Rifampicin In Plasma Research Articles

Naïve-pooled pharmacokinetic analysis of pyrazinamide, isoniazid and rifampicin in plasma and cerebrospinal fluid of Vietnamese children with tuberculous meningitis.

BackgroundAmong the various forms of TB, tuberculous meningitis (TBM) is the most severe, with about 30 % mortality and 50 % of survivors left with neurological sequelae. Children suffer more frequently from TBM than adults and outcomes are often poor due to difficulties in making the diagnosis and uncertainty regarding the best anti-tuberculosis drug regimen. The aim of this prospective study was to describe the pharmacokinetics of pyrazinamide, isoniazid and rifampicin in plasma and cerebrospinal fluid of children with tuberculous meningitis treated with the standard TBM regimen.MethodsWe performed a prospective observational study of 100 consecutively treated children (≤15 years of age) with tuberculous meningitis in Ho Chi Minh City, Vietnam. Children were treated according to the 2006 WHO recommended pediatric treatment regimen consisting of isoniazid (5 mg/kg), rifampicin (10 mg/kg) and ethambutol (15 mg/kg) for 8 months, with the addition of pyrazinamide (25 mg/kg) for the first 3 months and streptomycin (15 mg/kg) for the first 2 months. Pyrazinamide, isoniazid and rifampicin concentrations were measured in plasma at day 14 and in cerebrospinal fluid (CSF) at 1 month by HPLC-UV. A naïve-pooled non-compartmental data analysis was used to describe the pharmacokinetic properties of drugs in the two-age groups of children ≤ 4 years or > 4 years of age.ResultsYounger children, when compared to older children, presented a higher body weight-normalized clearance and volume of distribution, and lower median total plasma exposures for the three studied drugs with −14 %, −22 % and −16 % for Pyrazinamide, Isoniazid and Rifampicin, respectively. In CSF, individual concentrations of isoniazid and pyrazinamide were comparable to that in plasma in both age groups; but rifampicin concentrations were lower than the minimum inhibitory concentration of susceptible bacteria in all but two children.ConclusionsThere is an age-dependent variation in the plasma and cerebrospinal fluid pharmacokinetics of rifampicin, isoniazid and pyrazinamide. The safety and efficacy of higher doses of rifampicin should be investigated for the treatment of childhood tuberculous meningitis.

A simultaneous population pharmacokinetic analysis of rifampicin in Malawian adults and children.

Low rifampicin plasma concentrations can lead to treatment failure and increased risk of developing drug resistant tuberculosis. The objectives of this study were to characterize the population pharmacokinetics (popPK) of rifampicin in Malawian children and adults with tuberculosis, simulate exposures under revised WHO dosing guidelines that aim to reduce the risk of low exposures of rifampicin and examine predicted exposures using weight- and age-based dosing bands under new dosing recommendations. Patients were recruited at least two weeks after initiation of the intensive phase of treatment and received RIF in FDC of anti-TB drugs. A total of 5-6 rich and 1-2 sparse samples were collected. nonmem (v7.2) was used to build a population-PK model. A 165 TB patients, 115 adults and 50 children, aged 7 months to 65 years and weighing 4.8 to 87 kg, were included in the one compartment model with first order absorption best described the data. The mean population estimate for CL/F was 23.9 (lh(-1) 70kg(-1) ) with inter-individual variability of 46.6%. Exposure was unaffected by HIV status. Relative bioavailability in children was estimated at 49% lower compared to adults (100% relative bioavailability). Simulations showed significantly lower rifampicin exposure in children vs. adults. In children average AUC was 13.5mgl(-1) h, which was nearly half that was observed in adults (26.3mgl(-1) h). Using age as a surrogate for weight in dosing bands gave similar results compared with the weight bands. Increasing dose to approximately 15mg kg(-1) , increased AUC in children to an average of 22mgl(-1) h. bringing expected exposures in children closer to those predicted for adults. The popPK model developed can be used to optimize rifampicin exposures through dosing simulations. WHO dosing recommendations may not be achieved using currently licensed fixed dose combination formulations of TB therapy.

