Abstract
Artesunate (AS) is being developed as a potential agent for the treatment of severe and complicated malaria. A risk assessment of the therapeutic index and related hematological changes of AS and artelinate (AL) following daily intravenous injection for 3 days was conducted in Plasmodium berghei–infected and uninfected rats. The minimum doses of AS and AL for parasitemia suppression were 2.3 and 2.5 mg/kg, respectively, and the suppressive doses for half parasitemia (SD50) were 7.4 and 8.6 mg/kg, respectively. The maximum tolerated dose (MTD) for AS was 240 mg/kg with a therapeutic index of 32.6. The MTD for AL was 80 mg/kg with a therapeutic index of 9.3. Hematological changes were studied on days 1 and 8 after the final dosing. In both AS- and AL-treated rats, dose-dependent and rapidly reversible hematological changes (significant reductions in RBC, HCT, Hb, and reticulocyte levels) were seen in the peripheral blood. Bone marrow evaluation revealed a statistically significant reduction in the myeloid/erythroid ratio only at the highest dose of AS (240 mg/kg), albeit still within the normal ratio range (1.0–1.5:1.0). Looking at the respective therapeutic indices the authors have concluded that AS is much safer than AL. Both drugs induced hematological changes in rats that parallel the dose-dependent, reversible anemia and reticulocytopenia previously reported in animals and humans. However, no significant bone marrow depression was seen for either agent.
Severe malaria is initially treated parenterally to rapidly lower the level of parasitemia to a non–life-threatening level. A rapid decrease in peripheral parasitemia will also lead to reversal of any concomitant organ dysfunction. However, because end-organ damage may have already occurred and quinoline “schizonticides” do not kill early schizonts in sequestered erythrocytes, a rapid decrease in peripheral parasitemia may not translate into complete reversal of organ damage. Comparative studies of parenteral antimalarials, in which patients were subsequently administered the same oral antimalarial irrespective of the parenteral agent initially used, the major end points of death, coma recovery time, permanent neurologic sequelae, and parasite clearance time were strongly influenced by the efficacy of the initial parenteral therapy.
When the parasitemia level has dropped to where a patient is able to tolerate oral medications, parenteral therapy is stopped and oral antimalarials are given to kill any remaining parasites (i.e., to achieve cure). Oral antimalarials typically used include quinine, mefloquine, sulphadoxine-pyrimethamine, or, more recently, oral artemisinins.
Parenteral agents available for the worldwide treatment of severe malaria are primarily quinine (intramuscular or intravenous) or quinidine (intravenous). Despite appropriate medical care and prior parenteral quinine treatment, mortality from severe malaria remains approximately 20%.
Artemisinin, extracted from ‘qinghao’ or sweet wormwood (Artemisia annua L.) which has been part of traditional Chinese herbal medicine for centuries, was rediscovered and isolated in 1972 by Chinese scientists seeking new treatments for malaria. The structure of artemisinin was determined to be a sesquiterpene lactone with an internal peroxide linkage (China Cooperative Research Group on Qinghao and Its Derivatives as Antimalarials 1982). The biochemical mechanism of antimalarial action of the artemisinins has been hypothesized to involve an iron-mediated cleavage of the endoperoxide bridge, producing oxygen radicals.
The various artemisinins in clinical use differ according to substituents at position C-10. Dihydroartemisinin (DHA; recently named artenimol, and referred to as dihydroquinhaosu [DQHS] in the older medical literature) is the most active compound in the class used clinically. DHA is poorly water soluble and is only available in a tablet formulation for oral use. Artemether (AM) and arteether/artemotil (AE), the methyl and ethyl ethers of DHA, respectively, are available in sesame oil for intramuscular injection. Artesunate (AS), the hemisuccinate ester of DHA, is water soluble and can be administered intravenously, intramuscularly, intrarectally, or orally. AM, AE, and AS have been studied for the treatment of severe malaria; DHA, not being available in a parenteral formulation, has not.
