pharmacokinetic properties of cycloserine in the Chinese population have not been reported. The aim of our study was to evaluate the pharmacokinetic properties and tolerability of single and multiple oral administrations of cycloserine capsules in healthy Chinese volunteers. METHODS: This open-label, single- and multiple-dose 3-way crossover study was conducted in healthy Chinese volunteers. Subjects were randomized to receive a single dose of cycloserine (250, 500, or 1000 mg) in separate trial periods, with a 1-week washout between periods. Those allocated to the 250-mg dose continued into the multiple-dose phase, in which they received 250 mg BID for 5 consecutive days. During the single-dose phase, blood samples were collected at regular intervals from 0 to 72 hours after drug administration and the concentrations of cycloserine were determined using LC-MS/MS. During the multiple-dose phase, blood samples were obtained before drug administration on Days 4, 5, and 6 to determine the Cmin at steady state. On Day 6, blood samples were also collected from 0 to 72 hours after drug administration. Pharmacokinetic parameters were estimated using noncompartmental methods. Tolerability was determined using clinical evaluation and monitoring of adverse events. FINDINGS: The study enrolled 12 healthy Chinese volunteers (6 men: mean [SD] age = 23.0 [2.6] years, weight = 60.2 [6.2] kg, height = 170.0 [3.0] cm, and body mass index = 20.7 [1.7]; 6 women: mean [SD] age = 25.3 [1.4] years, weight = 51.5 [3.3] kg, height = 160.0 [4.0] cm, and body mass index = 20.1 [0.9]). After administration of a single dose, cycloserine was rapidly absorbed, reaching peak plasma concentrations approximately 0.84 hours after oral administration, and t½ in plasma was about 13.0 hours. The geometric mean (SD) Cmax value increased in proportion to cycloserine dose, from 19.42 (5.89) to 84.76 (21.74) mg/L, and the geometric mean (SD) AUC0-72h value increased from 264.16 (133.37) to 1153.87 (522.16) mg·h/L in the range of a 250- to 1000-mg dose. After administration of multiple doses of cycloserine 250 mg BID, the mean (SD) t½ was 13.56 (4.38) hours, the apparent total clearance of the drug from plasma after oral administration was 1.02 (0.42) L/h, and the apparent volume of distribution was 18.22 (5.25) L, which were comparable with those after single dosing. The accumulation index was 2.19 (0.51), and the fluctuation was 1.05 (0.35). Results of the t tests of Cmax and AUC found no significant differences between the male and female groups. No serious adverse events were reported, and there were no discontinuations due to adverse events. IMPLICATIONS: The pharmacokinetic properties of cycloserine were linear at doses from 250 mg to 1000 mg. After multiple doses, the pharmacokinetic properties of cycloserine were consistent with those after single doses. At the doses studied, cycloserine appears to be well tolerated in these healthy volunteers. Chinese Clinical Trials registration: ChiCTR-TTRCC-13003982.

Low Serum Concentrations of Moxifloxacin, Prothionamide, and Cycloserine on Sputum Conversion in Multi-Drug Resistant TB

PURPOSE: Low serum concentrations of drugs used to treat multi-drug resistant tuberculosis (MDR-TB) have occasionally been associated with treatment failure. We determined the frequencies of low serum concentrations of anti-MDR-TB drugs, and assessed the effects of these concentrations on 2-month sputum conversion. MATERIALS AND METHODS: The serum levels of moxifloxacin (MF), prothionamide (PTH), and cycloserine (CS) were determined for 89 serum samples by high-pressure liquid chromatography-tandem mass spectrometry. RESULTS: Low serum concentrations of MF, PTH, and CS below the minimal levels of the normal ranges were 83.3% (20/24), 59.2% (29/49), and 71.2% (47/66), respectively. There were no significant differences between the 2-month sputum conversion group (n=25) and the 2-month sputum non-conversion group (n=4) in median drug concentrations (microg/mL) of MF (1.46 vs. 1.60), PTH (0.91 vs. 0.70), and CS (14.90 vs. 14.90). However, a poor compliance rate was significantly greater in the 2-month sputum non-conversion group (75.0%, 3/4) than in the 2-month sputum conversion group (0%, 0/25) (p=0.001). CONCLUSION: The frequency of low serum concentrations of anti-MDR-TB drugs was substantial and might not affect the 2-month sputum conversion rate. Larger prospective studies with timely sampling are needed to investigate the role of therapeutic drug monitoring in MDR-TB.

Serum concentrations of cycloserine and outcome of multidrug-resistant tuberculosis in Northern Taiwan.