Development and validation of RP-HPLC method for simultaneous estimation of rifampicin, isoniazid and pyrazinamide in human plasma

A rapid, simple, sensitive and cost effective stability indicating high performance liquid chromatographic method for the simultaneous determination of rifampicin, isoniazid and pyrazinamide in human plasma was developed and validated in accordance to Food and Drug Administration (FDA) guidelines. The three drugs were eluted under isocratic mode using a 250 × 4.0 mm i.d., 5 μm Phenomenex ODS 2 C18 column. The mobile phase was composed of a mixture of acetonitrile, methanol and water in the ratio of 30: 5: 65 (v/v, pH adjusted to 5.2) at a flow rate of 1.0 mL/min. The limits of detection and quantification for rifampicin were 0.13 and 0.4 μg/mL, for isoniazid—0.6 and 1.8 μg/mL; and for pyrazinamide—0.5 and 1.6 μg/mL, respectively. The method can be successfully applied for pharmacokinetic, bioavailability or bioequivalence studies of rifampicin, isoniazid and pyrazinamide combination in human subjects.

Pharmacokinetic/pharmacodynamic analysis of an intensified regimen containing rifampicin and moxifloxacin for tuberculous meningitis

Recent data suggest that intensified antimicrobial treatment may improve the outcome of tuberculous meningitis (TBM). Considering that drug exposure is the intermediate link between dose and effect, we examined the concentration–response relationship for rifampicin and moxifloxacin in TBM patients. In an open-label, phase 2 clinical trial performed in Indonesia (ClinicalTrials.gov NCT01158755), 60 TBM patients were randomised to receive standard-dose (450mg oral) or high-dose rifampicin (600mg intravenous) plus either oral moxifloxacin (400mg or 800mg) or ethambutol (750mg). After 14 days, all patients continued with standard tuberculosis treatment. Pharmacokinetic sampling was performed once in every patient during the first three critical days. Differences in exposure between patients who died or survived were tested with independent samples t-tests. The relationship between drug exposure and mortality was examined using Cox regression. Compared with patients who died during the 2 weeks of intensified treatment, surviving patients had significantly higher rifampicin plasma AUC0–6h, plasma Cmax and CSF Chighest. Additionally, patients had a 32–43% lower relative likelihood of dying with an interquartile range increase in rifampicin exposure. Moxifloxacin exposure did not show a clear relationship with survival. From exposure–response curves, a rifampicin plasma AUC0–6h of ∼70mg·h/L (AUC0–24h of ∼116mgh/L) and a Cmax of ∼22mg/L were deduced as minimum target values for treatment. A strong concentration–effect relationship was found, with higher rifampicin exposure leading to better TBM survival. The current treatment dose of rifampicin is suboptimal; higher doses of rifampicin should be evaluated.

A Modified and Cost-Effective HPLC Method for Determination of Plasma Concentrations of Rifampicin in Pulmonary TB Patients

Objective: To evaluate the pharmacokinetics of standard doses of rifampicin (RMP) in fixed dose combination in pulmonarytuberculosis patients by a modified high performance liquid chromatography (HPLC) methodMaterials and Methods: This descriptive study was conducted after approval from Ethical Committee, Army Medical CollegeRawalpindi and was funded in part by National University of Sciences and Technology (NUST), Islamabad. Twenty adultpatients with newly diagnosed pulmonary TB consented to participate in the study. RMP plasma concentrations were assayedby a simple and sensitive HPLC method in the initial phase of pulmonary tuberculosis treatment. The method was modifiedto use naproxen as an internal standard and validated according to International Conference on Hormonization (ICH) guidelines.Results: The calibration curve of rifampicin was linear within the range of 0.781–50 μg/ml. Both intra-day and inter-dayvariability and accuracy demonstrated good reproducibility at all quality control levels. The developed method was found tobe simple, precise and accurate for estimation of rifampicin in plasma. The pharmacokinetic parameters of RMP showed markedinter-individual differences and sub-therapeutic levels.Conclusion: Evaluation of pharmacokinetics of standard doses of rifampicin in fixed dose combination in pulmonary tuberculosispatients by a modified high performance liquid chromatography (HPLC) method is precise, accurate and cost-effective. It maybe used for monitoring plasma RMP levels in TB patients who are slow to respond or are non-responders and have less availabilityof resources.Keywords: Tuberculosis,

