Isoniazid and rifampicin concentrations in children with tuberculosis with either a daily or intermittent regimen: implications for the revised RNTCP 2012 doses in India.

Suboptimal plasma drug concentrations in antitubercular therapy (ATT) may lead to delayed treatment response and the emergence of acquired drug resistance. This study aimed (i) to determine and compare plasma concentrations of isoniazid (INH) and rifampicin (RIF) in children treated for tuberculosis receiving a daily or intermittent ATT regimen and (ii) to study the effect of INH and RIF exposure on clinical outcome at the end of therapy (EOT). A total of 41 children aged 2-16 years initiated on either a daily or three-times weekly (intermittent) ATT regimen were recruited into the study. Towards the end of the intensive phase, blood specimens were collected pre-dose and at 0.5, 1, 1.5, 2, 2.5, 4 and 6 h post-dose. Concentrations of INH and RIF were analysed using validated liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography assays, respectively. The maximum plasma concentration (Cmax), the area under the concentration-time curve from 0-6 h (AUC0-6h) and treatment outcome were determined. Ninety-two percent of patients had an INH Cmax > 3 µg/mL. Seventy-seven percent of patients had a RIF Cmax < 8 µg/mL and 28% of patients had a RIF AUC0-24h < 13 mg ⋅ h/L. INH and RIF exposure did not differ between daily and intermittent ATT regimens on the day of administration. All children had a favourable outcome at EOT. Since 77% of children had low RIF exposure, we recommend routine use of therapeutic drug monitoring to prevent relapse and to support implementation of the revised RNTCP 2012 doses.

Determination of serum carbamazepine concentration using dried blood spot specimens for resource-limited settings.

OBJECTIVES: Carbamazepine (CBZ) is a commonly used anti-epileptic in rural hospitals in India. These hospitals lack the facilities to measure CBZ concentration; however, in larger hospitals this is performed using high performance liquid chromatography (HPLC). Dried blood spot (DBS) represents a feasible matrix for safe transportation by post/courier. This study was to determine whether the concentration of CBZ in serum can be predicted from that measured in DBS using an inexpensive HPLC method and inexpensive standard filter paper. METHODS: CBZ in serum and DBS from 80 epileptic patients were measured using a validated HPLC assay. The data was then randomly divided into two groups; simple Deming regression was performed with the first group and validation was performed using the second. RESULTS: There was a good correlation between the serum and DBS concentrations (r = 0.932) in the first group. The regression equation obtained was: predicted serum concentration = DBS concentration x 0.83 + 1.09. In the validation group, the correlation between the predicted and actual serum concentrations was also good (r = 0.958), and the mean difference between them was only 0.28 µg/ml (p = 0.8062). The imprecision and bias in both the groups were acceptable. CONCLUSION: Using inexpensive materials, serum CBZ concentrations can be accurately predicted from DBS specimens. This method can be recommended for the therapeutic drug monitoring of CBZ in resource-limited settings.

Cisplatin Concentrations in Long and Short Duration Infusion: Implications for the Optimal Time of Radiation Delivery.

INTRODUCTION: Cisplatin has radiosensitizing properties and the best sensitization to radiotherapy occurs with a higher plasma concentration of cisplatin. To our knowledge the optimal time sequence between chemotherapy and administration of radiation therapy, to obtain maximum effect from concurrent chemoradiation is unclear. AIM: The aim of this study was to measure the two cisplatin infusion regimens in order to determine the total and free cisplatin post infusion concentration changes over time. These changes may have clinical implications on the optimum time of administration of post infusion radiation therapy. MATERIALS AND METHODS: Two cohorts of patients were recruited and both, total and free plasma concentration of cisplatin following long and short durations of intravenous infusion was determined. Blood samples were collected at 0.5, 1, 1.5, 2, 3 and 5 hours from the start of the infusion in the 1hour infusion group and at 2, 3, 3.5, 4, 6 and 24 hours from the start of the infusion, in the 3 hour infusion group. Total and free cisplatin concentrations were measured using a validated HPLC-UV method. RESULTS: The highest concentration of total and free cisplatin was achieved at the end of the infusion in both regimens. Total cisplatin concentration declined 30 minutes after the end of infusion in both the groups. After 1hour of discontinuing cisplatin, the free cisplatin concentration also declined significantly. CONCLUSION: We conclude that radiation should be administered within 30 minutes of completion of the infusion irrespective of the duration of infusion.

