Single dose pharmacokinetics study of 97/63 (IND191710, 2004), a trioxane antimalarial developed by Central Drug Research Institute, Lucknow, India, was studied in rats following intravenous and oral administration. Serum samples were analysed by HPLC-UV assay. Separation was achieved on a RP-18 column attached with a guard using acetonitrile : phosphate buffer (70 : 30% v/v) with UV detector at wavelength 244 nm. Serum samples were extracted with
World Health Organization (WHO) has recommended artemisinin class of compounds as promising novel antimalarials for further clinical development [
Central Drug Research Institute (CDRI) developed a promising antimalarial compound, 97/63 (Racemate) (Figure
Chemical structure of parent compound 97/63 (a) and IS (b).
Analytical method was developed and validated in terms of sensitivity, specificity, accuracy, precision, and recovery. The study samples were analyzed using validated HPLC-UV assay developed for quantification of 97/63 in rat serum.
Pure reference standards (purity >99%) of 97/63 and 99/411 (Figure
HPLC system consisted of a pump (LC-10ATvp with SCL 10Avp system controller, Shimadzu, Japan) with quaternary flow control valve system (FCV-10ALvp) and a degasser (DGU-14A) to pump the mobile phase. The detection of 97/63 and IS was performed using SPD-10A UV-VIS detector at 244 nm. SIL-10ADvp Shimadzu autoinjector was used to inject samples. Chromatographic separation was performed isocratically on Spheri-5, RP-18 column (100 × 4.6 mm i.d. 5 mm) (Pierce Chemicals Co., Rockford, IL, USA) coupled with a guard column (30 × 4.6 mm i.d., 5 mm) packed with the same material. The mobile phase composed of acetonitrile : phosphate buffer (10 mM, pH 4.0) (70 : 30% v/v) at flow rate 1 mL min−1. Phosphate buffer was prepared by dissolving 1.36 g of potassium dihydrogen orthophosphate in one liter triple-distilled water and its pH 4.0 was adjusted with 40% orthophosphoric acid. Phosphate buffer was filtered with 0.22
Stock solution (1 mg mL−1) of 97/63 and IS was prepared by dissolving separately 10 mg of compounds in 10 mL acetonitrile. Working stock solutions of strengths 10
Calibration standards (CS) samples of 97/63 from 10–500 ng mL−1 in rat serum were prepared by adding appropriate volumes of working stocks in 200
CS, QC, and test samples were prepared separately using a simple and efficient two-step liquid-liquid extraction process using
Method development involved use of different columns (Cyano and RP-18) to check noninterference in the region of compound of interest with endogenous substances, drug metabolite, and so forth in the determination of concentration [
Matrix effect between analytes, IS, and coeluents from biometrices might probably exist. So matrix effect was evaluated by peak area ratio of the analytes dissolved in the supernatant of processed blank serum to that of analytical standard of same concentration. Five samples of each set at QC of 97/63 were evaluated. Moreover highly sensitive methods could be affected by carryover, visible in LC-UV detection. In this study carryover was evaluated by analyzing the mobile phase after plasma samples with the highest analyte concentration 2000 ng mL−1.
Linearity for CS (
The recovery of 97/63 prior to analysis was determined at QC samples of low (10 ng mL−1), medium (100 ng mL−1), and high (500 ng mL−1) concentrations. The extraction recoveries at three QC levels were determined by comparing peak areas obtained from plasma samples with those found by direct injection of standard solutions prepared in mobile phase of same concentrations. Recovery of IS was also calculated.
Accuracy was determined by injection of calibration standards and QC standards in five replicates on five different days (
Long-term stability of 97/63 was carried by preparing QC samples at low, medium, and high concentrations in three replicates for four different days and stored at −60°C. These sets of samples were analyzed after 0, 7, 15, and 30 days of storage and their concentrations were read from the respective calibration standard curve. The results are expressed as % deviation from 0 days concentration.
F-T stability of 97/63 in serum samples was determined by preparing QC of strengths low (10 ng mL−1), medium (100 ng mL−1), and high (500 ng mL−1) in three replicates for four different days. One set of three concentrations was analyzed on day of preparation (no F-T cycle) before storing the remaining sets at −60°C. Other sets were analyzed after 1, 2, and 3 F-T cycles. Thawing was achieved by keeping the sealed tubes at room temperatures for 30 min. The results are expressed as % deviation with initial concentration.