Novel on‐line column extraction apparatus coupled with binary peak focusing for high‐performance liquid chromatography determination of rifampicin in human plasma: A strategy for therapeutic drug monitoring

In order to develop a method that is completely suitable for the routine therapeutic drug monitoring, a sensitive and fully automated on-line column extraction apparatus in combination with high-performance liquid chromatography allowing binary peak focusing was developed and validated for the determination of rifampicin in human plasma. Rifapentine was used as an internal standard. The analytical cycle started with the injection of 100 μL of the sample pretreated by protein precipitation in a Venusil SCX extraction column. After the elution, the analytes were transferred and concentrated in an Xtimate C18 trap column. Finally, the trapped analytes were separated by an Xtimate C18 analytical column and were analyzed by an ultraviolet detector at 336 nm. With this new strategy, continuous on-line analysis of the compounds was successfully performed. The method showed excellent performance for the analysis of rifampicin in plasma samples, including calibration curve linearity (All r were larger than 0.9996), sensitivity (lowest limit of quantification was 0.12 μg/mL), method accuracy (within 6.6% in terms of relative error), and precision (relative standard deviations of intra- and interday precision were less than 7.8%). These results demonstrated that the simple, reliable, and automatic method based on on-line column extraction and binary peak focusing is a promising approach for therapeutic drug monitoring in complex biomatrix samples.

Determination of rifampicin in rat plasma by modified large-volume direct injection RAM-HPLC and its application to a pharmcokinetic study.

A direct large volume injection high-performance liquid chromatography (HPLC) method with homemade restricted-access media (RAM) pre-column and combined with a column-switching valve was established and developed for determination rifampicin (RIP) in rat plasma. The rat plasma samples (100 μL) were injected directly onto pre-column, where RIP was retained and pre-concentrated, while proteins were washed to waste using a methanol-water (5:95) as the mobile phase at a flow rate of 1 mL/min. Then, by rotation of the switching valve at 5 min, the RIP were eluted from the pre-column and transferred to an Luna C18 analytical column by the chromatographic mobile phase consisting of methanol-acetonitrile-10 mm ammonium format (60:5:35) at a flow rate of 1 mL/min. The total analytical run time was 15 min with UV detection wavelength at 254 nm. Carbamazepine was used as the internal standard. Excellent linear correlation (r = 0.9993) was obtained in the range of 0.25-8 µg/mL for rat plasma. The intra-day and inter-day precisions of RIP were all <5.0%. The recoveries were in the range of from 99.98-113.66% for plasma. This on-line RAM-HPLC method was successfully applied to the pharmacokinetic study of RIP in rat plasma.

Development and validation of a hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the simultaneous determination of five first-line antituberculosis drugs in plasma

A new, sensitive and fast method for the simultaneous determination of pyrazinamide, isoniazid, streptomycin, ethambutol, and rifampicin in human plasma was developed and validated. The method required only 100 μL of plasma and one step for sample preparation by protein precipitation. The drugs were separated by using a hydrophilic interaction liquid chromatography (HILIC) column. The mobile phase was methanol and water (0.1% formic acid and 5 mM ammonium acetate, pH 3.0 ± 0.1) in a ratio of 65:35 (v/v), which was eluted at an isocratic flow rate of 0.5 mL/min. Tandem mass spectrometry was performed with a triple-quadrupole tandem mass spectrometer. By use of the HILIC column, the detection was free of ion-pair reagents in the mobile phase, with no significant matrix effects. The total run time was less than 2 min for each sample. The method was validated by evaluating its selectivity, sensitivity, linearity, accuracy, and precision according to US Food and Drug Administration guidelines. The lower limit of quantification was 4.0 ng/mL for pyrazinamide, isoniazid, and rifampicin, 0.5 ng/mL for ethambutol, and 10.0 ng/mL for streptomycin. The intraday precision and interday precision were less than 9%, with the accuracy ranging between -9.3 and 7.3%. The method was successfully applied to therapeutic drug monitoring of 33 patients with tuberculosis after administration of standard antituberculosis drugs. The method has been proved to meet the high-throughput requirements in therapeutic drug monitoring.