A Nonparametric Pharmacokinetic Approach to Determine the Optimal Dosing Regimen for 30-Minute and 3-Hour Meropenem Infusions in Critically Ill Patients.

BACKGROUND: Pharmacokinetics of meropenem differ widely in the critically ill population. It is imperative to maintain meropenem concentrations above the inhibitory concentrations for most of the interdose interval. A population pharmacokinetic/pharmacodynamic model was developed to determine the probability of target attainment for 3-hour and 30-minute infusion regimens in this population. METHODS: This study was performed in an intensive care setting among adult patients who were initiated on meropenem at a dose of 1000 mg. Multiple blood specimens were collected at predetermined time points during the interdose period, and meropenem concentrations were measured using high performance liquid chromatography. Using Pmetrics, a pharmacokinetic/pharmacodynamic model was developed and validated. Monte Carlo simulation was performed, and probability of target attainment (100% T > minimum inhibitory concentration (MIC), with a probability >0.9) for doubling MICs was determined for different regimens of meropenem. RESULTS: A 2-compartment multiplicative gamma error model best described the population parameters from 34 patients. The pharmacokinetic parameters used in the final model were Ke (elimination rate constant from the central compartment), Vc (volume of distribution of central compartment), KCP and KPC (intercompartmental rate constants), and IC2 (the fitted amount of meropenem in the peripheral compartment). Inclusion of creatinine clearance (CLcreat) and body weight as covariates improved the model prediction (Ke = Ke0 × (Equation is included in full-text article.), Vc = Vc0 × Weight). The Ke and Vc [geometric mean (range)] of the individuals were 0.54 (0.01-2.61)/h and 9.36 (4.35-21.62) L, respectively. The probability of attaining the target, T > MIC of 100%, was higher for 3-hour infusion regimens compared with 30-minute infusion regimens for all ranges of CLcreat. CONCLUSIONS: This study emphasizes that extended regimens of meropenem are preferable for treating infections caused by bacteria with higher MICs. The nonparametric analysis using body weight and CLcreat as covariate adequately predicted the pharmacokinetics of meropenem in critically ill patients with a wide range of renal function.

Clinical efficacy and pharmacokinetics of tacrolimus in children with steroid-resistant nephrotic syndrome.

BACKGROUND: Tacrolimus has gained acceptance in the management of steroid-resistant nephrotic syndrome (SRNS) in children. Due to limited data, therapeutic range is extrapolated from pediatric renal transplant recipients. This study was designed to assess therapeutic efficacy of tacrolimus in children with SRNS and its correlation with inter-dose area under concentration curve (AUC0-12 h) and trough concentration (C0). METHODS: Pre dose, 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 8, and 12 h after drug administration blood samples were collected in 25 children who were on tacrolimus for a minimum of 3 months and AUC0-12 h was calculated. RESULTS: There was an 80% (20/25) response rate with 64% (16/25) children achieving complete remission. Median C0 in remission was higher than in relapse group (2.95 ng/ml, versus 1.20 ng/ml, p = 0.005). Median AUC0-12 h in remission was higher compared to those in relapse group (79.75 versus 35.15 µg × h/l; p = 0.025). Maximum concentration after drug administration (Cmax) among the groups was not significantly different. There was a significant correlation between C0 and AUC0-12 h (r = 0.79); and Cmax and AUC0-12 h (r = 0.84). Five patients had a rise in serum creatinine, of which four were still proteinuric and had lower C0 and AUC0-12 h. No other adverse effect was noted. CONCLUSIONS: Tacrolimus had beneficial clinical response in SRNS. Target C0 and AUC0-12 h level for treatment remission was higher than those in relapse in children with SRNS but was lower than required in transplant recipient.

Estimation of Monocrotophos renal elimination half-life in humans.

