Metabolism of antimalarial sesquiterpene lactones

Pharmac. Ther. Vol.48, pp. 345-355, 1990 Printed in Great Britain.All rights reserved

0163-7258/90$0.00+ 0.50 © 1991PergamonPress plc

Specialist Subject Editor: D. C. WARH~IRST

METABOLISM OF ANTIMALARIAL SESQUITERPENE LACTONES IK-SOO LEE a n d CHARLES D. HUFFORD Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MI 38677, U.S.A. Abstract--Metabolism of artemisinin derivatives, which are antimalarial sesquiterpenes,appeared to lead to the production of the more polar metabolites in general. Presence of the endoperoxide moiety in the A/B ring structure seems crucial for the expression of antimalarial activity of these compounds. Microbial models served as effective predictors for the mammalian metabolism of artemisinin derivatives as well as producing quantities of metabolites for referencestandards and structure elucidation studies. Combination of 2D-NMR and Thermospray HPLC/MS techniques was very useful for the structure elucidation of metabolites. CONTENTS 1. Introduction 2. Sesquiterpene Lactone Antimalarials 2.1. Artemisinin 2.2. Arteether 2.2.1. Introduction 2.2.2. Development of arteether 2.3. Artemether 2.4. Other sesquiterpene lactone derivatives 3. Model Systems for Drug Metabolism Studies 3.1. Mammalian metabolism studies 3.2. Microbial systems as predictors of mammalian metabolism 4. Metabolism of Antimalarial Sesquiterpene Lactones 4.1. Artemisinin 4.1.1. Mammalian metabolism studies 4.1.2. Microbial metabolism of artemisinin 4.2. Arteether 4.2.1. Mammalian metabolism of arteether 4.2.2. Microbial metabolism of arteether 4.3. Artemether 4.4. Artesunic acid and artelinic acid 4.5. Arteannuin B 5. Conclusions Acknowledgements References

1. I N T R O D U C T I O N For any drug to be approved for use in humans, safety and efficacy of the drug should be established through extensive studies. An important factor in the evaluation of the safety and efficacy of any drug is a knowledge of how the drug is metabolized. There was a realization that the processes of drug metabolism might be of importance in determining the pharmacological activity, clinical efficacy, and toxicological profile of drug molecules (Jenner and Testa, 1981). The knowledge of the pharmacological activity and amount of drug metabolites formed is important in determining the correct clinical parameters such as dosage levels and dosage intervals. Sometimes, a knowledge of drug metabolism may also offer insight into its biochemical mechanism of action. It is clear

345 345 345 346 346 347 347 347 348 348 348 349 349 349 349 350 350 350 35O 351 352 353 354 354

that a better understanding of drug metabolism is essential for the full knowledge of how a drug acts, why it exhibits toxicity, and even how it may be distributed, excreted and stored in the body. Ultimately, a full understanding of drug metabolism is necessary for the design of better drugs.

2. SESQUITERPENE LACTONE ANTIMALARIALS

2.1. ARTEMISININ Artemisinin (also called artemisinine, or qinghaosu, or artennuin; Fig. 1, [!]), a sesquiterpene lactone with an unusual endoperoxide linkage, is the clinically active antimalarial constituent isolated from

345

I.-S. LEE and C. D. HUrFORD

346

Is

14 H cH3 2 : .

3

H CHs

H CH~

.J~"~NI,(~

" :

HsC~

c I

H~C

HsC

O

.O~. H2.C.Hs xo .i./

OCH3

OH

[1]

[2]

[3]

[4]

. .c,s

H .CH~

, .C.,

H CH,,

~ k....I H

H CH3

I

:Vo

O

H

Hs

°

O

O CCH2C~COOH

H

Hs

O - CH2- - ~

n

[5] o

COOH

H,CO

O He

~ - O,~CH2

o

O

[6] H CHs

[8]

CH3

H CHs

140 H

O

H

O

0

H

CH~

3

OH o [10]

[11]

[12]

