Halofantrine Enantiomers Research Articles

Effect of serum lipoproteins on stereoselective halofantrine metabolism by rat hepatocytes

Experimental hyperlipidemia has shown to decrease cytochrome P450 3A4 and 2C11 expression and to increase liver concentrations and the plasma protein binding of halofantrine (HF) enantiomers. The present study examined the effect of hyperlipidemic (HL) serum on the metabolism of HF enantiomers by primary rat hepatocytes. Hepatocytes from normolipidemic (NL) and HL (poloxamer 407 treated) rats were incubated with rac-HF in cell media with or without additional rat serum (5%). In those incubations with rat serum, the hepatocytes were preincubated or coincubated with serum from NL or HL rats. Rat serum-free hepatocyte incubations served as controls. Stereospecific assays were used to measure HF and desbutylhalofantrine (its major metabolite) enantiomer concentrations in whole well contents (cells + media). Concentrations of desbutylhalofantrine were not measurable. The disappearance (apparent metabolism) of (-)-HF exceeded that of antipode, but HF metabolism did not differ between hepatocytes from NL and HL rats. Coincubation of HL rat serum with NL hepatocytes caused a significant decrease in the disappearance of (-)-HF, whereas in HL hepatocytes, a substantially decreased apparent metabolism was noted for both enantiomers. Compared with NL serum, (-)-HF disappearance was significantly lowered upon preincubation of NL hepatocytes with HL serum. A combination of factors including diminished drug metabolizing or lipoprotein receptor expression, and increased plasma protein binding in the wells, may have contributed to a decrease in apparent metabolism of the HF enantiomers in the presence of lipoproteins from HL rat serum.

Effect of experimental hyperlipidaemia on the electrocardiographic effects of repeated doses of halofantrine in rats

Halofantrine can cause a prolongation of the cardiac QT interval, leading to serious ventricular arrhythmias. Hyperlipidaemia elevates plasma concentration of halofantrine and may influence its tissue uptake. The present study examined the effect of experimental hyperlipidaemia on QT interval prolongation induced by halofantrine in rats. Normolipidaemic and hyperlipidaemic rats (induced with poloxamer 407) were given 4 doses of halofantrine (i.v., 4-40 mg·kg(-1)·d(-1)) or vehicle every 12 h. Under brief anaesthesia, ECGs were recorded before administration of the vehicle or drug and 12 h after the first and last doses. Blood samples were taken at the same time after the first and last dose of halofantrine. Hearts were also collected 12 h after the last dose. Plasma and heart samples were assayed for drug and desbutylhalofantrine using a stereospecific method. In the vehicle group, hyperlipidaemia by itself did not affect the ECG. Compared to baseline, QT intervals were significantly higher in both normolipidaemic and hyperlipidaemic rats after halofantrine. In hyperlipidaemic rats, plasma but not heart concentrations of the halofantrine enantiomers were significantly higher compared to those in normolipidaemic rats. Despite the lack of difference in the concentrations of halofantrine in heart, QT intervals were significantly higher in hyperlipidaemic compared to those in normolipidaemic rats. The unbound fraction of halofantrine appeared to be the controlling factor for drug uptake by the heart. Our data suggested a greater vulnerability to halofantrine-induced QT interval prolongation in the hyperlipidaemic state.

Enhanced oral absorption of halofantrine enantiomers after encapsulation in a proliposomal formulation.

In this study, we evaluated the ability of a coated, encapsulated formulation to increase the oral bioavailability of (+/-)-halofantrine (HF) enantiomers, a drug with low and erratic oral bioavailability. After encapsulation of HF in distearoylphosphatidylcholine, the dried particles were coated with cellulose acetate phthalate. A suspension of the product was made using methylcellulose as a dispersion agent, and the product was administered to Sprague-Dawley rats to provide a HF dose of 7 mg kg-1 as the HCl salt. HF HCl powder in 1% methylcellulose with or without liposomal product excipients was also administered to separate groups of rats, which served as control groups. Serial blood samples were obtained from the rats and plasma was assayed by stereospecific high-performance liquid chromatography. There were no significant differences in the area under the concentration-time curve (AUC) or maximum concentration (Cmax) between the two control groups. Plasma concentrations of both HF enantiomers were significantly higher in the rats given HF as an encapsulated proliposomal formulation compared with the control groups. Compared with methylcellulose control, the encapsulation product resulted in increases of 41 to 47% in the AUC of HF enantiomers, and 90 to 100% in Cmax. The ability of an encapsulated proliposomal product to significantly increase the oral absorption of HF was clearly demonstrated.