INTRODUCTION: Monocrotophos, implicated in about 1/4th of organophosphate poisonings in our centre, is associated with the highest mortality (24%). Yet data on its pharmacokinetics in humans is limited. We estimated the renal elimination half-life of monocrotophos. PATIENTS AND METHODS: Consecutive patients presenting with monocrotophos overdose over a 2-month period who had normal renal function were recruited. Monocrotophos in plasma and urine were quantitated by high-performance liquid chromatography. Urine was obtained from catheterised samples at 0-2, 2-4, 4-6, 6-8, 8-12 and 12-24 h. Plasma specimens were collected at the time of admission, and at the midpoint of the urine sample collections at 1, 3, 5, 7, 10, 15 and 21 h. Renal elimination half-life was calculated from the cumulative amount excreted in the urine. RESULTS: The cohort of 5 male patients, aged 35.8 ± 2.94 years, presented with typical organophosphate (cholinergic) toxidrome following intentional monocrotophos overdose. All patients required mechanical ventilation; one patient died. Plasma data was available from 5 patients and urine data from 3 patients. The median renal elimination half-life was 3.3 (range: 1.9-5.0 h). Plasma monocrotophos values, as natural log, fell in a linear fashion up to around 10 h after admission. After the 10-hour period, there was a secondary rise in values in all the 3 patients in whom sampling was continued after 10 h. CONCLUSION: A renal elimination half-life of 3.3 h for monocrotophos is consistent with a water-soluble compound which is rapidly cleared from the plasma. The secondary rise in plasma monocrotophos values suggests possible re-distribution. Determining the elimination profile of this compound will help develop better strategies for treatment.

Therapeutic Drug Monitoring of Levetiracetam and Lamotrigine: Is There a Need?

BACKGROUND: This study was a retrospective assessment of the therapeutic drug monitoring data collected for levetiracetam and lamotrigine from a clinical setting. The proportion of patients in relation to the therapeutic ranges for serum concentrations of lamotrigine and levetiracetam was estimated, and the influence of age and anticonvulsant comedications on their clearances were studied. METHODS: Information on levetiracetam (2011-2013) and lamotrigine (2008-2013) dose, trough concentration, age, sex, body weight, and anticonvulsant comedications prescribed was obtained from the therapeutic drug monitoring register and archived medical records. Patients were categorized into 4 groups based on anticonvulsant comedications and further divided into 3 subgroups based on age (a: <9 years; b: 9-17 years; c: ≥18 years). In each subgroup, the proportion of patients who achieved trough concentrations in the therapeutic range for levetiracetam and lamotrigine was computed. Apparent clearance (CL/F) was compared across subgroups by 1-way analysis of variance, and factors which significantly predicted CL/F were identified by stepwise multiple linear regression. RESULTS: Overall, 348 (330 patients) and 706 (493 patients) samples for levetiracetam and lamotrigine were included in the analysis. Of these, 56.9% and 72.4% were within, 43.1% and 23.9% below, 0% and 3.7% above the therapeutic range for levetiracetam and lamotrigine, respectively. A significant difference in CL/F was noted across subgroups for levetiracetam (P < 0.001) and lamotrigine (P < 0.001). Age <9 years, age ≥18 years, and inducer comedications significantly predicted CL/F for levetiracetam. For lamotrigine, inhibitor comedications, age <9 years, inducer comedications, and age 9-17 years significantly predicted CL/F. CONCLUSIONS: These findings emphasize the need to monitor relatively newer anticonvulsants, lamotrigine and levetiracetam, especially among children and when other anticonvulsant comedications are prescribed or discontinued in the treatment regimen.

Pharmacokinetics of concentration-controlled mycophenolate mofetil in proliferative lupus nephritis: an observational cohort study.

BACKGROUND: Mycophenolate mofetil (MMF) has variable pharmacokinetics. This study examines the pharmacokinetic and clinical correlations in proliferative lupus nephritis. METHODS: Thirty-four patients were started on MMF, and the area under the concentration-time curve (AUC) was measured by limited sampling strategies, and dosing was adjusted to achieve an AUC of 30-60 mg·h·L. Twenty-seven patients had at least 2 measurements, and renal response was assessed within 1 year. RESULTS: About 61.8% of patients had mycophenolic acid (MPA) AUC <30 mg·h·L with an empiric starting dose of 30 mg/kg. About 79.4% of patients achieved renal response by 1 year, and the median time to renal response was 111 days. MMF dose per body weight had a weak correlation with the AUC and did not correlate with trough concentrations. The median dose was 1.5 g/d at entry and 2 g/d after dose modification during the induction phase. Trough concentrations had a weak correlation with AUC. Patients with serum albumin ≥35 g/L had a greater chance of having an AUC ≥30 mg·h·L. The between-patient coefficient of variability for dose-normalized AUC was 37.9% at entry and 31% within 1 year, whereas repeated measurements over time in an individual had a good intraclass correlation of 0.78. Infections occurred in 11.8% and toxicities in 5.9%. MPA exposure was not significantly associated with adverse events. Patients with an AUC ≥30 mg·h·L had greater renal response at 1 year. CONCLUSIONS: Lupus nephritis patients induced with concentration-controlled MMF had excellent renal response and fewer adverse events with lower than usual dosing. MPA exposure had high interpatient variability, requiring measurements for personalized dosing, and fewer adverse events. Long-term cost reduction is achievable with lower doses and good renal response in the majority of patients.

