Neurotoxicity and Toxicokinetics of Artelinic Acid Following Repeated Oral Administration in Rats

Abstract

Neurotoxicity secondary to oil-soluble artemisinins has been reported in various animal species. The onset of neurotoxicity and toxicokinetics of oral artelinic acid (AL), a water-soluble artemisinin, were investigated. After dose range study, rats were dosed at either 160 mg/kg daily for 9 consecutive days or at 288 mg/kg once every other day for five doses, so that the total dose (1440 mg/kg) and duration (9 days) were identical. Neuronal damage of varying severity was identified beginning as early as 1 day after completing dosing and continued for up to 10 days post dosing. Neuronal injury was most severe 7 days after the last treatment in each of the two dosing regimens. The rats dosed with 160 mg/kg of AL daily showed moderate neurotoxicity and lost 22% of their body weight during treatment. Compared with the first dose, the toxicokinetic profile of this regimen changed significantly, with the elimination half-life increasing 3.82-fold and the volume of distribution increasing 5.23-fold on the last day of dosing. In the animals treated with AL at 288 mg/kg every other day for 5 doses, minimal neuronal degeneration (severity score 1.17) was identified and the body weight was only 8% loss. Furthermore, there were no obvious differences in the pharniacokinetic parameters between first and last dosing days with this regimen. Additionally, a progressively drug retention in stomach and drug accretion in blood were only found in rats treated with 160 mg/kg daily for 9 days. These results imply that delayed gastric emptying resulted in AL accumulation in blood and prolonged a neurotoxic exposure time (186 h) in 160 mg/kg rats when compared to that (75 h) in 288 mg/kg animals. Therefore, the drug exposure time is a key factor in the neurotoxicity induced by AL.

Various compounds of the artemisinin family are currently used for the treatment of malaria worldwide and these drugs include artemisinin (QHS), dihydroartemisinin (DHA), artemether (AM), arteether (AE), and artesunate (AS) (Meshnick 2002), as well as developed in preclinical stage such as artelinic acid (AL, Lin et al. 1987) (Figure 1). It is widely known that artemisinin drugs are fast-acting and are effective when given parenterally, orally, or by rectal suppository (Hien 1994; Karunajeewa et al. 2003). No serious adverse effects have yet been reported in human patients. However, potential neurotoxicity of the artemisinin compounds remains a concern for scientists and administrative authorities (Brewer et al. 1994b).

Neurotoxicity of artemisinin derivatives was first discovered by Brewer et al. (1994b) and has been clearly demonstrated in rodents, dogs and monkeys (Brewer et al. 1994a; Genovese et al. 1998; Kamchonwongpaisan et al. 1997; Nontprasert et al. 1998; Petras et al. 1997). An intramuscular (IM) injection of AE at 12.5 mg/kg/day for 7 days was shown to cause significant damage to rat brainstem nuclei (Genovese et al. 1998). An IM injection of AM at a dose of 40 mg/kg for 8 days induced consistent neuronal damage in dogs (Classen et al. 1999). Other studies have demonstrated that both 50 mg/kg/day of AE given IM for 5 days and 8 mg/kg/day given for 14 days cause brain stem pathology in rats and rhesus monkeys, respectively (Kamchonwongpaisan et al. 1997; Petras et al. 1997).

The substantial neuropathology induced by AM and AE in animal models was predominantly distributed to the caudal brain stem, especially the nucleus trapezoideus and superior olive nucleus (Brewer et al. 1994a; Genovese et al. 1998). Although the mechanism of neurotoxicity of the artemisinin derivatives is not fully understood, it appears to be related to its mechanism of antimalarial action. Cleavage of the endoperoxide bridge portion of the sesquiterpene lactone generates free-radical intermediates, which cause oxidative damage to neuronal cells (Gachot, Eliaszewicz, and Dupont 1997; Meshnick et al. 1993; Meshnick, Taylor, and Kamchonwongpaisan 1996).