In developing a potential antimalarial for human use the potential therapeutic and adverse responses (therapeutic index) must be considered before a drug is advanced to preclinical testing. The therapeutic index among the key criteria for drug selection and the Food and Drug Administration (FDA) recommends that therapeutic index assessments (efficacy and toxicity studies) be carried out in same animal species (ICH Harmonised Tripartite Guidance for Industry, 1994) as cross-species scaling is often inaccurate.
Efficacy studies performed in China have unambiguously shown that artemisinin and its derivatives are very potent compounds against various malarias in vitro and in vivo (China Cooperative Research Group 1982; Li, Arnold, and Guo 1984). In vitro studies show the relative antimalarial potency of artemisinin compounds as 1 (artemisinin, ART) < 1.2–1.5 (AL) < 1.9–2.3 (AM) < 2.2–2.6 (AE) < 3.9–4.8 (AS) < 3.6–5.8 (DHA) (Bustos, Gay, and Diquet 1994; Shmuklarsky et al. 1993). Although the above data indicate AL to have a low antimalarial potential in vitro, other studies have shown AL to have the same order of antimalarial activity as AE or AM in vitro (Basco and Bras 1993). The efficacy of artemisinin and its derivatives have been thoroughly documented in mice and rats infected with Plasmodium berghei (Shmuklarsky et al. 1993), aotus monkeys infected with Plasmodium falciparum (Kawai, Kano, and Suzuki 1993), and rhesus monkeys infected with Plasmodium cynomolgi and Plasmodium coatneyi (Meano, Brown, and Smith 1993).
Artemisinin compounds are in widespread use for the treatment of severe and complicated malaria and AS has been the most commonly used derivative for more than fifteen years; many clinicians feel intravenous (IV) AS to be the most effective treatment for severe malaria (de Vries and Dien 1996). The effectiveness of AS has been attributed to its rapid and extensive conversion to DHA (Batty et al. 1998, 1999; Li et al. 1998a, 1998b, 2002). DHA is three- to fivefold more potent than other artemisinin derivatives (Li et al. 2002) and can completely inhibit parasite growth within 2 to 4 h. It is the only artemisinin derivative with activity against all asexual blood stage parasites (Skinner et al. 1996). AS represents the gold standard against which any new candidate artemisinin derivative, specifically an IV injection agent such as AL, should be compared.
AL, a water-soluble artemisinin derivative, is currently under preclinical development for the treatment of multidrug-resistant and severe malaria by the U.S. Army. Recently, AS and AL were found to be similarly effective.
Published data show AS dosing at 100 mg/kg daily for 5 to 6 days results in proliferation of lymphocytes, increases phagocytic activity of peritoneal macrophages, and induces splenomegaly in mice (Guojun and Yi 1983). Toxicological results in monkey indicated the presence of leukopenia and neutropenia in all animals by 8 days’ dosing. Thrombocytopenia was observed in all surviving animals by days 8 to 10 with rebound. In similar studies up to 25% of the total red blood cell (RBC) count was lost in all surviving monkeys treated with AS. Reticulocytopenia was observed in 75% of animals with rebound on day 15 (our unpublished data).
AS hemotoxicity in the rats, and to a greater degree in rhesus monkeys, parallels the effects reported in human (0.6% reticulocytopenia in 4062 patients at low doses of 2 to 3 mg/kg; Ribeiro and Olliaro 1998). In humans AS caused rapid inhibition of hematopoiesis (Cao et al. 1997) and lower peripheral reticulocyte counts by day 5 of treatment compared to the quinine group (p = .011). These reductions in reticulocyte counts and anemia have also been confirmed by other investigators (Itoda et al. 2002; Same-Ekobo et al. 1999; Yoshizawa et al. 2002). Hematological changes induced by AS administered at therapeutic doses in humans are not completely clear. The present study was proposed to investigate safety evaluation and therapeutic index of AL and AS at various dosages in the P. berghei–infected and noninfected rats.