The effect of storage on dry residue stability was determined at QC 10 ng mL−1 (low), 100 ng mL−1 (medium), and 500 ng mL−1 (high) concentration levels. All the samples (three replicates of three concentrations
After sample preparation, stability of 97/63 in the autoinjector was evaluated for 4 h; the maximum duration for which the sample may have to wait in the autoinjector tray for pending analysis and results are expressed in % deviation with initial injection (
Male Sprague-Dawley (SD) rats weighing
Young and healthy male SD rats weighing
An oral dose of 72 mg kg−1 and intravenous dose of 18 mg kg−1 as suggested by the efficacy studies were, therefore, used in the study. The intravenous formulation for single dose, 18 mg kg−1, was a solution of 97/63 in DMSO. The oral formulation for single dose, 72 mg kg−1, was also a solution of 97/63 in DMSO. The formulations were subjected to quality control (QC) checks to ensure their strength and content uniformity prior to use. The strength of formulation for intravenous and oral administration was 18 mg mL−1 and 36 mg mL−1. Compound 97/63 formulated in DMSO was administered to overnight fasted rats by oral and intravenous route. The dose volume factor for intravenous administration was 1 mL kg−1, with 250 g rat receiving 0.25 mL, and for oral administration it was 2 mL kg−1, with 250 g rat receiving 0.5 mL of formulation. Sparse sampling approach was used to collect blood samples by cardiac puncture. The total volume of blood withdrawn within 24 hours was less than 5% of blood volume. Blood samples were collected in glass tubes at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 18, 24, 48, and 60 h after oral dose. Blood samples were stored at room temperature for 30 min to separate the serum. Serum was separated by centrifugation at 1500 ×g for 10 min and stored at –60°C until analysis. Analysis was performed within 30 days of sample collection.
Curve fitting and estimation of pharmacokinetic parameters of 97/63 were carried out using WinNonlin (5.1) software program (Pharsight Corp, USA). The data were tested using Gauss-Newton algorithm, with noncompartment, one compartment, and two-compartment models after applying different weighting factor to the observed values of the concentrations. Decision on the suitability of a particular compartment model in explaining the data was undertaken based on the Akaike or Schwarz information criterion, correlation coefficient, and scatter of residuals.
Data are reported as means ± SD and statistical analysis was performed using one way analysis of variance (ANOVA) at 95% confidence interval (CI).
The successful analysis of analytes in biological fluids using HPLC relies on the optimization of sample preparation, chromatographic separation, and postcolumn detection. Each of these steps was carefully optimized for developing sensitive, selective, and reproducible assay of 97/63 in rat serum.
The chromatographic conditions were modified to get better selectivity and sensitivity. Thus, molarity and pH of buffer and type of columns were optimized. The effect of molar strength and nature of buffers in mobile phase on peak shape of 97/63 was studied [
Sample clean-up techniques were also optimized to get rid of interfering endogenous substances without sacrificing the recovery of 97/63 and IS. A proper selection of solvent was essential for yielding maximum recoveries. Extraction solvents like
Absolute recoveries of 97/63 in rat serum.
Spiked concentration |
% absolute recovery |
---|---|
10 | 78.38 ± 4.24 |
25 | 87.78 ± 4.51 |
50 | 76.51 ± 0.02 |
100 | 76.14 ± 0.33 |
250 | 74.18 ± 0.29 |
500 | 74.72 ± 0.05 |
Representative chromatograms of (a) blank serum sample, (b) calibration standard at 25 ng mL−1 concentration, and (c) test sample of 1 h after oral (72 mg kg−1) dose.
No significant suppression for 97/63 by matrices was observed. No detectable carryover occurred in mobile phase runs after plasma samples determinations.
The peak area of 97/63 in rat serum varied linearly with concentration over the range 10–500 ng mL−1. The calibration model was selected on individual calibration data by linear regression with or without intercept and weighing factor. The linear regression equation was
Accuracy and precision of 97/63 in rat serum (
Spiked Concentration |
Accuracy (% bias) | Precision (% RSD) | ||
---|---|---|---|---|
Interday | Intraday | Interday | Intraday | |
10 | −14.00 | −0.70 | 14.47 | 14.8 |
100 | 9.64 | 6.83 | 14.06 | 5.56 |
500 | 13.42 | 6.83 | 5.74 | 4.71 |
97/63 was found to be stable over a period of 30 days in normal serum when stored at −60°C (Figure
Stability studies: (a) long-term stability, (b) freeze-thaw stability, and (c) dry residual stability.