Although studies on neurotoxicity of AM and AE are extensive, studies on the neurotoxicity of water-soluble artemisinin derivatives are limited and controversial. It has been demonstrated that AS, a water-soluble artemisinin, has the highest conversion rate to DHA (Li et al. 1998a). Further, DHA was shown to be the most toxic of the class to incubated neuronal cells (McLean and Ward 1998; Wesche et al. 1994). However, a study by Nontprasert et al. did not reveal any pathologic evidence of neuronal death in mice receiving either oral or intramuscular AS in doses of up to 300 mg/kg/day for 28 days (Nontparasert et al. 2002). Some authors believe that the capacity of different artemisinins to cause neurotoxicity might be due to their individual drug exposure times rather than the individual drug exposure levels (Li et al. 2002).

The present study was designed to investigate the extent to which the toxicokinetic (TK) factors of artemisinins are involved in the neurotoxic effects. Instead of investigating an oil-soluble artemisinin such as AE or AM, we chose to investigate a water-soluble artemisinin derivative, AL, which was reported not to cause any neuronal damage in all animals following single or multiple intravenous injection at a maximal tolerant dose of 40 mg/kg (Li et al. 2007). To best elucidate AL’s neurotoxicity and TK profiles, we designed two oral dosing regimens to replace intravenous administration in according to our dose range studies, which showed that daily oral dose at 160 mg/kg for 7 days gave rise to a mild or moderate neurotoxicity in rats (our unpublished data). Therefore, in one regimen we administered AL at 160 mg/kg daily for 9 successive days (160 mg/kg × 9) and in the second we administered AL at 288 mg/kg once every other day for 5 doses (288 mg/kg × 5). Both regimens had the same total dose (1440 mg/kg) and treatment duration (9 days).

MATERIALS AND METHODS

Chemicals

The AL used was a GMP product (MFG’s Code: NJ33-55-1) with 99% purity produced and checked by Stocks Company of New York. DHA was obtained from the Walter Reed Army Institute of Research (WRAIR) and was used as an external standard for high-performance liquid chromatography (HPLC) assay. Artemisinin was used for internal calibration in the quality control process for the HPLC assay. Carboxymethylcellulose (CMC), Tween 80, and sodium hydroxide were purchased from Sigma Chemical (St. Louis, MO). Methanol, n-butyl chloride, acetonitrile, and ethyl acetate were obtained from Burdick and Jackson (Muskegee, MI). All chemicals purchased were of analytic grade or the highest grade available.

Animals

Ten-week-old Sprague-Dawley (SD) male rats were purchased from Charles River Laboratories (Raleigh, NC). All animals were individually housed and maintained in a stable environment at 21°C with 50% to 60% humidity and 12-h day/night cycles. Standard rodent feed (Text Diet 5800; TestDiet, Richmond, IN) was provided during the day cycle from 8:00 AM to 4.00 PM and deprived at all other times. Tap water was provided ad libitum. All rats were randomly assigned to a treatment group. Research was conducted in compliance with the Animal Welfare Act, as well as other federal statutes and regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council Publication, 1996 edition).

Drug Administration and Toxicokinetic Study

Drug suspension was prepared as AL in 1% CMC and 0.2% Tween 80 at a concentration of 80 mg/ml daily. Based on results of the pilot tests in the dose range and toxicokinetic studies, a total AL dose of 1440 mg/kg was selected as the treatment in the definitive phase of the study and as the comparator dose of the two regimens. Adult male rats were administered AL by oral gavage to evaluate neurotoxicity, absorption from the gastrointestinal tract and TK parameters based on daily body weight. There were two regimens in the neurotoxicity and toxicokinetic studies. Regimen 1 was 160 mg/kg per day for 9 days (daily treatment) and regimen 2 was 288 mg/kg given once every other day for five doses (alternate day treatment). The total dose of AL was 1440 mg/kg for both regimens. The vehicle (1% CMC and 0.2% Tween 80) was administered to the control animals once daily for 9 days. The body weight and food and water consumption were monitored every day including clinical observation days after last dosing.