FIG. I. Antimalarial sesquiterpene lactones and their derivatives. the Chinese medicinal herb, Qinghao or Artemisia annua L. [Family; compositae] (Klayman, 1985). Traditionally this herb has been used in Chinese folk medicine, for many centuries, as a treatment for fever and malarial diseases (China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials, 1982b). This compound, artemisinin, has been successfully used in several thousand malaria patients in China, including those with both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. Recently, the importance of this compound as an antimalarial has been reemphasized in a series of efforts to find alternative drugs effective against Plasmodium falciparum strains showing resistance to classical antimalarial agents (Klayman, 1985). On the other hand, the production of resistance to artemisinin in P. yoelii in mice has been observed (Warhurst, 1986). It was suggested that a possible mechanism of resistance to blood schizontocides is a change in membrane composition. Many derivatives of artemisinin have been synthesized and considered for evaluation as antimalarial agents due to their potential effectiveness in the treatment of chloroquine- and multi-drugresistant P. falciparum malaria, and cerebral malaria (Li et al., 1982). Investigation of its thermal stability and other chemical behaviors, rearrangement and decomposition studies (Lin et aL, 1985; Luo et al., 1985) as well as chemical transformation (Liu et aL,

1979; Zhu et al., 1980) have been reported. Also in consideration of the analysis of artemisinin in plant extracts, medicine and biological fluids, u.v.-absorption and HPLC characteristics were investigated after a modification which makes it u.v.-detectable (Zhao and Zeng, 1985). Luo and coworkers (1984) reported an analytical method which can detect the metabolites of artemisinin in nanomolar quantities. Previous studies on the chemistry, pharmacology and clinical applications of artemisinin have been extensively reviewed by Luo and Shen in their recent publication (1987). 2.2. ARTEETHER 2.2.1. Introduction Arteether (Fig. 1, [2]) is the ethyl ether derivative of dihydroartemisinin, a sodium borohydride reduction product of artemisinin. It has become an increasingly important new drug candidate as a treatment for the erythrocytic stages of chloroquine-resistant strains of P. falciparum and for cerebral malaria. It has been chosen by SWG-CHEMAL (the Steering Committee of the Scientific Working Group on the Chemotherapy of Malaria of the World Health Organization in Geneva, Switzerland) for development with a view to use in high-risk malaria patients including those with cerebral malaria. The I H- and ~3C-NMR spectral assignments of arteether have

Metabolism of antimalarial sesquiterpene lactones been reported (Hufford and E1Sohly, 1987). Brossi and coworkers have recently published an article on the synthesis and antimalarial properties of arteether (Brossi et al., 1988). And more recently, studies on the pharmacological activity of arteether have been carried out in experimental animals (Kar et al., 1989). Antimalarial efficacy studies on arteether have also been reported (Dutta et al., 1989). Idowu and coworkers have proposed an analytical method for determining arteether in blood plasma using HPLC and u.v. (Idowu et al., 1989a). Since there have been no previous reports on the metabolism of arteether, a series of comprehensive studies on its metabolism have recently been undertaken (Hufford et al., 1990; Lee et al., 1990; Baker et al., 1989). 2.2.2. Development o f arteether The objective of CHEMAL's program since March, 1985 has been the development of arteether as a single dose parenteral treatment of severe and complicated forms of falciparum malaria (Report of CHEMAL, 1986). Ethers have good storage stability and are highly soluble in oils and well suited for the preparation of injectable formulations. The decision to develop arteether rather than artemether was based on the following reasoning although both compounds are equally more potent and more soluble than artemisinin (Gu et al., 1980). Arteether is more lipophilic and its metabolic breakdown gives ethanol and not methanol, thus avoiding problems of methanol toxicity which might arise from the metabolic formation of formaldehyde and formic acid (Ritchie, 1975). The development of this drug for the treatment of complicated and severe cases of malaria was considered to be an acceptable objective since the benefits should outweigh the reported problems of toxicity, particularly those related to fetotoxicity of the artemisinin series of compounds (China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials, 1982b). 2.3. ARTEMETHER Artemether (Fig. 1, [3]) is the methyl ether derivative of dihydroartemisinin. This compound has been synthesized to enhance the antimalarial activity and solubility of artemisinin. Artemether was found to be slightly more effective than artemisinin, in terms of MIC, but the difference was quite minor (Thaithong and Beale, 1985). Artemether, which was shown to have markedly superior activity to dihydroartemisinin in vivo (Gu et al., 1980) was not, however, more active in vitro. It was also found that dihydroartemisinin is more active in vitro than artemisinin (Li et al., 1983). Based on these observations, it was speculated that intrinsic activity against the parasite is enhanced by reduction of the keto group, but modifications where further substitutions are made on the alcoholic group have advantages in vivo (Li et al., 1983). 2.4. OTHER SESQUITERPENELACTONEDERIVATIVES