The effect of experimental hyperlipidemia on the stereoselective tissue distribution, lipoprotein association and microsomal metabolism of (±)-halofantrine

The effect of hyperlipidemia on the biodistribution of (+/-)-halofantrine (HF) was studied in rats. Plasma, adipose, and highly perfused tissues heart, lung, liver, kidney, spleen and brain were harvested for up to 48 h after dosing animals with 2 mg/kg (+/-)-HF intravenously by tail vein. Stereospecific HPLC was used to measure HF and desbutyl-HF (DHF) enantiomer concentrations. Plasma concentrations of both HF enantiomers in hyperlipidemic (HL) exceeded those in normolipidemic (NL) rats by 11- to 15-fold. Significant increases in AUC of both HF enantiomers were noted in HL spleen tissue whereas decreases were seen in HL lung and fat. In rest of the tissues either decreases or no changes were noted in HL. The concentrations of DHF were very low in NL and HL plasma but were much higher in all highly perfused tissues. Both HF and DHF enantiomers shifted from lipoprotein deficient fraction to triglyceride-rich fractions in HL plasma following in vitro incubation of the respective racemic compounds. Compared to NL, no significant differences were noted in HF metabolism to DHF in HL liver microsomes. It would appear that both reduced plasma unbound fraction and lipoprotein associated directed uptake of lipoprotein-bound drug by tissues play roles in enantiomer biodistribution.

Effect of bile and lipids on the stereoselective metabolism of halofantrine by rat everted‐intestinal sacs

The everted rat intestinal-sac model was utilized to assess the effect of post-prandial conditions on the stereoselective intestinal metabolism of halofantrine to its active metabolite desbutylhalofantrine. Everted intestinal sacs were incubated with (+/-)-halofantrine HCl in the presence of simulated bile solution (containing lecithin, lipase and cholesterol) and lipids to mimic post-prandial conditions in the small intestine. The halofantrine enantiomer concentrations in intestinal sacs were relatively constant in the presence of bile, but decreased significantly on addition of lipids to the incubation media. Formation of desbutylhalofantrine enantiomers was inversely proportional to bile concentration whereas addition of lipids in the presence of bile caused a significant decrease in desbutylhalofantrine:halofantrine ratio of (-) enantiomers. Pre-treatment of rats with peanut oil had no significant effect on desbutylhalofantrine formation in the incubated sacs or microsomal preparations, nor did it affect the expression of intestinal cytochrome P450. Addition of extra cholesterol to the bile incubations caused a significant increase in tissue halofantrine and desbutylhalofantrine concentrations, which as for lower cholesterol, were diminished on addition of other lipids. The results were consistent with previous in vivo evaluations showing that the desbutylhalofantrine to halofantrine ratio was decreased by the ingestion of a high fat meal.

Stereoselective halofantrine and desbutylhalofantrine disposition in the rat: cardiac and plasma concentrations and plasma protein binding.

Halofantrine (HF) is a chiral antimalarial drug known to cause cardiac arrhythmias in susceptible patients. In this study, the cardiac uptake and plasma protein binding of HF and desbutylhalofantrine (DHF) enantiomers were examined in the rat. Rats were given 2 mg/kg of either HF HCl or DHF HCl intravenously, then sacrificed at various times after dosing. Specimens were assayed using stereospecific methods. Uptake of HF and DHF enantiomers into heart was rapid. Substantial concentrations of both HF and DHF enantiomers were observed in rat heart, with stereoselectivity being noted for both in plasma and heart. Stereoselectivity was more pronounced for HF (AUC (+):(-) ratio= 1.58) than DHF (AUC (+):(-) ratio =1.16) in heart tissue. Heart:plasma AUC ratios of 6.8-8.0, and 9.3-21, were observed for HF and DHF enantiomers, respectively, indicating that DHF has greater cardiac uptake than HF itself. Plasma protein binding was extensive for both HF and DHF (>99.95%), and was stereoselective for DHF, with a 38% higher unbound fraction for (-)-DHF than antipode. In contrast, binding of HF enantiomers was non-stereoselective. The lower degree of stereoselectivity for DHF in heart tissues was attributable to its greater stereoselectivity in plasma protein binding.

The stereoselective distribution of halofantrine enantiomers within human, dog, and rat plasma lipoproteins.