In groups of the neurotoxicity and TK studies, blood samples for each time point were collected from each rats treated with the two regimens of oral AL. On treatment days 3, 5, 7, and 9, selected rats from both regimens their stomachs had been also removed at either 8 or 24 h post dosing. The stomach contents were emptied into plastic vials and weighed. A portion of the stomach contents then underwent drug extraction.

Both TK blood and stomach content samples were analyzed by HPLC-ECD (electrochemical detection) assay. At predetermined time points after dosing, rats were anesthetized with isofluorane at 5% and maintained at 2% for sample collection. Blood sample (7 ml) from each rat for each time point was drawn from the inferior vena cava and then the plasma was separated by centrifuge. On the first and last day of dosing, blood samples were collected at 10 time points for PK analysis to establish the timing of the absorption, distribution, and elimination phases. Trough levels were determined by drawing a blood sample immediately prior to dosing on all the other days. Thus, a total of 24 samples were collected (at 0, 10, 30, and 60 min, and 1.5, 2, 3. 5. 8. 24. 48, 72, 96, 120, 144, 168. 192. 192.5, 193, 195, 197, 200. 216. and 240 h) in tubes with dry heparin. All plasma samples were stored at −20°C prior to processing. Sample extraction was performed as previously described (Li et al. 1998b).

HPLC-ECD Assay

HPLC with reductive BCD was performed for determination of both AL and DHA. Artemisinin was used as an internal standard. A Waters μBonda-pack C-18 column (30 cm × 4.6 mm i.d.; Waters Associates, Milford, MA) was used with 30:70 acetonitrile-acetic acid (0.1 M, pH 3.75) as the mobile phase. All runs were isocratic with a flow rate of 1.5 ml/min. Data were acquired and analyzed using a Waters 820 chromatography data system. Recovery rates varied between 81% and 89% for AL and DHA. The parent compound and DHA were eluted within 20 min. The limits of the assay were 5 and 10 ng/ml for DHA and AL, respectively. The intra- and interday coefficients of variation were within ±10% in accuracy and precision on all measurements.

Neurohistopathology

On preselected days, rats were deeply anesthetized with intraperitoneal injections of sodium pentobarbital. The pericardium and heart were then exposed by thoracotomy. After incising the pericardium, an 18-gauge cannula was used to obtain a transcardiac perfusate by going through the left ventricle and aorta. This perforates was then chilled using heparinized saline as a clearing solution. The right atrium was then pierced to permit exsanguination and removal of any remaining perfusion fluids. After vascular clearing, perfusate was fixed with chilled Bouin’s solution for 5 to 10 min. Next, the head and cranium were carefully removed to avoid applying pressure on the brain. To avoid the development of neuronal hyperchromatosis, the brain was left in place for several hours before its removal from the skull. Next, the whole brain was removed and immersed in fresh Bouin’s fixative overnight. The brains were then blocked transversely at the caudal aspect of the pons for macroscopic evaluation.

The brains were then immersed in 70% ethanol, which was changed daily to remove any excess picric acid. The brains were dehydrated using ascending grades of ethanol and then embedded in paraffin. Using a rotary microtome, 5-micron sections were cut. Serial sections were acquired and stained with a standard hemotoxylin and eosin stain. Cresyl violet was used to confirm neuronal chromatolysis in selected cases. Serial sections through the brain were examined blindly to pathologist and for microscopic evidence of cellular pathology in various target nuclei (nucleus trapezoideus. nucleus superior olive, nucleus ruber, and nucleus nervi ascialis). Cell injury (chromatolysis) and death (necrosis) were assessed using a standardized scoring system in which the scores were linked to the severity of pathology observed. A score was assigned to each individual case based on examination of a slide set of four different sections. Scores were assigned to slides according to the following schedule: 0 (normal or no neurons affected), 1(1–2 neurons affected/target nucleus), 2 (3–4 neurons affected/target nucleus), 3 (5–10 neurons affected/target nucleus), 4 (11–20 neurons affected/target nucleus), and 5 (≥21 neurons affected/target nucleus). Two bilateral target nuclei were evaluated per brain stem section. Av scores were determined using all nuclei evaluated.