Dihydroartemisinin (Fig. 1, [4]) is obtained (Liu et al., 1979) by reducing artemisinin with sodium

347

borohydride, and the iactone group is converted to a lactol (Hemiacetal function). Dihydroartemisinin itself has good antimalarial activity, as it was reported to be therapeutically more active than its parent compound (China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials, 1982a), but because of its poor stability in water environment, it is normally used after derivatization. For subnanogram detection of dihydroartemisinin, the use of diacetyldihydrofluorescein (DADF) was proposed (Luo et al., 1987) for derivatization of dihydroartemisinin. Artesunic acid (Fig. 1, [5]) and artelinic acid (Fig. 1, [6]) are both semisynthetic water-soluble derivatives (sodium salts) of dihydroartemisinin. Artesunic acid, the hemi-succinic ester of dihydroartemisinin, is prepared by esterification of dihydroartemisinin with succinic acid. It has been used longer than artelinic acid. It was found that the usefulness of sodium artesunate is impaired by its poor stability in aqueous solution due to the lability of the ester linkage to hydrolysis. And thus to overcome the ease of hydrolysis of the ester group, artelinic acid was prepared as a new series of derivatives, in which the solubilizing moiety is joined to dihydroartemisinin by an ether rather than an ester linkage (Lin et aL, 1987). Recently, an HPLC method for determination of artelinic acid in blood plasma has been reported (Idowu, 1989b). Artemisitene (Fig. 1, [7]), an endoperoxide closely related to artemisinin, and arteannuin B (Fig. 1, [8]) have also been isolated from Artemisia annua (Acton and Kiayman, 1985; Jeremic et al., 1973, respectively). Recently a hypothesis that a requirement for the cyclic peroxide function to display antimalarial activity might involve the unique C--O-O-C--O-C-O-C = O moiety found in artemisinin, has been conceived (Imakura et al., 1988) from earlier studies (Tani et aL, 1985) on structure-activity relationships and a program of synthesis directed at the preparation of more active, simple analogs of artemisinin. Desethanoquinghaosu (Fig. 1, [9]) was such an example synthesized (Imakura et al., 1988) based on this hypothesis. Later, deoxoartemisinin (Fig. 1, [10]) which is devoid of the carbonyl function at C-12, and yet retains the biologically active endoperoxide, was prepared by Jung and coworkers (1989). It was found to show approximately eight times the antimalarial activity of artemisinin in vitro against chloroquineresistant malaria. 9,10-Dehydrodeoxoartemisinin (Fig. 1, [11]) is another such compound, closely related in structure to deoxoartemisinin and devoid of the carbonyl function at C-12. 9,10-Dehydrodeoxoartemisinin was prepared synthetically by Cao and coworkers (1982), and recently proved to be active in the antimalarial activity test (Lin et aL, 1989). Derivatives of artemisinin lacking the carbonyl function were projected to possess increased stability and thus longer half-life in the body (Jung et aL, 1989). Yingzhaosu A (Fig. 1, [12]), although not a sesquiterpene lactone, is an example of another rare natural product which has a peroxide linkage and antimalarial activity. Yingzhaosu was isolated from Artabotrys uncinatus (Lam.) Merr. (Liang, 1985; Liang et al., 1979).