Comparison of the effect of lignocaine instilled through the endotracheal tube and intravenous lignocaine on the extubation response in patients undergoing craniotomy with skull pins: A randomized double blind clinical trial.

BACKGROUND: A desirable combination of smooth extubation and an awake patient after neurosurgical procedures is difficult to achieve in patients with skull pins. Lignocaine instilled into endotracheal tube has been reported to suppress cough by a local mucosal anesthetizing effect. We aimed to evaluate if this effect will last till extubation, if given before pin removal. MATERIALS AND METHODS: A total of 114 patients undergoing elective craniotomy were divided into three groups and were given 1 mg/kg of intravenous (IV), 2% lignocaine (Group 1), placebo (Group 2) and 1 mg/kg of 2% lignocaine sprayed down the endotracheal tube (Group 3) before skull pin removal. The effectiveness of each to blunt extubation response was compared. Plasma levels of lignocaine were measured 10 min after administration of the study drug and at extubation. Sedation scores were noted, immediately after extubation and 10 min later. RESULTS: Two percent of lignocaine instilled through endotracheal route was not superior to the IV route or placebo in attenuating cough or hemodynamic response at extubation when given 20-30 min before extubation. The plasma levels of lignocaine (0.8 µg/ml) were not high enough even at the end of 10 min to have a suppressive effect on cough if given IV or intratracheally (IT). Lignocaine did not delay awakening in these groups. CONCLUSION: IT lignocaine in the dose of 1 mg/kg does not prevent cough at extubation if given 20-30 min before extubation. If the action is by a local mucosal anesthetizing effect, it does not last for 20-30 min to cover the period from pin removal to extubation.

In vitro study of elution kinetics and bio-activity of meropenem-loaded acrylic bone cement.

BACKGROUND: Use of antibiotic-loaded acrylic bone cement to treat orthopaedic infections continues to remain popular, but resistance to routinely used antibiotics has led to the search for alternative, more effective antibiotics. We studied, in vitro, the elution kinetics and bio-activity of different concentrations of meropenem-loaded acrylic bone cement. METHODS: Meropenem-loaded bone cement cylinders of different concentrations were serially immersed in normal saline. Elution kinetics was studied by measuring the drug concentration in the eluate, collected at pre-determined intervals, by high-performance liquid chromatography. Bio-activity of the eluate of two different antibiotic concentrations was tested for a period of 3 weeks against each of the following organisms: Staphylococcus aureus ATCC 2593 (MSSA), Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, S. aureus ATCC 43300 (MRSA) and Klebsiella pneumoniae ATCC 700603 (ESBL). RESULTS: Meropenem elutes from acrylic bone cement for a period of 3-27 days depending on the concentration of antibiotic. Higher doses of antibiotic concentration resulted in greater elution of the antibiotic. The eluate was found to be biologically active against S. aureus ATCC 2593 (MSSA), P. aeruginosa ATCC 27853, E. coli ATCC 25922 and K. pneumoniae ATCC 700603 (ESBL) for a period of 3 weeks. CONCLUSIONS: The elution of meropenem is in keeping with typical antibiotic-loaded acrylic bone cement elution characteristics. The use of high-dose meropenem-loaded acrylic bone cement seems to be an attractive option for treatment of resistant Gram-negative orthopaedic infections but needs to be tested in vivo.

A possible simplification for the estimation of area under the curve (AUC0₋12) of enteric-coated mycophenolate sodium in renal transplant patients receiving tacrolimus.