Statistical Analysis

For TK analysis, the concentration-time data of AL collected during first day and last day were fitted to a two-compartment open model using a nonlinear, extended least-square fitting procedure (WinNonlin 5.0; Scientific Consulting, Apex, NC). The area under the curve (AUC) was determined by the linear trapezoidal rule with extrapolation to infinity based on the concentration of the last time point divided by the terminal rate constant. Extrapolations to time 0 were done using zero concentration for intragastric dosing and using C₀ values determined from the two-compartment model equation at time 0 by intravenous (IV) route. Mean clearance rate (CL) was determined by dividing the dose by the AUQ_inf for oral administration. Mean residence time (MRT) was determined by dividing the area under the first moment curve (AUMC) by AUC.

Body weights were analyzed using analysis of variance (ANOVA) and student’s t test. The bioavailability of oral AL was calculated as AUC_oral× Dose _IV /AUC _IV × Dose_oral using the AUC _IV and Dose _IV data as listed in our previous AL pharmacokinetic study (Li et al. 2005). Severity scores were assigned after assessment of the severity of the injured neurons using the above scoring system. Differences between the dosed and control groups in the severity of damaged neurons were assessed using a homoscedastic student’s t test (TTEST function, Excel; Microsoft, Redmond, WA). Statistical significance was designated as p ≤ .05.

RESULTS

In this study, we found that animals treated with repeated, high-dose oral AL developed neurotoxicity due to drug detention in stomach, which prolonged the drug exposure time and absorption of drug in intestines, leading to drug accumulation in blood. The prolonged exposure time may be related to the moderate neurotoxicity in the present study. This consequence is very similar with intramuscular AE or AM detention in the injection sites (Li, Brewer, and Peggins 1998). The details of this evaluation are described as follows.

Neuronal Damage Identified with Microscopy

Serial section sets through the brain were examined microscopically for evidence of cellular pathology in the target nuclei. Cell injury (chromatolysis) and death (necrosis) were assessed using a standardized scoring system (see Neurohistopathology). Each case was based on a slide set consisting of four slides from different sections. Each brain stem section was evaluated for two bilateral target nuclei. An average score was computed based on all the nuclei examined.

On day after finishing 9 days of daily oral AL treatment at 160 mg/kg, two of four rats exhibited minimal (severity score of 0.5) brain damage, which consisted of an average of 0.75 injured neurons in the superior olive complex (SOC) affected and multifocal neuronal chromatolysis in the brain stem. Mild neuronal damage with a severity score of 1.75 was noted 3 days after completing treatment, with an average of 3.0 neurons affected in half of animals. On the 7th day after the last dose of AL, moderate to marked damage, defined as a severity score of 3.25, was noted. Damage was widespread, with an average of 9.25 neurons affected by multifocal chromatolysis or necrosis in the SOC and trapezoid neurons in the brain stems of the rats in this dosing group. The histopathologic lesions were characterized by scattered chromatolysis, swelling of perikaryon, increased cytoplasmic eosinophilia, nuclear eccentricity, occasional shrunken, angular nuclei, and dissolution of the Nissl substance. Additionally, two rats in this group had moderate to marked neuronal changes including rare neuronophagia within the vestibular nuclei and reticular formation. Ten days after completing the 9-day dosing regimen, neuronal damage was found to be less severe, with an average of 3.5 chromatolytic neurons affected in the SOC and minimal neuronal necrosis in the brain stem (severity score 1.63) in four of six rats from this group (Table 1).

Rats treated with 288 mg/kg of AL every other day for five doses did not have abnormalities in histopathology 1 day after completing the regimen. Three days after dosing, some minimal damage (severity score 0.75) was noted, with an average of 1.5 neurons, showing evidence of chromatolysis in half the animals. Seven days after treatment, the severity of neurotoxicity in the alternative day dosing group was much lower (severity score 1.17) than that in the rats treated daily (severity score 3.25). These results show that neurons from rats in the 288 mg/kg every other day dosing regimen had significantly less severe damage than those from the 160 mg/kg daily dosing group in all severity assessments done. See Table 1 for data comparison of the two regimens.