348

I.-S. LEE and .C.D. HUFFORD 3. MODEL SYSTEMS FOR D R U G METABOLISM STUDIES

Drug metabolism studies traditionally have relied on the use of model systems to predict metabolic pathways in humans. For this purpose, either in vivo whole animal systems (usually utilizing small laboratory animal models) or in vitro enzyme systems (using microsomal preparations, tissue cultures, or perfused organ systems) have been widely employed. In either case, there are certain limitations and disadvantages to these systems: (a) Animals can be expensive to maintain. (b) Microsomal fractions, with which the majority of in vitro studies are conducted, are often influenced by the manner of preparation. The reliability and reproducibility of such factors as the methods of homogenization and the choice of media can be very important (Testa and Jenner, 1976). (c) Although mammalian tissue preparations occasionally may be utilized in synthesizing preparative quantities of metabolites, there are difficulties related to the stability of such systems (Orrenius and Ernester, 1974). (d) Biological specimens obtained from mammalian systems usually yield mere microgram quantities of metabolites, which are not sufficient for complete structure elucidation and biological evaluation. The use of microbial systems as alternative in vitro models to the traditional use of mammalian systems for initial drug metabolism studies in humans overcomes most, if not all, of these obstacles (Clark et al., 1985). Smith and Rosazza (1974) proposed the use of microbial systems as models for mammalian metabolism of xenobiotics. These authors proposed that microorganisms could serve as convenient and reliable models of mammalian metabolism since there was a significant similarity in certain microbial enzyme systems and mammalian liver enzyme systems, an observation which was also noted by Ferris and coworkers (1973). 3.1. MAMMALIANMETABOLISMSTUDIES

Traditionally, in vivo drug metabolism studies have involved the administration of the drug to laboratory animals which serve as model systems (i.e. dog, rat, cat, mouse, guinea pig and rabbit). The biological fluids and tissues of these animals are then examined for the presence of the parent drug and its metabolites. Such metabolites are usually present in very small quantities and, as a result, are often very difficult to isolate and identify chemically. Approaches to metabolite identification have often involved speculation of the type of metabolic changes which may occur, chemical synthesis of possible metabolites, and finally direct comparisons. As now undertaken, metabolism studies are time-consuming processes which many times miss important metabolites. Further, a large number of animals are required, which today is quite expensive.

Coutts and Jones (1980) state that there are four major steps in most drug metabolism studies, which include the isolation of a given drug and its metabolites from biological media, the separation of the components that have been isolated, the identification of each component, and the quantification of recovered drug and metabolites. Generally there is only a small quantity of an unknown material present; therefore, it is often quite difficult to detect the metabolite, since there are a multitude of naturally occurring substances that might interfere with identification of metabolites. Often speculations of what processes may occur will lead to the synthesis of proposed metabolites which can serve as standards by which to develop analytical methodology. If, however, the organism does not carry out the expected or predicted transformations, an important metabolite(s) may be missed, since many of the more sensitive analytical techniques are also very selective. After a metabolite has been detected, the next step would be the isolation and identification of the metabolite. Even with the currently available sensitive analytical techniques, this can often be a problem if the metabolite is a minor one or present in small quantities. This is usually the case with in vivo studies since many drugs cannot be tolerated in large doses. Although isolation from biological fluids such as plasma and urine is usually cleaner than isolation from tissues, there are often a number of endogenous substances which may coextract and interfere with analysis. Once the metabolite is isolated, identification is usually accomplished on small quantities using sensitive spectroscopic techniques. Finally once the metabolite is isolated and identified, there is usually a desire to determine its pharmacological activity and/or toxicity. This can be accomplished only with significant quantities and usually requires chemical synthesis of the metabolites, and there are many with complex structures, including asymmetric centers, that would be difficult, at best, to prepare chemically. 3.2. MICROBIAL SYSTEMSAS PREDICTORSOF MAMMALIANMETABOLISM

The methodology used in microbial transformation studies has been reviewed by Smith and Rosazza (1975), and also by Goodhue (1982). Microbial transformations of nonsteroid cyclic compounds were discussed in detail by Kieslich (1976). Microbial drug metabolism studies are conducted in the same manner, and more recently the use of microorganisms for the study of drug metabolism was extensively reviewed with selected examples (Clark et al., 1985). Generally, a large number of microorganisms will be selected for preliminary or initial screening for their ability to metabolize a drug substrate. The selection of microorganisms is based on a number of factors including literature precedent, experience and intuition. Usually a two-stage fermentation procedure as outlined by Smith and Rosazza (1975) is used for microbial metabolism studies. Smith and Rosazza have reported good success in microbial metabolism studies using a soybean meal-glucose medium. In other cases, the use of a peptone-glucose medium has been very successful and has the added