BACKGROUND: Therapeutic drug monitoring (TDM) has been advocated to promote the efficacy of anti-tuberculosis agents. Cycloserine (CS) is a second-line anti-tuberculosis drug whose serum concentrations in tuberculosis (TB) patients are largely unknown. OBJECTIVES: To investigate serum CS concentrations after drug ingestion in TB patients. METHODS: Multidrug-resistant TB patients who were taking CS in a tertiary care centre in northern Taiwan between 1 April 2009 and 31 October 2009 were enrolled in the study. Serum CS concentrations were measured at 2 and 6 h after drug administration. RESULTS: Of 32 patients enrolled, 23 were males and 9 females. The mean CS dose was 8.8 ± 1.3 mg/kg. The mean serum concentrations at 2 and 6 h were respectively 19.7 ± 8.3 and 18.1 ± 8.7 µg/ml. Seven patients (22%) had serum drug concentrations that were higher at 6 h than at 2 h, 12 (38%) had peak serum concentrations within the recommended range of 20-35 µg/ml; 18 patients (56%) had concentrations <20 µg/ml at both 2 h and 6 h; and 2 patients (6%) had at least one measurement >35 µg/ml. CONCLUSION: Lower than recommended serum CS concentrations and delayed absorption were common. It is essential to develop practical TDM to maintain proper serum drug concentrations.

D-cycloserine augmentation of exposure therapy for post-traumatic stress disorder: a pilot randomized clinical trial.

Viewing post-traumatic stress disorder (PTSD) as a disorder of emotional learning, this study used a cognitive enhancer synergistically with virtual reality exposure (VRE) therapy for the treatment of PTSD. The main objective was to determine if a novel pharmacotherapy, D-cycloserine (DCS), enhanced the efficacy of the psychotherapy. Pre-clinical studies suggest that when fear extinction occurs during DCS administration, neuroplasticity may be enhanced. VRE therapy is a particularly promising format to test the hypothesis that DCS enhances extinction learning, as sensory fear cues are standardized across patients. In a pilot randomized, double-blind, placebo-controlled trial, 100 mg of DCS or placebo was administered 90 min before each weekly VRE session, to ensure peak plasma concentrations during the sessions in 25 patients with chronic PTSD. The primary outcome measure was the Clinician Administered PTSD Scale (CAPS). Secondary outcome measures included the Beck Depression Inventory-II and the State-Trait Anger Expression Inventory-2. Assessments occurred at pre-treatment, following sessions 3, 6, 10, post-treatment, and at 6 months. The difference in CAPS between the VRE-DCS (n=13) and VRE-placebo (n=12) groups increased over time beginning at 6 weeks, with medium to large between-group effect sizes immediately post-treatment and 6 months later (d=0.68 and d=1.13, respectively). A similar pattern was observed for depression, anger expression, and sleep. PTSD remission rates were significantly greater for the VRE-DCS group (46% vs 8% at post-treatment; 69% vs 17% at 6 months). Patients in the VRE-DCS group showed earlier and greater improvement in PTSD symptoms compared with the VRE-placebo group. These results suggest a promising new treatment for PTSD.

Plasma drug activity in patients on treatment for multidrug-resistant tuberculosis.

Little is known about plasma drug concentrations relative to quantitative susceptibility in patients with multidrug-resistant tuberculosis (MDR-TB). We previously described a TB drug activity (TDA) assay that determines the ratio of the time to detection of plasma-cocultured Mycobacterium tuberculosis versus control growth in a Bactec MGIT system. Here, we assess the activity of individual drugs in a typical MDR-TB regimen using the TDA assay. We also examined the relationship of the TDA to the drug concentration at 2 h (C2) and the MICs among adults on a MDR-TB regimen in Tanzania. These parameters were also compared to the treatment outcome of sputum culture conversion. Individually, moxifloxacin yielded superior TDA results versus ofloxacin, and only moxifloxacin and amikacin yielded TDAs equivalent to a -2-log killing. In the 25 patients enrolled on a regimen of kanamycin, levofloxacin, ethionamide, pyrazinamide, and cycloserine, the C2 values were found to be below the expected range for levofloxacin in 13 (52%) and kanamycin in 10 (40%). Three subjects with the lowest TDA result (<1.5, a finding indicative of poor killing) had significantly lower kanamycin C2/MIC ratios than subjects with a TDA of ≥1.5 (9.8 ± 8.7 versus 27.0 ± 19.1; P = 0.04). The mean TDAs were 2.52 ± 0.76 in subjects converting to negative in ≤2 months and 1.88 ± 0.57 in subjects converting to negative in >2 months (P = 0.08). In Tanzania, MDR-TB drug concentrations were frequently low, and a wide concentration/MIC range was observed that affected plasma drug activity ex vivo. An opportunity exists for pharmacokinetic optimization in current MDR-TB regimens, which may improve treatment response.