Body Weight Changes Following AL Treatment in Rats

As illustrated in Figure 2, rats treated with vehicle only had a gradual increase in body weight during the experiment. Compared with the control animals, animals treated with either dosing regimen of oral AL weighed significantly less. However, when end-of-study weights were compared to initial body weights, rats in the daily AL dosing regimen (160 mg/kg × 9) had a 21.9% reduction in mean weight. This is significantly more severe (p = .0051) than the 8.1% loss in mean weight of the rats in treated with another regimen at 288 mg/kg in every other day for five doses. In addition, the animals dosed with 288 mg/kg of AL appeared to stop losing weight at about day 7 and to recover back the lost weight, but that did not happen to the rats treated with 160 mg/kg of daily AL (Figure 2). This suggests that the animals treated with 160 mg/kg daily suffered significantly more severe toxicity (p =.0051) than the rats treated with 288 mg/kg every other day, even though the same total dose was reached.

Toxicokinetics

AL was administered to rats orally at 160 mg/kg daily for nine doses. AL’s TK profile was determined by measuring drug levels by HPLC-ECD. Next, the computer-fitted plasma concentration-time curves following multiple oral administrations of AL were plotted (Figure 3. top graph). Evaluation of individual AL plasma concentration-time curves showed a biphasic pattern of drug disposition, so that a two-compartment open TK model was used to fit the data on day 1. The same biphasic pattern of disposition was obtained on the last dosing day, so that a two-compartment open TK model was also used to fit the data. The TK parameter estimates from days 1 and 9 are summarized in Table 2. Comparison of the day 1 and day 9 results revealed that the TK parameters were very different. A significantly longer (3.82-fold) elimination half-life time was noted on day 9 compared to that on day 1. Similarly, the observed mean volume of distribution (V_ss) was 5.23-fold greater on day 9 than on day 1. This prolonged elimination results in drug accumulation and a longer drug exposure.

Although the peak plasma concentration (C_max) was not changed much during the 9-day dosing, the mean AUC of AL was revealed to be greater on the last dosing day (168.01 μg·h/ml) than that (128.38/μg·h/ml) on day 1. The corresponding bioavailability of oral AL was 47.67% on day 1, but increased to 62.38% on day 9. Furthermore, the conversion of AL to DHA is very poor. The ratio of DHA/AL was only 0.012 following multiple doses of oral AL in rats (Table 2). The total AUC for DHA over the 9-day treatment was only 17.26 μg·h/ml, which is all conversion value of AL to DHA in this dosing regimen.

In the other regimen, animals were treated with 288 mg/kg of AL every other day for five doses and had plasma concentrations of drug determined at various time points. Next, the computer fitted plasma concentration-time curves were plotted (Figure 3, bottom graph). The TK parameter estimates for this regimen are summarized in Table 2 for days 1 and 9. Evaluation of the results showed that the TK parameters did not significantly differ between the beginning and end of the study. Further, the bioavailabilities were similar too with 44.58% on day 1 and 52.02% on day 9. Conversion of AL to DHA is presented in Table 2. The total area under the curve for DHA (10.29/μg·h/ml) for the treatment period was compared to that of AL (1302 μg·h/ml) and the ratio was found to be 0.0098.

AL Detection in Rat Stomach Contents

The stomach contents were examined in the rats belonging to the daily 160 mg/kg dosing regimen for AL at 8 and 24 h post dosing at various time points in the study. The quantity of AL detected in the stomach was expressed as both the amount of drug per gram of stomach contents and as a percentage of a single dose (Table 3). On days 3, 5, 7, and 9, we detected 0, 46.52, 178.59, and 486.21 μg of AL per gram of stomach contents 8 h after the last dose. The increasing amount of drug remaining in stomach over the course of dosing suggests that gastric emptying is inhibited. This inhibition of gastric emptying progresses from mild inhibition on day 5 to severe inhibition on day 9. This inhibition persisted even at 24 h post dosing. Twenty-four hours post dosing on days 5, 7, and 9, we found 5.74, 25.50, and 29.11 μg of AL/g contents remained in the stomach. It is postulated that this decrease in gastrointestinal (GI) motility could result from a decrease in vagal tone from a decrease in sympathetic outflow (Hull and Maher 1990).