Enteric-coated mycophenolate sodium (EC-MPS) is widely used in renal transplantation. With a delayed absorption profile, it has not been possible to develop limited sampling strategies to estimate area under the curve (mycophenolic acid [MPA] AUC0₋12), which have limited time points and are completed in 2 hours. We developed and validated simplified strategies to estimate MPA AUC0₋12 in an Indian renal transplant population prescribed EC-MPS together with prednisolone and tacrolimus. Intensive pharmacokinetic sampling (17 samples each) was performed in 18 patients to measure MPA AUC0₋12. The profiles at 1 month were used to develop the simplified strategies and those at 5.5 months used for validation. We followed two approaches. In one, the AUC was calculated using the trapezoidal rule with fewer time points followed by an extrapolation. In the second approach, by stepwise multiple regression analysis, models with different time points were identified and linear regression analysis performed. Using the trapezoidal rule, two equations were developed with six time points and sampling to 6 or 8 hours (8hrAUC[0₋12exp]) after the EC-MPS dose. On validation, the 8hrAUC(0₋12exp) compared with total measured AUC0₋12 had a coefficient of correlation (r²) of 0.872 with a bias and precision (95% confidence interval) of 0.54% (-6.07-7.15) and 9.73% (5.37-14.09), respectively. Second, limited sampling strategies were developed with four, five, six, seven, and eight time points and completion within 2 hours, 4 hours, 6 hours, and 8 hours after the EC-MPS dose. On validation, six, seven, and eight time point equations, all with sampling to 8 hours, had an acceptable r with the total measured MPA AUC0₋12 (0.817-0.927). In the six, seven, and eight time points, the bias (95% confidence interval) was 3.00% (-4.59 to 10.59), 0.29% (-5.4 to 5.97), and -0.72% (-5.34 to 3.89) and the precision (95% confidence interval) was 10.59% (5.06-16.13), 8.33% (4.55-12.1), and 6.92% (3.94-9.90), respectively. Of the eight simplified approaches, inclusion of seven or eight time points improved the accuracy of the predicted AUC compared with the actual and can be advocated based on the priority of the user.

Can mycophenolic acid dose requirement during the first transplant help predict dosing for the second transplant?

We describe the pharmacokinetic profile of mycophenolic acid (MPA) in a patient receiving Mycophenolate mofetil (MMF) during her first and second renal transplantations. The MMF dose required to achieve a therapeutic range of MPA-AUC(0)(-)(12)(h) early following the second transplantation was 10 times greater than that required late following the first transplantation. Her MMF requirement then declined and continued to decrease even beyond 1 year. Intra-individual variability in MPA profiles precluded the ability to predict MMF dosing for the second transplant based on that during the first. Therapeutic drug monitoring of MMF should be continued beyond 1 year of transplantation.

Mycophenolic acid estimation by pooled sampling: a novel strategy.

The aim of the study was to determine the reliability of estimating area under the curve from 0 to 6 hours (AUC0-6) of mycophenolic acid (MPA) by pooling the blood samples from different sampling time points. Eighty 6-hour concentration-time profiles were obtained from 68 patients on mycophenolate mofetil and the MPA AUC0-6 was calculated. In the pooled strategy, each of the equally spaced time point samples was pooled into two samples. Two rectangles were created instead of multiple trapezoids and the sum of their areas equal to the MPA AUC0-6. The linear correlation (r), intraclass correlation, bias, and precision were calculated between the pooled MPA AUC0-6 and the MPA AUC0-6 derived from measurements at different time points. Pharmacokinetic profiles of an additional 20 patients were obtained to study the possibility of using fewer time points to create a single pooled sample to obtain MPA AUC0-6. The linear correlation (r) and intraclass correlation between pooled and measured MPA AUC0-6 was 0.982 and 0.979, respectively. There was a highly significant correlation (r) of 0.978 between the pooled versus measured for both MPA AUC0-3 and MPA AUC3-6. The mean bias and precision (95% confidence interval) for pooled with total measured MPA AUC0-6 was -6.4% (-7.8% to -4.94%) and 7.37% (6.21%-8.54%), respectively. The pooled sample approach using only five time points to estimate MPA AUC0-6 had an unacceptable bias and precision. Pooling 10 samples to a set of two samples gave a highly accurate measure of MPA AUC0-6. The advantages for a central laboratory are the high throughput of samples and the transportation of only two specimens from other centers, all of which leads to a reduction in cost. This approach is extremely useful for studies aimed at examining the bioavailability of mycophenolate mofetil in different ethnic populations within India.

A reliable limited sampling strategy for the estimation of mycophenolic acid area under the concentration time curve in adult renal transplant patients in the stable posttransplant period.