To study the in vitro distribution of the enantiomers of the antimalarial drug halofantrine in human, dog and rat plasma lipoprotein-fractions. Plasma was spiked with racemic halofantrine (1,000 ng/ml) and incubated for 1 h at 37 degree C. The fractions (high and low density lipoproteins, triglyceride-rich lipoproteins and lipoprotein deficient plasma) were separated using density gradient ultracentrifugation. Fractions were assayed for halofantrine enantiomer using stereospecific high performance liquid chromatography. The (-) enantiomer of halofantrine displayed higher affinity for the lipoprotein-deficient fraction than the (+) enantiomer in all three species. The (+) enantiomer was predominately located in the lipoprotein rich fractions of dog and human plasma (the (+):(-) ratio ranging from 1.2-9.6). In contrast, the (+):(-) ratio was consistently < 1 in lipoprotein-deficient fractions. Dog displayed a large magnitude of stereoselectivity in halofantrine distribution to the plasma fractions tested. There were substantial interspecies differences in the pattern of distribution of halofantrine enantiomers within the different fractions. A significant positive relationship was observed between halofantrine uptake into lipoprotein-rich fractions and the percent of apolar core lipid in those fractions. There was also a strong negative correlation between total protein concentration and the enantiomeric ratio in the lipoprotein-deficient plasma fraction. Distribution of halofantrine enantiomer to plasma lipoprotein-fractions is stereoselective and species specific. This differential binding of halofantrine enantiomers to lipoproteins may need to be considered in viewing pharmacokinetic and pharmacodynamic data involving the drug.

Pharmacokinetics of halofantrine in the rat: stereoselectivity and interspecies comparisons.

The antimalarial drug, halofantrine, is chiral and is administered clinically as the racemate. In order to define the pharmacokinetic properties of halofantrine enantiomers in the rat, male Sprague-Dawley rats (264-311 g) were given halofantrine HCl orally (n = 5; 14 mg/kg) or intravenously (i.v.) (n = 5; 2 mg/kg). Plasma samples were collected over a 72 h period, and these were assayed for halofantrine enantiomer concentrations using a stereospecific reverse phase HPLC assay. After dosing by both routes of administration the (+) enantiomer was found to have significantly higher AUC, and higher Cmax after oral dosing. Pharmacokinetic analysis indicated that in the rat, the (+) enantiomer is cleared slower, and is less extensively distributed than its antipode. The bioavailability of the enantiomers after oral administration was less than 27%. Urinary excretion was a negligible route of elimination of unchanged drug. Using allometry, the pharmacokinetics of (+/-)-halofantrine in rats scaled nicely with literature data from dogs and humans. The pharmacokinetic properties of halofantrine enantiomers in the rat resembled those seen in humans, indicating that the rat is a good model for the study of halofantrine pharmacokinetics.

In vitro effects of racemates, separate enantiomers and major metabolites of mefloquine and halofantrine on metoprolol biotransformation by rat liver microsomes

1. The effects of the anti-malarial drugs mefloquine and halofantrine and of their major metabolites on metoprolol metabolism by rat liver microsomes have been investigated. 2. The observed Km and Vmax, and the formation kinetics of alpha-hydroxymetoprolol and O-demethylmetoprolol, two major metoprolol metabolites, were in keeping with published data. 3. In vitro, mefloquine competitively inhibited metoprolol biotransformation, whereas halofantrine did so in a mixed fashion. The mefloquine Ki of metoprolol alpha-hydroxylation and O-demethylation were 3.4 and 5.8 muM respectively, whereas those of halofantrine were 0.15 and 0.32 muM respectively. 4. The main metabolites, N-debutylhalofantrine and carboxymefloquine, were 4-10- fold less inhibitory than the parent drugs. The difference in inhibitory potency of parent drugs and metabolites was higher for halofantrine than for mefloquine. The potency order for metoprolol metabolism inhibition was halofantrine mefloquine N-debutylhalofantrine carboxymefloquine. 5. A preliminary study with anti-malarial enantiomers showed a weak difference, in metoprolol metabolism inhibition between the enantiomers of halofantrine or mefloquine. 6. It is concluded that halofantrine is a potent inhibitor of metoprolol metabolism and that halofantrine metabolites or its enantiomers may have a different inhibitor potency than the parent drug: (1) the inhibition potency of these compounds should be studied in vitro and (2) their in vivo elimination half-life and plasma concentrations should be taken into be account to extrapolate this experimental results to in vivo.

Chiral chromatographic method to determine the enantiomers of halofantrine and its main chiral desbutyl metabolite in erythrocytes

We describe a direct liquid chromatographic method with spectrofluorimetric detection to quantify the two enantiomers of halofantrine and the two enantiomers of its main chiral N-monodesbutylated metabolite in erythrocyte pellets. The method involves a Chiralpak AD column and a rapid one-step extraction procedure with acetonitrile. The method was validated for the four enantiomers within the range 0–1000 ng/ml. The absence of stereoconversion was studied in samples stored frozen for up to eight months. The optical rotation of the halofantrine and metabolite enantiomers was determined after separation on a semi-preparative Chiralcel OD column with polarimetric detection.