EXPERIMENTAL DESIGN
Materials and Drugs
Artesunic acid (4-(10′-dihydro-artemisinin-oxymethyl) succinate) and artelinic acid (4-(10′-dihydro-artemisinin-oxymethyl) benzoic acid hemihydrate) were manufactured as
Lysine was chosen as the vehicle for both AS and AL for the therapeutic index study to ensure the study was being performed in the same animal species and under the same laboratory conditions. The preformulated AS/lysine or AL/lysine salt was prepared with 1:1 molecular weight of AL or AS with lysine. The salt weight was equivalent to the real weight of AS or AL for study use. The salt was dissolved in the solution containing 0.45% NaCl/0.1%
AS/NaHCO3 injection—a Chinese formulation—was selected for comparison with AL/lysine in the definitive efficacy study. AS was purchased from Thailand containing 60 mg AS powder (per vial) and 1 ml of 5% sodium bicarbonate. A stock IV solution was prepared daily by dissolving 60 mg of AS powder in 0.6 ml of 5% sodium bicarbonate and 1.04 ml 5% glucose injection or normal saline injection so that each milliliter contained 36.7 mg/ml of AS. The dosing solution was prepared fresh every day before the experiments.
Animals
The ANKA strain of P. berghei used in this study was rat-adapted from a mouse strain by three successive 4-week passages through 7-week-old rats. Parasitized blood from these animals was cryopreserved in a large batch and used to inoculate donor animals for the efficacy and toxicity experiments. Seven-week-old (young adults; body weight of 186 to 213 g) P. berghei–infected and uninfected Sprague-Dawley rats were randomly assigned into study groups of 6 or 10 animals. Animal protocol was approved by the Institutional Animal Care and Use Committee, Walter Reed Army Institute of Research, and the research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals, and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council Publication, 1996 edition.
All animals were quarantined (stabilized) for at least 7 days prior to infection. Rats were individually housed with food and water supplied ad libitum. Rats were inoculated intraperitoneally (IP) with cryopreserved P. berghei–infected rat blood (2 × 107/rat in 0.5 ml Glucose-citrate (Q.C.) solution) obtained from donor rats infected 1 week earlier with cryropreserved parasites. Two pretreatment smears were taken from all animals for parasitemia analysis. Animals with > 5% parasitemia were selected for efficacy and maximum tolerated dose (MTD) studies. Eighteen post-treatment smears were obtained from each rat at 0, 3, 5, 8, and 12 h on day 6, and at 0, 3, and 6 h on days 7 and 8. From day 9 until day 28 blood smears were obtained from each animal once daily. Infected rats were used for efficacy, toxicity, and therapeutic index studies. Only uninfected rats were used for the hemotoxicity studies.
Hematology and Blood Chemistry
At 24 and 168 h after the final dose of AS/lysine or AL/lysine, animals were anesthetized by isofluorane inhalation anesthesia (Abbott Labs, North Chicago, IL) delivered by a small-animal anesthesia system (Euthanex Corp, Palmer Park, PA). Each animal was initially placed in a chamber primed with 5% isofluorane, and upon induction, maintained in a non-rebreathing mask at 2% isofluorane delivered with 0.5 ml O2 per minute. Animals were bled from the abdominal inferior vena cava after an abdominotomy. One milliliter of the blood sample was placed in K3 EDTA tubes (Beckton Dickinson, Franklin Lakes, NJ) for complete blood count (CBC) analysis using the ABX Pentra 60 hematology analyzer (ABX diagnostics, Irvine CA). Another 2 ml of blood was placed in serum separator tubes (Becton Dickinson) for biochemistry analysis using commercial assay kits on the Vitros 250 Chemistry analyzer (Johnson & Johnson, Rochester, NY). Reticulocyte analysis was performed with light microscopy. After sample collection, rats were euthanized by cervical dislocation.