High-Performance Liquid Chromatographic Quantification of Rifampicin in Human Plasma: Method for Therapeutic Drug Monitoring

High-Performance Liquid Chromatographic Quantification of Rifampicin in Human Plasma: Method for Therapeutic Drug Monitoring

The pharmacokinetics of enteral antituberculosis drugs in patients requiring intensive care

There is a paucity of data on the pharmacokinetics of fixed-dose combination enteral antituberculosis treatment in critically ill patients. To establish the pharmacokinetic profile of a fixed-dose combination of rifampicin, isoniazid, pyrazinamide and ethambutol given according to weight via a nasogastric tube to patients admitted to an intensive care unit (ICU). We conducted a prospective, observational study on 10 patients (mean age 32 years, 6 male) admitted to an ICU and treated for tuberculosis (TB). Serum concentrations of the drugs were determined at eight predetermined intervals over 24 hours by means of high-performance liquid chromatography. The therapeutic maximum plasma concentration (Cmax) for rifampicin at time to peak concentration was achieved in only 4 patients, whereas 2 did not achieve therapeutic Cmax for isoniazid. No patient reached sub-therapeutic Cmax for pyrazinamide (6 were within and 4 above therapeutic range). Three patients reached sub-therapeutic Cmax for ethambutol, and 6 patients were within and 1 above the therapeutic range. Patients with a sub-therapeutic rifampicin level had a higher mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score (p=0.03) and a lower estimated glomerular filtration rate (GFR) (p=0.03). A fixed-dose combination tablet, crushed and mixed with water, given according to weight via a nasogastric tube to patients with TB admitted to an ICU resulted in sub-therapeutic rifampicin plasma concentrations in the majority of patients, whereas the other drugs had a more favourable pharmacokinetic profile. Patients with a sub-therapeutic rifampicin concentration had a higher APACHE II score and a lower estimated GFR, which may contribute to suboptimal outcomes in critically ill patients. Studies in other settings have reported similar proportions of patients with 'sub-therapeutic' rifampicin concentrations.

Troubleshooting carry-over of LC–MS/MS method for rifampicin, clarithromycin and metabolites in human plasma

Clarithromycin and rifampicin are used for the treatment of Mycobacteria. Pharmacokinetic drug interaction is possibly due to the influence of the two drugs on the liver enzymes. Using a Hypurity Aquastar C18 column (50mm×2.1mm×5μm) for liquid chromatography including a polar end-capped phase for the determination of clarithromycin, rifampicin and their metabolites together in plasma using LC-MS/MS resulted in a substantial carry-over. As a consequence, the throughput of the method is not assured. Using a step-by-step troubleshooting procedure, such carry-over was found originating from column memory effect. With the use of another type of C18 column, the carry-over is eliminated. Due to the absence of carry-over, the analytical concentration ranges are extended and are therefore more appropriate for the analysis of patient samples. The method was re-validated for linearity, reproducibility and dilution integrity.

Quantification of rifampicin in human plasma and cerebrospinal fluid by a highly sensitive and rapid liquid chromatographic–tandem mass spectrometric method

A highly sensitive and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed to measure the levels of the antitubercular drug rifampicin (RIF) in human plasma and cerebrospinal fluid (CSF). The analyte and internal standard (IS) were isolated from plasma and CSF by a simple organic solvent based precipitation of proteins followed by centrifugation. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 25–6400 ng/mL with intra- and inter-day precision of <7% and <8%, respectively. The validated method was applied to the study of RIF pharmacokinetics in human CSF and plasma over 25 h period after a 10 mg/kg oral dose.