A liquid chromatographic assay for the stereospecific quantitative analysis of halofantrine in human plasma

A stereospecific liquid chromatographic (LC) assay was developed for the quantification of the antimalarial drug, halofantrine, in human plasma. Following protein precipitation with acetonitrile, the enantiomers of halofantrine were extracted from human plasma using ammonium hydroxide and tert-butyl methyl ether-hexane. A precolumn derivatization step was employed using (+)- di-O- acetyl- l-tartaric acid anhydride to form diastereomeric derivatives of the halofantrine enantiomers. Chromatographic resolution of the diastereomers was performed using reversed-phase LC with UV detection at 254 nm. The recovery of (±)-halofantrine from human plasma at 25 and 2000 ng ml −1 was 68.2 and 61.4%, respectively. The derivatization yield following extraction and derivatization of 2000 ng ml −1 of (±)-halofantrine was 95.6%. Using 0.5 ml of plasma, the limit of quantification for each halofantrine enantiomer was 12.5 ng ml −1. Linear responses in analyte/internal standard peak height ratios were observed for analyte concentrations ranging from 12.5 to 1000 ng ml −1. Chromatograms of drug-free plasma showed no interfering peaks with retention times similar to those for (+)- (−)-halofantrine or internal standard. Based on the validation data, the assay performed well over the enantiomer concentration range of 12.5–500 ng ml −1.

Direct determination of the enantiomers of the antimalarial drug halofantrine and its active metabolite N-desbutylhalofantrine in human plasma

The plasma concentrations vs. time profiles of the enantiomers of halofantrine and its main metabolite N-desbutylhalofantrine have been determined in two healthy volunteers after a single oral administration of 500 mg of racemic halofantrine hydrochloride (Halfan). A solid-phase extraction method was used for the purification of the samples. The enantiomers of halofantrine and N-desbutylhalofantrine were determined simultaneously using chiral high-performance liquid chromatography on chiral stationary phases with fluorescence detection.

Plasma concentrations of the enantiomers of halofantrine and its main metabolite in malaria patients.

The plasma concentrations of the enantiomers of halofantrine and its N-desbutyl metabolite in six patients with malaria were measured after oral administration of 3 x 750 mg doses of micronised, racemic halofantrine hydrochloride given at 6-hour intervals. Significant differences were observed between the plasma concentrations of the enantiomers both of halofantrine and its N-monodesbutyl metabolite. AUC(0)84h values were higher for (+)halofantrine (9917 micrograms.ml-1.h) than for (-)-halofantrine (6127 micrograms.ml-1.h). The clinical significance of these observations is not known. The isomers have equipotent activity in vitro but their relative toxicity has not yet been assessed.

Direct determination of the enantiomers of halofantrine and its pharmacologically active metabolite N-desbutylhalofantrine by high-performance liquid chromatography

The enantiomers of halofantrine and its main metabolite, N-desbutylhalofantrine, were separated on two different polysaccharide-based chiral stationary phases. The stereoselective biotransformation of halofantrine was determined using rat liver homogenates. (−)-Halofantrine is metabolized preferentially.

Chiral resolution of some antimalarial agents by sub‐ and supercritical fluid chromatography on an (S)‐naphthylurea stationary phase

AbstractThe behavior of mefloquine, halofantrine, enpiroline, quinine, quinidine, chloroquine and primaquine is studied by subcritical fluid chromatography on a (S)‐naphthylurea column (250 mm × 4.6 mm ID) with a subcritical mobile phase composed of carbon dioxide, methanol and triethylamine (flow rate of 3 ml/min). Except for primaquine and chloroquine, each enantiomer was separated at a temperature between 40 and 60°C, and at a pressure below 15 MPa. A 98/2, v/v CO2/methanol 0.1% triethylamine mixture allowed the separation of halofantrine enantiomers while the enantiomers of the more polar metabolite (N‐desbutylhalofantrine) were separated with a 80–20 v/v mixture as used for mefloquine, enpiroline, quinine and quinidine. The influence of temperature, pressure and of the nature of the mobile phase is discussed. © 1993 Wiley‐Liss, Inc.

The determination of the enantiomers of halofantrine and monodesbutylhalofantrine in plasma and whole blood using sequential achiral/chiral high-performance liquid chromatography.

The determination of the enantiomers of halofantrine and monodesbutylhalofantrine in plasma and whole blood using sequential achiral/chiral high-performance liquid chromatography.