Pathological Evaluation
The half and MTD of AS and AL were selected. Negative-control groups (dosed with vehicle lysine) were compared with the test groups. All doses were repeated daily for 3 days, and the animals were sacrificed at 24 or 168 h after the final dose. Pathological evaluation was conducted on all animals following sacrifice. Tissues that were of interest in our efforts were harvested, including sternum (bone and bone marrow), thymus, lymph nodes, spleen, heart, lungs, liver, and kidneys. Harvested tissues were placed in 10% neutral-buffered formalin for proper fixation and routinely processed for production of hematoxylin and eosin stained slides for histopathological examination. For cytological bone marrow evaluation, a small amount of marrow from the femur or tibia of each rat was harvested with a small brush moistened with phosphate-buffered saline (PBS). Bone marrow smears were prepared on glass slides and stained with Diff-Quik stain (Dade Behring, Newark, DE). The smears were examined to determine the percentage of cells in the various cell lineages and maturation stages, and to observe any degenerative or reactive changes in cell populations. Cell differential counts were made by counting, in single cell layer areas of the slide at 1000× magnification, a minimum of 250 cells in six locations on each slide.
For histological bone marrow evaluation, sections of sternum from each rat were routinely processed, sectioned at 4 to 6 μ m, stained with hematoxylin and eosin, and evaluated by light microscopy. The marrow was examined to produce an estimate of erythrocytic and myelocytic series cellularity, myelocytic:erythrocytic ratio, megakaryocytic series cellularity, and for evidence of degeneration, necrosis, or reactive changes. The other harvested tissues were similarly processed, stained, and evaluated by light microscopy.
Therapeutic Index Determination
In efficacy experiments, the parasite suppression, clearance, malaria cure, parasite clearance time (PCT), duration days of clearance, and time to recrudescence were calculated as described previously (Li et al. 2003a). Parasitemia suppression was deemed to have occurred if parasitemia initially fell, but were not cleared by drug treatment. The minimum suppression dose in half parasitemia (SD50) was measured. The clearance effect of each drug was determined as the CD100, which was the minimum dose that could clear parasites in 100% of animals, without causing obvious clinical toxicity. Clearance was defined as two negative blood smears, taken 4 to 24 h apart, prior to day 12 post infection. The detection limit for a negative thin smear was defined as the failure to observe a parasite after examination of 10,000 RBCs (i.e., 0.01% parasitemia). Area under the effect curve (AUEC) was defined as the area under the effective curve via parasitemia counts. In addition, after treatment, the parasitemia in most rats remained unchanged for several hours (lag phase) was defined as lag time. The lag time was calculated by using TableCurve 6.0 Program (Advanced Graphics Software, Encinitas, CA).
MTD was defined as the dose that caused clinical toxicity in 100% of animals but did not cause death. To evaluate the overall therapeutic index (a numerical estimate of the relationship between the toxic dose of a drug and its therapeutic dose), the MTD was divided by SD50 and CD100. The data was generally found to fit a normal distribution. Means and standard deviations were calculated. Coefficients of variation (CVs) were calculated as percentage of standard deviation divided by mean value. Statistical analysis was conducted with Microsoft Excel using a Student’s t test for dependent samples to compare means of paired and unpaired samples between treatment groups.
Statistical Analysis
Data were analyzed for homogeneity of variance using Bartlett’s test. Homogenous data observed at the level of 5% (w/w) were analyzed using the parametric one-way analysis of variance (ANOVA), and the significance of differences was assessed using Scheffe’s method to compare the values between the control group and each drugs administered group. Mean ± standard deviations are shown.
RESULTS
Rat Malaria Model
Similar to our previous studies (Li et al. 2003a), treatments with chosen antimalarial agents were carried out on days 6, 7, and 8. Clinical observation on all animals lasted for 21 days following infection (Figure 1). Parasitemia suppression, clearance, and curative rates were evaluated during the treatment period in the rat malaria model. Infection of 208 rats with an achieved infection rate of 98.6% and 6.7% of delayed infection rate in the present study is similar to hyperparasitemia observed in our previous efficacy study (Li et al. 2003a). In the infected animals mean parasitemia slowly increased during the first 1 to 4 days from 0% to 6%, and then plateaued at 6% to 9% for another 2 to 3 days (Figure 1). After day 6 parasitemia increased rapidly to reach the mean peak of 41% ± 13% (CV 40%) by day 11. The model was given a long therapeutic time window of within 5 to 6 days (from day 5 to day 11 post inoculation). Parasitemia declined (self cleared) to undetectable levels over the next 7 to 11 days. A 16.7% mortality rate was noted in the control rats (n = 24).