Development and validation of a LC-MS/MS method for D-cycloserine determination in human plasma for bioequivalence study.

A reliable and high throughput high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for determining levels of the antitubercular drug-D -cycloserine in human plasma. Plasma samples analyte with an internal standard (IS) (niacin) were prepared by solid-phase extraction using Waters Oasis MCX cartridges. The chromatographic separation was performed using the HILIC mode on a YMC-Pack SIL-06 column (150 × 4.6 mm; 3 µm) under isocratic conditions. The run time of analysis was 5 min. The mobile phase consisted of methanol, propanol-2 and 0.075 % trifluoroacetic acid (66.5:28.5:5, v/v/v). Protonated ions formed by turbo ion spray in positive mode were used to detect the analyte and the IS. MS/MS detection was used to monitor the fragmentation of 103-75 m/z for cycloserine and 124 to 80 m/z for niacin (IS) on an API 4000 (AB Sciex) triple quadrupole mass spectrometer. A linear dynamic range of 0.3-30 µg/mL was established for cycloserine using 0.2 mL human plasma and a 1 µL injection volume. The mean relative recovery of cycloserine and niacin were 77.2 and 82.4 %, respectively. The procedure of sample preparation was consistent and reproducible (precision, 0.8-3.4 %; accuracy, 93.8-104.9 %). The method was validated in accordance with requirements of the European Medicines Agency and successfully applied to a bioequivalence study of 250 mg tablet formulations in 23 healthy human subjects.

A rapid and sensitive liquid chromatography-tandem mass spectrometric assay for cycloserine in 50µL of human plasma: Its pharmacokinetic application.

This paper describes a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry assay for the determination of cycloserine in human plasma using carbamazepine as internal standard (IS). Analyte and the IS were extracted from the 50µL of human plasma via protein precipitation using acetonitrile. The chromatographic separation was achieved on a C(18) column by using a mixture of acetonitrile-0.5% formic acid buffer (60:40, v/v) as the mobile phase at a flow rate of 0.8mL/min. The calibration curve obtained was linear (r(2)≥0.99) over the concentration range of 50-15,000ng/mL. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5min for each sample made it possible to analyze more number of samples in short time, thus increasing the productivity. The proposed method was found to be applicable to pharmacokinetic studies.

Development and validation of a selective and sensitive LC-MS/MS method for determination of cycloserine in human plasma: application to bioequivalence study.

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of cycloserine in human plasma is developed using niacin as internal standard (IS). The analyte and IS were extracted from 500 µL of human plasma via solid phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on a Peerless Basic C18 (100 mm × 4.6mm, 3 µm) column under isocratic conditions. Detection of analyte and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for cycloserine and niacin were at m/z 103.1 → 75.0 and 124.1 → 80.1 respectively. The method was fully validated for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.0013 and 0.20 µg/mL respectively with a linear dynamic range of 0.20-30.00 µg/mL for cycloserine. The intra-batch and inter-batch precision (%CV) across six quality control levels was less than 8.0% for cycloserine. The method was successfully applied to a bioequivalence study of 250 mg cycloserine capsule formulation in 24 healthy Indian male subjects under fasting condition.

Characterization of the interactive effects of glycine and D-cycloserine in men: further evidence for enhanced NMDA receptor function associated with human alcohol dependence.

Reduced responses to N-methyl-D-aspartate (NMDA) glutamate receptor antagonists in alcohol-dependent animals and humans provided evidence that chronic alcohol consumption increased NMDA receptor function. To further probe alterations in NMDA glutamate receptor function associated with human alcohol dependence, this study examined the interactive effects of agents acting at the glycine(B) coagonist site of the NMDA receptor. In doing so, it tested the hypothesis that raising brain glycine concentrations would accentuate the antagonist-like effects of the glycine(B) partial agonist, D-cycloserine (DCS). Twenty-two alcohol-dependent men and 22 healthy individuals completed 4 test days, during which glycine 0.3 g/kg or saline were administered intravenously and DCS 1000 mg or placebo were administered orally. The study was conducted under double-blind conditions with randomized test day assignment. In this study, DCS produced alcohol-like effects in healthy subjects that were deemed similar to a single standard alcohol drink. The alcohol-like effects of DCS were blunted in alcohol-dependent patients, providing additional evidence of increased NMDA receptor function in this group. Although glycine administration reduced DCS plasma levels, glycine accentuated DCS effects previously associated with the NMDA receptor antagonists, ketamine and ethanol. Thus, this study provided evidence that raising glycine levels accentuated the NMDA receptor antagonist-like effects of DCS and that alcohol-dependent patients showed tolerance to these DCS effects.