Neurotoxic Exposure Time

Given by the oral route, AL showed a moderate neurotoxicity in rats treated with 160 mg/kg daily for 9 days. To calculate the neurotoxic exposure time, we used the minimum detectable neurotoxic effect level (MDNEL), defined as the minimal inhibition of drug retention in the stomach on day 5, as this seems to relate to a neurotoxic effect induced by various artemisinins, including: AM, AE, and DHA (Li, Brewer, and Peggins 1998; Li et al. 2002). The corresponding plasma concentration of AL on day 5 was 346 ng/ml, which was a minimal concentration to induce the drug retention in stomach of the rats. Therefore, in our judgment, the MDNEL should be 346 ng/ml and any exposure above this level be considered a neurotoxic exposure. In this study, we calculated the neurotoxic exposure time to be 186 h for the animals dosed at 160 mg/kg daily for nine doses, which induced a moderate neurotoxicity (Table 2). In contrast, rats that were treated with AL at 288 mg/kg every other day for five doses showed minimal neuronal degeneration and had a neurotoxic exposure time of only 75 h (Figure 3).

DISCUSSION

Oil-soluble artemisinins, such as AM and AE, possess the ability to induce fatal neurotoxicity. This neurotoxicity likely results from their delayed and prolonged absorption following intramuscular injection with a sesame oil vehicle, which leads to drug accumulation. The excess accumulated drug increases the amount and length of toxic drug exposure time. The neurotoxic potential of water soluble or gastric applied artemisinin derivatives is controversial (Kissinger et al. 2000). However, in this study we found that the water-soluble artemisinin AL, with short half-life and produced limited drug exposure times, also induced the neurotoxicity following repeated, high-dose oral AL. The dosing regimens had the same total dose given, and we found moderate toxicity in the rats treated with 160 mg/kg of AL daily for nine doses and minimal neurotoxicity in animals treated with 288 mg/kg of AL every other day for five doses. The significant difference is dependent on drug exposure time, especially on the neurotoxic exposure time, as described below.

In our evaluation of the TK parameters, we found that the mean elimination half-life of AL in the rats treated with daily drug was markedly extended from 2.84 to 10.68 h and that the volume of distribution (V _ss ) was also considerably increased from 489 to 2519 ml/kg. One theory to help explain the physical changes of the pharmacokinetic parameters may relate to the gastrointestinal toxicity induced by daily oral AL. To investigate further, the amount of AL retained in the stomach have been determined at 8 and 24 h post dosing at various time points in the study. Following five doses, AL began to be retained in the stomach of rats treated with AL at 160 mg/kg daily for 9 days. Normally, food is emptied from rat stomach in 3 h or less (van der Velde, Koslowsky, and Koopmans 1999; Kaplan, Spector, and Grill 1992). Because the drug was still detectable at 8 and 24 h post dosing, that the repeated dosing of AL significantly inhibits gastric emptying has been suggested (Li, Brewer, and Peggins 1998). Although retention of drug in the stomach could account for some of the observed gastrointestinal toxicity, similar gastrointestinal toxicity has been demonstrated following intramuscular injection of the other three artemisinins (AE, AM, and DHA) in rats following a week of daily injections (Li, Brewer, and Peggins 1998).