Simultaneous determination of efavirenz, rifampicin and its metabolite desacetyl rifampicin levels in human plasma

A simple and rapid isocratic, high performance liquid chromatography (HPLC) assay employing solid phase extraction (SPE) for the simultaneous determination of the anti HIV drug, efavirenz, the anti-tuberculosis drug, rifampicin and the desacetyl metabolite of rifampicin in plasma from HIV/tuberculosis infected patients has been developed. Using a Zorbax SB-Phenyl reverse-phase analytical column with UV detection, good separation and detection of the drugs was attained within a 10 min run time. Intra- and inter-assay precision RSD values were found to be less than 15% at the concentrations examined (0.1–20 μg/mL). The LOQ was found to be 0.1 μg/mL for each agent and the assay was found to generate a linear response up to 20 μg/mL. This low cost assay can accurately detect efavirenz and rifampicin concentrations within a clinically relevant concentration range using standard chromatography equipment, making it particularly applicable to resource-limited settings.

Development and validation of liquid chromatography-Mass spectrometry method for the estimation of rifampicin in plasma

A selective, rapid and sensitive liquid chromatography-mass spectrometry method was developed for the quantitative estimation of rifampicin in plasma. With phenacetin as internal standard, sample pretreatment involved a one-step extraction with ethyl acetate from plasma. The sample was analyzed using methanol: 2mM ammonium acetate: 80:20 v/v as mobile phase. Chromatographic separation was achieved on a BDS Hypersil Gold C18 column which was followed by detection with mass spectrometry. Linear calibration curves were obtained in the concentration range of 5.021-1008.315 ng/ml. The inter- and intra-day accuracy values were below 15% at all quality control levels. Percent recoveries for rifampicin at high, middle and low quality control samples was obtained 55.15, 48.65 and 49.62%, respectively and for internal standard was 60.22%. Rifampicin was found stable through all validation parameters. Developed method was found to be simple, precise, accurate and rapid for estimation of rifampicin in plasma. Thus, the method can be employed for routine pharmacokinetic and bioequivalence studies.

Simultaneous Determination of Isoniazid, Pyrazinamide, Rifampicin and Acetylisoniazid in Human Plasma by High-Performance Liquid Chromatography

A sensitive and accurate high-performance liquid-chromatography method was developed and validated for the simultaneous determination of pyrazinamide (PZA), isoniazid (INH), rifampicin (RFP) and acetylisoniazid (AcINH) in human plasma. Separation was performed on a Max-RP C(12) column using gradient elution and a flow-rate program. The mobile phase was methanol-acetonitrile-buffer (20 mM of heptanesulfonic acid sodium, pH 2.5) with a ratio of 10:8:82 (v/v/v) at the initial phase. All calibration curves had good linearity (r(2) > 0.99) between the test ranges. The intra- and inter-day precision was less than 8.8% in good accuracy (<15%). The limit of detection with a signal-to-noise (S/N) of 3 was 0.014, 0.009, 0.023 and 0.054 µg mL(-1) for PZA, AcINH, INH and RFP, respectively. The method was selective, sensitive and reliable, and is a good alternative for routine therapeutic drug monitoring of the four compounds during the treatment of tuberculosis patients.

Development of a single‐tube, low‐cost, analytical process to extract, separate and determine efavirenz and rifampicin plasma concentrations in HIV/TB co‐infected patients

7‐11 November 2010, Tenth International Congress on Drug Therapy in HIV Infection, Glasgow, UK

Influence of Moringa oleifera on pharmacokinetic disposition of rifampicin using HPLC‐PDA method: a pre‐clinical study