A six-month, placebo-controlled trial of D-cycloserine co-administered with conventional antipsychotics in schizophrenia patients.

RATIONALE: D-Cycloserine, a partial agonist at the glycine site of the N-methyl-D-aspartate receptor, has demonstrated inconsistent efficacy for negative and cognitive symptoms of schizophrenia. The strongest evidence for efficacy has come from studies using D-cycloserine at a dose of 50 mg/day added to conventional antipsychotics in trials of 8 weeks duration or less. OBJECTIVE: To assess the efficacy for negative symptoms and cognitive impairment of D-cycloserine augmentation of conventional antipsychotics in a 6-month trial. METHODS: Fifty-five schizophrenia patients with prominent negative symptoms, treated with conventional antipsychotics, were randomly assigned to treatment with D-cycloserine 50 mg/day or placebo for 6 months in a double-blind, parallel group design. RESULTS: Twenty-six subjects completed the 6-month trial; drop-out rates did not differ between treatment groups. D-Cycloserine treatment did not differ from placebo treatment on any primary outcome measure at 8 or 24 weeks, including response of negative symptoms and performance on a cognitive battery. Serum D-cycloserine concentrations did not correlate with response of negative symptoms. CONCLUSION: D-Cycloserine did not exhibit therapeutic effects in this trial, possibly reflecting the high drop-out rate, a narrow range of therapeutic serum concentrations, a modest magnitude of therapeutic effect for the selected outcome measures, or loss of efficacy over time. Because D-cycloserine is a partial agonist with relatively low affinity for the glycine site, the magnitude of potential therapeutic effect may be smaller than that achieved by the higher-affinity full agonists, glycine and D-serine.

Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection, using derivatization with p-benzoquinone.

A new method for determining cycloserine in plasma samples is described. This method is based on the derivatization of cycloserine with p-benzoquinone, a reaction that takes place at the same time as the process of plasma deproteinization due to the presence of ethanol as solvent in the solution of the derivatization reagent. Four derivatives are obtained from this reaction. The main derivative is well correlated with the cycloserine concentration. The ratio between the volumes of the plasma sample and the reagent solution is 1:2 for a p-benzoquinone concentration of 1000 microg/mL. Elution from a C18 column was isocratic, using a mobile phase containing (v/v) 85% aqueous 0.1% formic acid solution, and 15% (v/v) of a mixture of methanol and acetonitrile (1:1), with a flow-rate of 1 mL/min, at 25 degrees C. Determinations by fluorescence detection were achieved with excitation at 381 nm and emission at 450 nm, with a detection limit of 10 ng/mL for an injection volume of 5 microL. This method was validated and applied to the determination of cycloserine in blood plasma samples of several healthy volunteers.

Influence of D-cycloserine on the anticonvulsant activity of some antiepileptic drugs against audiogenic seizures in DBA/2 mice.

D-Cycloserine (DCS; 1-100 mg/kg, intraperitoneally (i.p.)) was able to antagonise the audiogenic seizures in DBA/2 mice in a dose-dependent manner. DCS, 2.5 mg/kg i.p. did not significantly affect the occurrence of audiogenic seizures in DBA/2 mice, but potentiated the anticonvulsant activity of carbamazepine, diazepam, felbamate, lamotrigine, phenytoin, phenobarbital and valproate against sound-induced seizures in DBA/2 mice. The degree of potentiation induced by DCS was greatest for diazepam, phenobarbital, phenytoin and valproate, less for carbamazepine and least for lamotrigine and felbamate. The increase in anticonvulsant activity was usually associated with a comparable increase in motor impairment. However, the therapeutic index of the combined treatment of the above drugs+DCS, was more favourable than the same drugs+saline with the exception of DCS+carbamazepine and DCS+lamotrigine. Since DCS did not significantly influence the total and free plasma levels of the anticonvulsant drugs studied, pharmacokinetic interactions, in terms of plasma levels, are not probable. The possibility that DCS can modify the clearance from the brain of the anticonvulsant drugs studied cannot be excluded. DCS did not significantly affect the hypothermic effects of the anticonvulsants tested. In conclusion, DCS potentiates the anticonvulsant action of some classical antiepileptic drugs, most notably diazepam, phenobarbital, phenytoin and valproate.