In the previously referenced study, the gastric retention ratio, which defined as the weight of the stomach pouch contents of the treated animals divided by the weight of the stomach pouch contents of the control animals, was significantly increased for all three of the drugs tested. This increased gastric retention ratio was twofold higher in AE than in AM and 11- to 16-fold higher in DHA than in AM (Li, Brewer, and Peggins 1998). Although the mechanism responsible for this inhibition of the stomach emptying has not been definitively established for AL but demonstrated for AE, AM, and DHA (Li, Brewer, and Peggins 1998), we hypothesize that it results from a decreased sympathetic outflow from the central nervous systems (CNS), which results in a decreased vagal tone and consequent decrease in GI motility (Hull and Maher 1990). In this regard, it is known that lesions in certain nuclear regions of the brain stem, specifically the solitary nucleus, dorsal motor nucleus, and nucleus ambiguus, can have profound effects on gastric motility via increases or decreases in vagal activity (Gillis et al. 1989). Histopathological evidence of lesions in the brain stem have previously been reported in AE and AM studies (Brewer et al. 1994a, 1994b; Genovese et al. 1998) and were also seen in the present study using AL.

The drug retained in the stomach appears to be a reservoir, which delays and prolongs the absorption of AL and results in drug accumulation (high AUC) and prolongs elimination with long t_1/2. This reservoir is very similar to the depot effect seen with the oil-soluble artemisinins (AM and AE) intramuscularly in the injection sites. This accumulation increases the time that the drug levels are above a toxicity threshold and can thereby induce neurotoxicity. In this study, we attempt to correlate the neurotoxic exposure time to the observed histopathological findings to the two dosing groups. We found that the neurotoxic exposure time to be significantly extended for animals in the daily AL treatment cohort (186 h) compared to those in the every other day dosing cohort (75 h). To determine the neurotoxic exposure time for the two regimens, we calculated the time that the AL plasma level was above the minimum detectable neurotoxic effect level (MDNEL), which is defined in this study as the plasma level at the time of onset of the drug retention effect. Although the MDNEL is not a minimum detectable pathological effect level (MDPEL), which is defined as the first observation of neuronal effects, as was used in a previous study where AE was shown to produce neurotoxicity when given IM at 12.5 mg/kg daily for 7 days (Li et al. 2002). Its estimation can give us an initial estimation of neurotoxic exposure time and allow us to correlate this exposure time with observed histopathology from different AL dosing regimens. It is clear that in the present study the delayed gastric emptying resulted in AL accumulation in blood and prolonged a neurotoxic exposure time (186 h) in the 160 mg/kg × 9 rats when compared to that (75 h) in 288 mg/kg × 5 animals. Therefore, the neurotoxic exposure time is a key factor in the neurotoxicity induced by oral AL in rats.

As shown in Table 1, morphologic abnormalities of rat neurons occurred as early as the first day after finishing the AL daily regimen. These abnormalities progressively worsened through day 7 post dosing, at which time the number of abnormalities began to lessen. Only mild abnormalities were noted by the 10th day after last dosing. This suggests that AL has a “delayed” neuropathological characteristic. Similar delayed neurotoxicity was observed with other artemisinin derivatives in cultured neuronal cells (Wesche et al. 1994) and in rats (Brewer et al. 1994a; Genovese et al. 1998). An additional, yet unexplained, finding was that the neuronal pathology of the daily treated group of rats became much less severe by 10 days after treatment (Table 1). Nontprasert and his colleagues similarly reported that no gross abnormalities were identified in mice brains 120 days after completing a 28-day treatment in which all mice developed abnormal equilibrium (Nontprasert et al. 2002).

The result suggests that morphologic evidence of AL-induced neuronal degeneration and necrosis is substantially decreased at 10 days versus 7 days post dosing. The neurotoxicity, not the neuropathology, is reversible. Necrotic neurons can be removed from the brain rapidly; likewise, reversibly injured neurons can recover a normal histological appearance rapidly after the injuring stimulus is removed. It is our interpretation that some neurons that showed morphologic changes consistent with injury or death at 7 days may have been either recovered to normal morphologic appearance (those with reversible injury) or completely removed from the tissue (necrotic neurons) by day 10. Present methods allowed us to look at several snapshots in time; we did not measure cumulative neuronal injury over time. Our study did not include morphometric attempts to quantify the decrement of the total neurons in the target nuclei over time. These findings seem to suggest that artemisinin induced neurotoxicity may be reversible with a “drug free” recovery period. However, more detailed observations are needed before a conclusion can be made with regards to this finding.