In renal transplant patients, there is an established relationship between mycophenolate area under the curve and clinical outcome. The authors have developed and validated a limited sampling strategy to estimate mycophenolic acid area under the curve to 12 hours (MPA AUC0-12) in a stable renal transplant Indian population prescribed a formulation of mycophenolate mofetil (Mofilet) along with prednisolone and tacrolimus. Intensive pharmacokinetic sampling was performed in 29 patients to measure mycophenolate concentration from trough to 12 hours postdose. Subsets of different timed concentrations against total measured 12-hour area under the curve were analyzed by linear regression. Three models were identified and linear regression analysis done. After all subset regression analysis, three, four, and five time point limited sampling strategies (LSS) were developed having correlation coefficients above 0.92. Validation of the models was performed using the jackknife method and their predictive performances were tested. After validation, the correlation coefficients for all three models were above 0.901. The five-point LSS had the best predictive performance with a bias (95% confidence interval) of 0.67% (-3.45 to 4.79) and mean precision 7.73%. In all patients except one, the five-point LSS estimation for total area under the curve was within +/- 20% of the total measured AUC0-12. Trough concentration had a significant correlation with AUC0-12 (r = 0.69). However, if dosing in routine clinical practice was adjusted based only on trough concentration, 41% of our patients would require a different dose compared with monitoring using AUC0-12. The five-point LSS uses half-hourly samples from trough to 1.5 hour postdose with an additional sample at 3 hours. Ninety-three percent of our patients had a Cmax within 1.5 hour and inclusion of all the time points up to1.5 hour gave a better estimate of AUC0-12. This model simplifies area under the curve measurement with high precision in stable adult renal transplant patients.

A limited sampling strategy for tacrolimus in renal transplant patients.

AIMS: To develop and validate limited sampling strategy (LSS) equations to estimate area under the curve (AUC(0-12)) in renal transplant patients. METHODS: Twenty-nine renal transplant patients (3-6 months post transplant) who were at steady state with respect to tacrolimus kinetics were included in this study. The blood samples starting with the predose (trough) and collected at fixed time points for 12 h were analysed by microparticle enzyme immunoassay. Linear regression analysis estimated the correlations of tacrolimus concentrations at different sampling time points with the total measured AUC(0-12). By applying multiple stepwise linear regression analysis, LSS equations with acceptable correlation coefficients (R(2)), bias and precision were identified. The predictive performance of these models was validated by the jackknife technique. RESULTS: Three models were identified, all with R(2) > or = 0.907. Two point models included one with trough (C(0)) and 1.5 h postdose (C(1.5)), another with trough and 4 h postdose. Increasing the number of sampling time points to more than two increased R(2) marginally (0.951 to 0.990). After jackknife validation, the two sampling time point (trough and 1.5 h postdose) model accurately predicted AUC(0-12). Regression coefficient R(2) = 0.951, intraclass correlation = 0.976, bias [95% confidence interval (CI)] 0.53% (-2.63, 3.69) and precision (95% CI) 6.35% (4.36, 8.35). CONCLUSION: The two-point LSS equation [AUC(0-12) = 19.16 + (6.75.C(0)) + (3.33.C1.5)] can be used as a predictable and accurate measure of AUC(0-12) in stable renal transplant patients prescribed prednisolone and mycophenolate.

Preparation of monoclonal antibodies reactive to the endogenous small molecule, anandamide.

The development of an easy and inexpensive immunoassay to measure the limited quantities of endogenous cannabinoids found in the body would be beneficial for both cannabinoid researchers and clinicians. This report describes the hapten design and carrier molecule strategy that we used to generate a panel of monoclonal antibodies (mAB) to the endogenous cannabinoid anandamide (N-arachidonylethanolamide, AEA). We designed and successfully prepared a hapten, N-arachidonyl-7-amino-6-hydroxy-heptanoic acid (AHA), which retained the basic characteristic features of anandamide--the carboxamide, the hydroxyl and the lipophilic arachidonyl moiety with its skipped double bond system, while still allowing attachment to protein. In addition, a secondary alcohol structure was added to reduce the potential for biological hydrolysis of the hapten. Because of the diverse responses obtained after coupling this hapten to four different carriers, we determined that the type of carrier molecule used was particularly important for generating anti-anandamide antibodies. Described in this report are the characteristics of a panel of 11 mAB, generated from four separate fusions, with a range of relative affinities and cross reactivities. Excellent selectivity for anandamide vs. two other endogenous cannabinoids and arachidonic acid was achieved this strategy (cross-reactivities <5%). In addition, at least one mAB maintained specificity for anandamide compared to two very closely related fatty acid amide molecules. However, the IC50 values in a standard enzyme-linked immunosorbent assay (ELISA) format (ca. 2-3 microM) indicate that improvement in antibody affinities or assay format will be required for an immunoassay to measure endogenous levels. Such work is underway.