Therapeutic Indices of AS and AL
The efficacy of AS/lysine following 3-day IV injection was evaluated through suppression and clearance of parasitemia in malaria-infected rats (Table 1). The minimum suppression dose of AS was 2.3 mg/kg, whereas SD50 for AS was 7.4 mg/kg. The CD100 for AS was 60 mg/kg. All animals had parasite recrudescence even at the MTD of 240 mg/kg. All animals survived the 240 mg/kg dose of AS; however, four out of five rats died at dosage of 480 mg/kg. MTD of AS was 240 mg/kg daily for a 3-day therapy in malaria-infected rats. Therefore, the therapeutic indices based on the MTD, suppression, and clearance end points were calculated as 32.4 (240 mg/7.4 mg = 32.4) for SD50 data and as 4 (240 mg/60 mg = 4) for the CD100 data (Table 1).
Efficacy of AL/lysine following 3-day IV injection was evaluated by investigating suppression, clearance, and possible cure of parasitemia in P. berghei–infected rats (Table 1). The minimum suppression dose for AL was 2.5 mg/kg and the suppression dose in half parasitemia (SD50) was 8.6 mg/kg. The minimum 100% clearance dose (CD100) for AL was 40 mg/kg. All animals suffered parasite recrudescence without curative effects even the MTD of 80 mg/kg. All animals survived the 80 mg/kg AL dose but two out of six rats died following a single dose of 160 mg/kg AL. In malaria-infected rats the MTD of AL for a 3-day therapy was 80 mg/kg. In accordance with the MTD, suppression, and clearance measurements, a therapeutic index of 9.3 (80 mg/8.6 mg = 9.3) was calculated for the SD50 data. The therapeutic index of 2 (80 mg/40 mg = 2) was noted for CD100 data. Therapeutic indices suggest that AS/lysine was 2–3 times safer than AL/lysine in treating malaria-infected rats.
Definitive Efficacy of AS and AL
Previous dose ranging studies of AS/lysine and AL/NaHCO3 were performed in malaria-infected rats at equimolar doses of 6, 12, 24, 48, 96, and 192 μ moles/kg daily for 3 days (Li et al. 2003a).
At an equimolar dose of 96 μ moles/kg AS (36.7 mg/kg) 7 out of 18 rats cleared parasitemia (Figure 2, bottom ). At this dose level the PCT of AS was 52.8 h with clearance lasting for only 2.7 days. On day 6 all animals revealed parasite recrudescence. No animal died during the treatment (Table 2). The lag time for AS was 4.0 ± 2.5 h. Computer fitting showed parasitemia at time 0 h before the drug effect (E0) was 8.1% ± 2.7%, whereas at the time of maximum drug effect (E max) parasitemia was noted at 0.339% ± 0.039%. Due to the limitations of microscopy, 0.01% parasitemia was the lowest reading. Accordingly, AS did not clear 0.339% (E max) of rats treated at this dose level. The EC50 was 8.86 h, and the AUEC was 536% of parasitemia·hour for this dosage of AS/NaHCO3 (Table 2).
At an equimolar dose of 96 μ moles/kg AL (40 mg/kg) parasitemia cleared in all of the 19 rats (Figure 2, top ). At this dose level the PCT of AL was 57.2 h with clearance lasting for 3.2 days before recrudescence. By day 6.5 all animals exhibited parasite recrudescence. No animal died during the treatment (Table 2). In most rats, parasitemia remained unchanged during the lag phase, which lasted for 3.4 ± 1.6 h. Computer fitting revealed that E0 was 6.2 ± 2.3%, whereas E max was measured at 0.008% ± 0.01%. The EC50 was 10.9 h, and the AUEC was 313% of parasitemia·hr for this dosage of AL/lysine (Table 2).