Following oral dose administration in male rats, the levels of 97/63 showed biexponential decay up to 48 h. A two-compartment 1st-order pharmacokinetic model without lag time and first-order elimination rate was considered to be the best fit to explain the generated data. Mean serum concentration-time profile of 97/63 after single oral dose of 97/63 in male rats is shown in Figure
Pharmacokinetic parameter of 97/63 after oral administration at 72 mg kg−1 and intravenous administration at 18 mg kg−1 dose of 97/63 in male rats (mean ± SD,
PK parameter | Oral | Intravenous |
---|---|---|
|
* | 1799.99 ± 330.24 |
|
229.24 ± 64.26 | * |
|
1.00 ± 0.7 | * |
|
0.84 ± 0.14 | 0.45 ± 0.08 |
|
10.61 ± 0.2 | 10.57 ± 0.16 |
|
1268.97 ± 27.04 | 2025.75 ± 574.3 |
Vd (L kg−1) | 172.31 ± 28.59 | 134.7 ± 11.17 |
Cl (L h−1 Kg−1) | 11.2 ± 0.07 | 8.89 ± 0.11 |
% bioavailability | 15.66 | — |
Log-linear scale of mean serum concentration (ng mL−1) versus time profile of 97/63 following (a) oral at 72 mg kg−1 and (b) intravenous at 18 mg kg−1 dose administration in male rats (mean ± SD,
Serum concentration-time profile of 97/63 in male rats after intravenous administration at 18 mg kg−1 dose has been depicted in Figure
The absolute bioavailability of compound 97/63 was calculated to be 15.66%. Poor bioavailability may be due to poor solubility. No abnormal clinical symptoms were noted following oral and intravenous administration of 97/63 in male rats. Rats showed normal activities same as untreated rats during sampling period.
Compound analysis by HPLC-UV method is a traditional technique for quantification and quantitation as well as easily available at affordable cost. A well developed and validated HPLC-UV method is more effective as compared to LC-MS method which is a costly affair. So various parameters responsible for development of a new assay to quantify drugs by HPLC were optimized. The chromatographic conditions such as molarity and pH of buffer and type of columns were optimized. The results suggested that the bioanalytical method is accurate, as the bias is within the acceptance limits of ±20% of the theoretical value at LLOQ and ±15% at all other concentration levels. The precision around the mean value never exceeded 15% at any of the concentrations studied. Data from quality control samples revealed that the proposed method shows adequate specificity, sensitivity, accuracy, and precision.
No significant matrix suppression and/or enhancement as well as no endogenous peak interference were observed with peaks of interest. The absolute recoveries of 97/63 over the range 10–500 ng mL−1 as well as IS from serum were more than 74%. The result showed that the analytical method was accurate and precise over the linearity concentration range.
97/63 was subjected to long-term, F-T cycle, dry residual, reinjection stability studies and the compound showed good stability. No stability problems were encountered during storage and processing of pharmacokinetic samples.
Following oral and intravenous dose administration in male rats, the levels of 97/63 could be monitored in serum from the first dose sampling point, that is, 5 min. After oral administration 97/63 was rapidly absorbed from the gastrointestinal tract, attaining maximum serum level concentration (
The low bioavailability may be due to first-pass metabolism of drug which might be excreted through the bile or variable rate of gastric emptying and motility of the gastrointestinal tract along the alimentary canal. Different degrees of drug response to the absorption site also influence the bioavailability. Metabolism in intestinal membrane during course of absorption as well as in liver is of major concern with the oral administration of many drugs, since it can reduce the systemic bioavailability of the drug. During gastrointestinal transit, the drug gets exposed to various pH conditions, gut flora, and enzymes due to which its systematic bioavailability may be reduced [
The low bioavailability of 97/63 can be improved by modifying it as produrg. Prodrug concepts at present are established tools for better fabrication of the physicochemical, biopharmaceutical, or pharmacokinetic properties of pharmacologically active agents [
The present bioanalytical assay was found to be specific, accurate, and precise over the linearity range of 10–500 ng mL−1. Liquid-liquid extraction method for sample preparation was efficient which shows extraction recovery of 97/63 from serum more than 74%. There were no stability problems for 97/63 during storage and sample processing hence fulfilling the criteria for bioanalytical methods. The relevance of pharmacokinetics in drug development has been well recognized and systematic interpretation of disposition behavior is of considerable use in reducing the expenditure and time involved in drug development besides optimization of drug therapy. Thus, a suitably validated method in different species can lead to rapid pharmacokinetic studies. This method will be applicable for further development and pharmacokinetic studies of 97/63. The observed low bioavailability (15.66%) and high variability of 97/63 can be attributed to lower log
The authors have no conflict of interests to disclose.
The authors wish to express their gratitude to Dr. Chandan Singh, Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow, for the authentic samples of 97/63. The authors are thankful to Council for Scientific and Industrial Research, New Delhi, India, for providing research fellowships and Director of CDRI for providing facilities and infrastructure for the study. This is CDRI Communication Number: 8752.