Metabolism of antimalarial sesquiterl~ne lactones

349

Artemisinin was shown to pass the blood-brain and blood-placenta barriers after i.v. injection. Very little unchanged artcmisinin was found in the urine and feces in 48 hr regardless of administration route. Human metabolism of artemisinin was studied by Zhu and coworkers (1980, 1983), and by China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials (1982c). Four compounds have been isolated as human metabolites of artemisinin. After oral administration of artcmisinin to humans, three metabolites were isolated from the urine samples. The structures of these three compounds have been identified (Zhu et al., 1980) as deoxyartemisinin, deoxydihydroartemisinin, and the so called 'crystal 7', which is only a tentative structure (Fig. 2). In fact, it resembles the rearranged microbial product AEM1. Later, another compound was isolated from human urine and was identified as 9,10-dihydroxydeoxyartemisinin (Zhu et al., 1983). None of the chemical structures of these metabolites have retained the endoperoxide linkage intact, and as is typical of compounds in this group that lack the peroxide moiety, these metabolites were found to be inactive against Plasmodium berghei in the mouse (Klayman, 1985). Shown in Fig. 2 are the chemical structures of the artemisinin metabolitcs isolated from human urine.

advantage of being a completely solubilizcd medium (Hufford et al., 1981a,b; Clark et al., 1981). Clearly there are a number of practical advantages offered by the use of microbial systems as models for drug metabolism. The culture media used are usually easily prepared and relatively inexpensive. The cultures, once initiated, require basically no maintenance during the metabolic studies, and the fermentation sampling process is usually very straightforward and can easily be done in large numbers. This means that relatively large number of microorganisms can be screened simultaneously for their ability to metabolize the drug substrate. In addition, the concentrations of the drug substrate used are usually much higher than could be obtained from in rive animal or in vitro enzyme or tissue culture systems. The higher drug substrate concentration coupled with the 'cleaner' extracts from microbial cultures leads to easier detection, isolation, and identification of metabolites. Finally the cost of maintaining a culture collection for metabolic studies is only a fraction of the cost of maintaining laboratory animals or even tissue culture systems.

4. METABOLISM OF A N T I M A L A R I A L SESQUITERPENE LACTONES 4.1. ARTEMISININ

4.1.2. Microbial Metabolism o f Artemisinin 4.1.1. Mammalian Metabolism Studies

Microbial metabolism studies of artemisinin were conducted (Lcc et al., 1989) based on the standard two-stage procedure (Smith and Rosazza, 1975). Screening scale studies have shown a number of microorganisms capable of metabolizing artcmisinin. Scale-up fermentation with Nocardia corallina (ATCC 19070) and Penicillium chrysogenum (ATCC 9480) have resulted in the production of two major microbial metabolites that have been characterized with the use of 2D-NMR techniques. These

The metabolic fate of artemisinin was investigated (Niu et al., 1985) using a TLC densitometric method. Artemisinin was shown, in this study, to be completely and rapidly absorbed after oral administration in rats. However, a very low plasma level was obtained even after a dose of 300 mg/kg. Liver was found to be the chief site of its inactivation. When artemisinin was given intramuscularly, significant and more persistent plasma levels were detected. , .c~ " :

H

3

C

. c~ i :

~

H

,

C

~

.

HgC~

H~ 0 Artemisinin

o

H CH3 " :

~

c.,,

= :

H3

\

0 Deoxyartemisinin

OH Deoxydihydroartemisinin

" 'H- ~O,CHs ~

H

o

o

'Crystal-7'

9,10-Dihydroxydeoxyartemisinin

FIG. 2. Humanmetabolites of Artemisinin.