Hemotoxicity
Between 15 mg/kg and 240 mg/kg AS there were significant reductions in RBC and total reticulocyte counts in uninfected male rats. A significant reduction in hematocrit (HCT) and hemoglobin (Hb) parameters (Table 3, Figure 3) were also noted in male rats. Similarly, a dose-related significant reticulocyte reduction was noted in female rats at 7.5 mg/kg AS and higher (Figure 3). However, there were no significant effects on the RBC, HCT, and Hb, suggesting that female rats are less sensitive than male animals with respect to some of their hematological parameters. There was a significant increase in platelet (Plt), neutrophil (Neut), and lymphocyte (Lymph) counts in the female animals. A significant decrease in body weight gain was noticed in both male and female rats at 60 mg/kg and above (Table 3).
Between 10 mg/kg and 80 mg/kg AL, significant reductions in RBC, WBC, HCT, and Hb counts were seen in male uninfected rats. AL at 5 mg/kg and above caused significant reductions in total reticulocyte counts (Table 4, Figure 3) for both male and female rats. Low-dose AL (between 2.5 mg/kg and 5 mg/kg) also significantly reduced RBC, WBC, HCT, and Hb levels in the female animals. A significant decrease in body weight was observed with high dose levels in both sexes (Table 4).
In multiple AS 240 mg/kg rats in the nonrecovery (day 1) group, there is evidence of lymphocyte necrosis within the thymus. Changes included loss of cortical lymphocytes and the presence of cellular nuclear debris phagocytized by resident macrophages—so called tingible body macrophages. One of four rats had a loss of the cortical lymphocyte population to a degree that the normally well-demarcated interface between the thymic cortex and medulla was indistinct. These thymic changes are considered treatment related. Similar effects were noted in one of the deceased rats of the infected group dosed at 240 mg/kg. One of the three infected rat treated at this dose level also showed diffused, moderate lymphoid depletion with marked cortical necrosis. These thymic changes are also considered treatment related.
Uninfected group treated with 120 mg/kg AS did not reveal any change in either the myeloid to erythroid lineage (M:E) ratio or thymic lymphocyte populations. However, all three rats in the infected group treated at this dose level revealed minimal to moderate amounts of thymic cortical depletion, with no apparent effects on the M:E ratio. Animals in the vehicle-control group (both infected and uninfected) revealed M:E ratio at a range of 1.25 to 1.50. None of the animals in the control group revealed thymic lesions.
Most animals treated with AS 240 mg/kg revealed mild to moderately decreased cellularity of the bone marrow 1 day after the last dosing. However, by day 8 after the last dose, AS 240 mg/kg recovery group revealed an increased M:E ratio of (3:0:1), suggesting a recovery in the bone marrow myeloid lineage. Cytologically, there were particular increased percentages of band and segmented neutrophils in the AS 240 mg/kg recovery group (day 8). This finding is consistent with the finding of peripheral neutrophilia noted on the CBCs. Comparing recovery and nonrecovery groups, there was not only recovery of the normal marrow cellularity (both myeloid and erythroid cells) 7 days following cessation of treatment, but also increased production and release of neutrophils, presumably due to increased peripheral demand.
Bone Marrow Test
Most rats had normal appearing bone marrow (M:E ratio 1.5:1), except animals treated with 240 mg/kg AS (Table 5). At this dose level five out of six uninfected rats had abnormal bone marrow biopsies with a mean myeloid:erythroid (M:E) ratio of 1.06:1, which was significantly reduced (p < .05) compared to vehicle control rats (mean ratio of 1.5:1). Changes seen in this group are suggestive of either myeloid hypoplasia or erythroid hyperplasia. Two of the three uninfected rats treated with AS 240 mg/kg had a M:E ratio of 1.0:1.