The influence of active fraction isolated from pods of an indigenous plant, Moringa oleifera (MoAF) was studied on the pharmacokinetic profile of the orally administered frontline anti-tuberculosis drug rifampicin (20 mg/kg b.w.) in Swiss albino mice. The antibiotic rifampicin alone and in combination with MoAF (0.1 mg/kg b.w.) was administered orally and heparanized blood samples were collected from the orbital plexus of mice for plasma separation at 0, 1, 2, 3, 4 and 5 h, post treatment. Plasma rifampicin concentration, pharmacokinetic parameters and drug metabolizing enzyme (cytochrome P-450) activity were determined. The pharmacokinetic data revealed that MoAF-treated animals had significantly increased rifampicin plasma concentration, C(max), K(el), t(½(a)), t(½(el)), K(a) and AUC as well as inhibited rifampicin-induced cytochrome P-450 activity. In conclusion, the result of this study suggested that the bioavailability-enhancing property of MoAF may help to lower the dosage level and shorten the treatment course of rifampicin.

Simultaneous determination of isoniazid, rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry

A rapid and selective high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250 mm × 4.6 mm, 5.0 μm, USA) at 25 °C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0 ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11–5.42 μg/g in tissues and 0.18–9.04 μg/ml in plasma, for RFP were 0.12–1200 μg/g in tissues and 4.0–200 μg/ml in plasma, and for LVX were 0.13–26.2 μg/g in tissues and 0.09–4.53 μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13 μg/g and for those in mouse plasma were 18.1, 20.0 and 21.8 ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05 μg/g and for those in mouse plasma were 5.5, 6.0 and 6.6 ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.

Development and validation of a simultaneous extraction procedure for HPLC-MS quantification of daptomycin, amikacin, gentamicin, and rifampicin in human plasma

A simultaneous extraction method to measure daptomycin, amikacin, gentamicin, and rifampicin in human plasma, by high-performance liquid chromatography, was developed and validated. The method involved a rapid sample preparation by protein precipitation with acetonitrile followed by direct injection into a high-performance liquid chromatography system coupled with mass detection. Drug retention times were 10.00 +/- 0.25, 2.00 +/- 0.25, 3.50 +/- 0.25, 11.50 +/- 0.25, and 12.50 +/- 0.25 min for daptomycin, amikacin, gentamicin, rifampicin, and quinoxaline, respectively. Good linearity (mean r(2) = 0.998) was obtained for all drugs quantified over the range of clinically relevant concentrations in human plasma and the use of the internal standard quinoxaline improves accuracy (RSD% <14.9%) and intra-day (RSD% <11.56) and inter-day (RSD% <12.10) precision for the analytical procedure. The limits of quantification for daptomycin, amikacin, gentamicin, and rifampicin were 1.56, 2.34, 0.63, 0.63 microg/ml, respectively. Moreover, the addition of ion pair trifluoroacetic acid in the sample allowed the majority of gentamicin and amikacin separation. A rapid, specific, sensitive, accurate, and reproducible HPLC method was developed and validated to measure daptomycin, amikacin, gentamicin, and rifampicin in human plasma. This method is suitable for clinical pharmacokinetic studies.

Simultaneous quantification of daptomycin and rifampicin in plasma by ultra performance liquid chromatography: Application to a pharmacokinetic study

A rapid and simple method based on ultra performance liquid chromatography (UPLC) with ultra violet detection has been developed for the determination of daptomycin (DPT) and rifampicin (RFM) in rabbit plasma using 4-nitrophenol as internal standard (IS). Sample preparation involved protein precipitation with an acetonitrile:methanol mixture and centrifugation. Chromatographic separation was achieved on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with methanol and 0.1% aqueous TFA. The total analysis time was 4.5 min with DPT and RFM eluting at 1.9 and 2.1 min, respectively. The method was fully validated with a lower limit of quantitation (LLOQ) of 2 μg mL −1 for both DPT and RFM. The intra- and inter-day precision, measured as % relative standard deviation, were less than 12.1 for DPT and 10.7 for RFM, respectively. This validated method was successfully applied to a pharmacokinetic study involving intravenous administration of 14 mg kg −1 DPT and 30 mg kg −1 RFM to rabbits.