350

I.-S. LEE and C. D. HIdFFORD H .ell,

HsC~

HO

°o o H O

Hs

O

Deoxyartemisinin

HsC

H .e,,

°o o H O

Hs

O

3c~-Hydroxydeoxyartemisinin

FiG. 3. Major microbial metabolites of Artemisinin. metabolites (Fig. 3) have been identified as deoxyartemisinin, a previously identified mammalian metabolite (Zhu et al., 1980), and 3~-hydroxydeoxyartemisinin, previously prepared only as a thermal rearrangement product (Lin et al., 1985). Dihydroartemisinin, which was expected as a possible metabolite, was not detected as a microbial metabolite for any of the microorganisms used in this study. 4.2. ARTEETHER 4.2.1. Mammalian Metabolism of Arteether Arteether has been previously reported (Baker et al., 1989) to be converted by rat liver microsomal enzyme system to dihydroartemisinin, as a major metabolite, and deoxydihydroartemisinin, 3~hydroxydeoxydihydroartemisinin, 3~-hydroxydeoxyarteether, and several isomers of monohydroxylated arteether with unknown stereochemistry (Fig. 4). In a recent study (Hufford et al., 1990), it was found that all of the isomers of the hydroxyarteether obtained from the microbial fermentation isolations had thermospray mass spectra that were essentially identical to that for arteether except that all of the lines were displaced upwards by 16m/z units (indicating the presence of hydroxyl group). In a comparison study using microbial arteether metabolites as reference standards, employing the same thermospray HPLC/MS procedure, the rat liver microsome incubations of arteether were also found to contain all four hydroxyarteether metabolites found in the microbial system. Deoxyarteether, expected as a potential metabolite, was not found in the isolates of incubation mixture. The chemical structures of these metabolites are shown in Fig. 4. 4.2.2. Microbial Metabolism of Arteether Microbial metabolism studies of the antimalarial drug arteether have shown that arteether is metabolized by a number of microorganisms. Large-scale fermentations with Aspergillus niger (ATCC 10549) and Nocardia corallina (ATCC 19070) have resulted in the isolation of four metabolites (Lee et al., 1990). Based on the chemical and spectroscopic data, especially 2D-NMR techniques, these four metabolites have been identified as 'AEM 1' (a rtee ther m etabolite 1), the structure of which is very similar to that of a thermal rearrangement product of artemisinin (Lin et aL, 1985), 3~-hydroxydeoxyarteether, a compound closely related to deoxyartemisinin which was obtained as a microbial (Lee et al., 1989) and a

mammalian metabolite of artemisinin (Zhu et al., 1980; China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials, 1982c), 3~-hydroxydeoxydihydroartemisinin, and deoxydihydroartemisinin, which is identical with the material prepared earlier by Brossi and coworkers (Brossi et al., 1988). In addition to these previously reported metabolites, six new microbial metabolites of arteether were characterized in more recent studies (Hufford et al., 1990) using large-scale fermentations with Cunninghamella elegans (ATCC 9245) and Streptomyces lavendulae (L-105). These six metabolites were identified as 2~-hydroxyarteether, 9~-hydroxyarteether, 9fl-hydroxyarteether, 14-hydroxyarteether, 3~-hydroxy-I 1-epi-deoxydihydroartemisinin, and an A/Bring rearrangement metabolite (Fig. 5). An in vitro antimalarial activity test conducted recently showed that 9fl-hydroxyarteether had an activity comparable to dihydroartemisinin and arteether. Thermospray Mass Spectroscopy/High Performance Liquid Chromatographic analyses have shown that four of these metabolites (hydroxylated arteethers) are also present in rat liver microsome preparations. Neither dihydroartemisinin, a major mammalian metabolite, nor deoxyarteether, a potential metabolite, were identified as microbial metabolites. The chemical structures of those microbial metabolites of arteether which were not detected in mammalian systems are shown in Fig. 5. 4.3. ARTEMETHER In a recent publication about the absorption and distribution of artemether in mammalian systems, Jiang and coworkers (1989) reported that the highest level of artemether was found in brain 5 min after i.v. injection of artemether to rat. Moderate levels of artemether were found in heart, lung and skeletal muscles, whereas the levels in liver and kidney were low. Based on their current study, these authors also discussed that artemether was shown to have a high affinity to brain tissues, and to be able to cross the blood-brain barriers. In a previous study using ~4C-labeled artemether, it was reported that certain amounts of demethylation (31% for i.v. and 15% for i.m.) occurred in artemether within 24 hr after injection to mice, and that the degree of demethylation was enhanced by phenobarbital induction of liver drug-metabolizing enzymes (Jiang et al., 1983). Mammalian metabolism studies have also been conducted recently using the isolated rat liver

351

Metabolism of antimalarial sesquiterpene lactones H CH~, = :

°Vc H

CHs

HO %

H - =

°o o

O

H

H3

O

H,

OCH2CHs

OCI.I=CH3

~

Arteether

ydroxydeoxyarteether

H CH., = HO

H CH~

H,,C

HsC

~

".= ".

i

H

CHa :

Hsc 0 H

0 Hs

OH

Dihydroartemisinin (mammalian metabolite only)

9~-Hydroxyarteether H CH3 . = H3C

OCl'lfCHs

OCH2CHs

2a-Hydroxyart~ether

4"

~ H CI.I:OH

H CHa H

, , , , :O H "

O

H3C o.