Recovery Study
Selected data are present in Table 5. On day 1 after final dose, rats treated with 120 mg/kg AS showed dose-related hematological changes for most parameters. There were statistically significant reductions in RBC, white blood cell (WBC), HCT, Hb, and reticulocyte counts. A similar pattern was seen in rats treated with AL at 40 and 80 mg/kg. Animals treated with AS and AL at same dose levels were also sampled at day 8 post dosing (Table 5). Erythrocyte parameters (RBC, Hb, and HCT), WBC and total reticulocyte counts were normal in most groups treated with AS and AL. Only slightly lower than normal numbers of reticulocyte cells were measured in rats dosed with 240 mg/kg AS.
Bone marrow depression is seemingly absent in rats treated with either of the two water-soluble agents. Before recovery, the M:E ratios of all treated animals were within the normal range (1.0 to 1.5). Although AS at 240 mg/kg caused a significant decrease in M:E ratio (1.06 for AS versus 1.5 for control; p < .05) at day 1 post dosing due to agranulocytosis and erythroid hyperplasia, the deviation was not threefold lower than the control to suggest an abnormal diagnosis. After the recovery period (7 days post dosing), M:E ratios of rats treated with AS 240 mg/kg were significantly higher than that of control animals. Previous studies have cited similar effects of high-dose AS in other animals. For instance, proliferation of lymphocytes, increased phagocytic activity of peritoneal macrophages, and induced splenomegaly were noted in mice (Guojun and Yi 1983).
Body Weight Changes
AS and AL were administered for 3 days at various dose levels. Body weights were measured on day 1 and day 8 after last dosing. Total body weight changes (percent loss versus percent gain) from day 1 until the end of treatment are shown on Tables 3, 4, and 5. Compared to the control, all treatment groups revealed a significant decrease in mean body weights during the nonrecovery group. However, when treatment was withdrawn during the recovery study, mean body weights increased compared to the first day of dosing, although AS 240 and AL 80 mg/kg revealed a more meager gain as compared to the control (Table 5).
DISCUSSION
Through appropriately optimizing age, inoculum size, and number of prior passages, an adult rat P. berghei ANKA model was developed with high incidences and consistency of infection and low rates of delayed infection (Li et al. 2003a). Under these conditions, a 98% infection rate was achieved while the rate of delayed infection remained low (11%). The principal measure of efficacy was the ability of the selected drugs to clear and cure parasitemia prior to day 21. Suppression of parasitemia provided a useful secondary measurement of efficacy.
The efficacy model of choice in preclinical drug development program for malaria is usually the mouse model because they are highly susceptible to infection by rodent plasmodia and develop fulminating infections with high parasitemia and mortality. These traits enable convenient assessment of the blood schizonticidal and curative effects of test drugs. However, rats are the more commonly used species for standardized toxicity testing necessary for the development of most pharmaceuticals and is also recommended by the FDA (ICH Harmonised Tripartite Guidance for Industry 1994). To obtain a good indication of efficacy and therapeutic index of a new antimalarial, it is imperative to investigate, in parallel, the efficacy and toxicity of candidate antimalarials in a rat malaria model. Therefore, a therapeutic index assessment (ratio of toxic dose/efficacious dose) was carried out in the same animal species (rat) for both efficacy and toxicity studies.
The studies demonstrated that the antimalarial potency of AL is about two- to fourfold higher than that of AS in rat malaria model, which is absolutely different to the in vitro data. In vitro results have indicated that AL possesses lower potency in its antimalarial effects than AE and AM, and an even lower potency than AS and DHA (Bustos, Gay, and Diquet 1994; Milhous et al. 1996; Shmuklarsky et al. 1993). The reason(s) for the differing antimalarial potency data between the in vivo and in vitro model remains to be determined.