Deoxydihydroartemisinin

H O

+

H3

O HO

OCH~CHs HO,

%

H CH3 :

9(z-Hydroxyarteether

=

Hs OCH2CHs

14-Hydroxyarteether

HsC

HO

Hs OH

3cc-Hydroxydeoxydihydroartemisinin FIo. 4. Mammalian and microbial metabolites of Arteether.

perfusion and the liver microsome preparation. After perfusion or incubation for 2 hr, the samples were subjected to normal extraction, isolation, and purification procedures, which resulted in the isolation of four metabolites. These compounds have recently been reported as dihydroartemisinin, deoxydihydroartemisinin, 9~-hydroxyartemether, and 9fl-hydroxyartemether, respectively.* Of these, 9-hydroxyartemether was reported to have significant antimalarial activity in preliminary in vitro experiments, although it is not clear whether both *Y.-L. Zeng, personal communication regarding a paper Recent advances in artemether metabolism studies presented

at the SWG-CHEMAL meeting, April, 1989, Beijing, Peoples' Republic of China.

of the isomers are equally active or not. Figure 6 shows the chemical structures of these four metabolites. As of this writing, there have been no reported metabolism studies on artemether using microbial enzyme systems. 4.4. ARTESUNICACID AND ARTELINICACID Previous studies on the pharmacokinetics of artesunic acid after intravenous administration of the sodium salt suggested that artesunic acid is rapidly hydrolyzed to dihydroartemisinin in rats (China Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials, 1982c), and therefore dihydroartemisinin was measured rather than

352

I.-S. L ~ and C. D. HUFFORD H :

CHs

HO.~

=

H CH3

p . . . ~ = _ j ~ ~ -.

.=

HaCm,~,,,,O. "P "1 o H O Its

OCH2CHs

OH

Arteether

3 a - H y d ~ - I 1-epideoxydihy~m-~misimn

H CH3

H CI~

CHsCO0, ~ O H

C~CO0 ,

~ It O "

~'~c~ OCH2CHs

OCH2CHa

'AEMI'

913-Hydroxy'AEMl'

FIG. 5. Microbial metabolites of Arteether not found in mammalian metabolism. artesunic acid. Dihydroartemisinin was measured by thin-layer chromatography, and the sensitivity of the method was reported to be 0.5 #g. In a more recent study with artelinic acid as a substrate, using rabbits and dogs, it was demonstrated that artelinic acid is also rapidly hydrolyzed to dihydroartemisinin in rive and could be detected with 2 ng/ml limit (Theoharides et aL, 1988). In both cases, dihydroartemisinin is considered to be the active antimalarial compound, and artesunate and artelinic acid are utilized only for purposes of water solubility and intravenous administration,

and should be considered as prodrugs of dihydroartemisinin (Fig. 7). 4.5. ARTEANNUINB Microbial transformation studies on arteannuin B were undertaken (E1Marakby et al., 1987) to examine its possible microbiological conversion into some useful metabolites. The microbial fermentation of the sesquiterpene lactone arteannuin B using Aspergillus flavipes produced dihydroarteannuin B as the main metabolite. Preparative scale fermentation of arteannuin with Beauvaria bassiana, on the other

.- .c., :

CH3 H - :.

H~C~

HsC H O

H O Hs

. c,,

I.I3

OH

I%C

"

OH

I)ihydrom't~misinin

O It O

Deoxydihydroarge~isimn

Hs H

Artemether

CHs

H CHs

- :

HsC~ , , , , , O H

OH

HsC~

H O

H O H3

3

OCH3 9a-Hydro:~axl;emet~er

OCH3 9~-Hydroxyartemether

FIG. 6. Mammalian metabolites of Artemether.

Metabolism of antimalarial sesquiterpcne lactones

353

H CHs = i o It

o cIt 3

O- CCI'I2CI'~COOH

OH

0 - ¢H2 ~

COOH

II

o

Artesunic acid

Dihydroartemisinin

Artelinic acid

FIG. 7. Dihydroartemisinin, a common metabolite of Artesunic acid and Artelinic acid. hand, has resulted in the production of two metabolites which were identified as 3fl-hydroxyarteannuin B and 13-hydroxy-I 1-epi-dihydroartcannuin B.