The efficacy study demonstrated that antimalarial potency of AL was superior to that of AS at the same molecular dosage. AL was found to be more potent than AS despite the fact that the metabolic conversion of AL to DHA is minimal (Li et al. 1998a), whereas the metabolic conversion of AS to DHA is rapid and extensive. DHA is three- to fivefold more potent than AS and AL (Li et al. 2002). The possible reasons for AL’s superior potency seen here may relate to (1) its greater transfer into the erythrocytes, (2) higher plasma concentrations, and (3) longer residence time in the animal (Li et al. 1998a, 1998b). AL has a greater stability in in vitro metabolic systems and biological fluids (Grace, Skanchy, and Aguilar 1999; Lin, Klayman, and Milhous 1987). Finally, it may also be possible to attribute the higher antimalarial activity of AL to enterohepatic circulation and moderate renal failure (Li et al. 2003b; Xie et al. 2003). Animals suffering from renal insufficiency often exhibit severe and even fatal side effects due to drug accumulation and retention that lead to pharmacokinetic alterations (Fabre and Balant 1976; Subach and Marx 1998).
An inquiry into drug-associated hemotoxicity is critically important in preclinical toxicology assessment. As the major haematopoietic organ of body, bone marrow is a primary target organ for hemotoxicity. Cytological examinations of peripheral blood and/or bone marrow to determine M:E ratios have been completed manually (Schomaker, Clemo, and Amacher 2002). In this study, sections of rat sternum were routinely processed and stained with hematoxylin and eosin. The marrow was examined primarily to produce an estimate of erythrocytic and granulocytic series cellularity, granulocytic:erythrocytic ratio, megakaryocytic series cellularity, and for evidence of degeneration, necrosis, or reactive changes.
In the AS 240 mg/kg group, the increased numbers of megakaryocytic series cells observed likely represents a relative increase compared to the other hematopoietic elements, i.e., normal numbers of megakaryocytic series cells and a decrease in myeloid and erythroid series cells. These findings suggest a treatment-related decrease in myeloid and erythroid cells in the rat marrow.
Artemisinins contain an endoperoxide bridge that has been linked to the production of reactive oxygen species (Brossi et al. 1988). Most free radical–generating drugs cause “oxidant damage” by producing oxygen free radicals, such as superoxide, that cause indiscriminate damage to the cell (Halliwell and Gutteridge 1990). Artemisinin derivatives were at first thought to act in this manner (Krungkrai and Yuthavong 1987; Meshnick et al. 1989). AS was shown to induce lipid peroxidation (Meshnick et al. 1989) in both infected and uninfected erythrocytes, as well as membrane protein thiol oxidation in isolated erythrocyte membranes (Meshnick et al. 1991). These artemisinin-induced membrane effects could underlie other observed effects of artemisinin derivatives, such as hemolysis (Gu, Warhurst, and Peters 1986; Scott et al. 1989), decreased erythrocyte deformability (Scott et al. 1989), and premature lysis of infected erythrocytes (Gu and Inselburg 1989). However, lipid peroxidation, membrane thiol oxidation, and most of the other effects could be observed only at very high drug concentrations (> 100 μ M), suggesting that these nonspecific oxidant reactions may not be related to killing of parasites but toxicity. These observations are very comparable to our findings in present studies.
In conclusion, the findings indicate that AL has a superior antimalarial potency than AS in malaria-infected rats despite lower conversion of AL to DHA. Greater transfer of AL into erythrocytes and pharmacokinetic alterations due to the AL-induced renal insufficiency (Xie et al. 2003) may be possible explanations for such superior potency. With both AS and AL, a dose-related and rapidly reversible hemotoxicity was seen in peripheral blood without any significant bone marrow suppression in malaria-infected and uninfected rats. AS had a higher therapeutic index and faster clearance activity than AL. Although both drugs have their own advantages based on the data from the rat malaria model, AS seems to be safer and a faster agent than AL when their respective therapeutic indices and toxicity profiles are compared. Although the reversible anemia and reticulocytopenia previously reported in animal species and humans, no drugs caused significant bone marrow depression in present study.
Footnotes
Figures and Tables
This study was supported by the United States Army Research and Materiel Command. The authors also thank the Medicines for Malaria Venture (MMV) for financial support in this work. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official, or as reflecting true views of the Department of the Army, Department of the Navy, or the Department of Defense.