5. CONCLUSIONS Metabolism studies on artemisinin have resulted in the isolation of four metabolites from human urine (Fig. 2). These metabolites were identified as deoxyartemisinin, deoxydihydroartemisinin, 9,10-dihydroxydeoxyartemisinin, and the so called 'crystal 7', although rigorous structure proofs have not been reported. Microbial metabolism studies also revealed that deoxyartemisinin is the major microbial metabolite. All of these metabolites proved to be inactive against P. berghei. Recent metabolism studies using rat liver microsomes and microbial enzymes have identified a number of metabolites of arteether. Metabolism studies on arteetber have resulted in the identification of a number of microbial metabolites which have been provided as reference standards for metabolism studies in rat liver microsomes. These microbial metabolites included 'AEMI', 3~-hydroxydeoxyarteetber, 3g-hydroxydeoxydihydroartemisinin, deoxydihydroartemisinin, 2a-hydroxyarteether, 9~-bydroxyarteether, 9fl-hydroxyarteetber, 14-hydroxyarteetber, 3~hydroxy-11-epi-deoxydihydroartemisinin, and another A/B-ring rearrangement metabolite similar to AEMI (Figs 4 and 5). Metabolism of artcetber in a rat liver microsome preparation indicated that the major mammalian metabolite of arteether was dihydroartemisinin which resulted from the oxidative Odealkylation of arteether. Oxidation of the ring system of artcether gave 9a-hydroxyarteether, 9fl-hydroxyartcetber, 2~-hydroxyartcether, and 14-hydroxyarteether as mammalian metabolites. Other minor metabolites arising from multistep transformation found to be present included 3g-hydroxydeoxyartcetber, deoxydihydroartemisinin, and 3~-hydroxydeoxydihydroartemisinin. Deoxyarteetber was not found either as a microbial metabolite or as an/n vitro metabolic product using the rat liver microsomes. Dihydroartemisinin, the major metabolite in the rat liver microsome, was not detected in any of the microbial cultures. Further microbial and mammalian metabolism studies on arteether are in progress. Metabolism studies conducted using rat liver perfusion and microsomal preparations revealed four compounds as mammalian metabolites of artemether. These compounds were identified as

dihydroartemisinin, dcoxydihydroartemisinin, 9ahydroxyartemetber, and 9fl-hydroxyartemether, respectively. Previous pharmacokinetic studies suggested that the sodium salt of artesunic acid was rapidly hydrolyzed to dihydroartemisinin. It was demonstrated that artelinic acid is also hydrolyzed rapidly to form dihydroartemisinin. Before any generalized conclusion about the metabolic behavior of antimalarial sesquiterpene lactones can b¢ drawn in detail, more metabolism studies on many different types of related compounds still remain to be performed. However, there are certain observations regarding the metabolites so far identified, from the currently available metabolism studies of antimalarial scsquiterpene lactone derivatives. (1) Metabolism of artemisininoids (artemisinin derivatives as a whole; natural or synthetic) tends to lead to the production of the more polar metabolites, which could be more water-soluble, and so more easily excreted after metabolic transformation. (2) Ether and ester derivatives of dihydroartemisinin tend to produce dihydroartemisinin, an O-dealkylated product, as a major metabolite. Dihydroartemisinin is still active as itself, and so the substrates leading to this compound seem to serve as prodrugs for dihydroartemisinin which shows in vivo activity. (3) Ether derivatives of dihydroartemisinin also tend to produce C9-hydroxy metabolites with retention of the intact endoperoxide and ether linkages. (4) All the metabolites with the intact endoperoxide moiety were active in the /n vitro antimalarial activity test, whereas none of the metabolites which had undergone the cleavage of endoperoxide linkage were found active. (5) Microbial metabolisms tend to serve well as model systems for the prediction of mammalian metabolism, and for the production of microbial metabolites as reference standards which can aid mammalian metabolism studies and structure elucidation studies. (6) Metabolism studies oftentimes provide metabolites with higher potency than their parent drugs, which can, in themselves, serve as models for future drug design or for further development of newer drugs. In this category are artcetber, which produces a high concentration of dihydroartemisinin and 9flhydroxyarteether, and artemetber which produces 9-hydroxyartemetber.

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Acknowledgements--The authors thank Dr Wilbur K. Milhous of Walter Reed Army Medical Center, Washington, DC, for testing the microbial metabolites for in vitro antimalarial activity. The full results of these studies will be published in the future. The authors also thank Dr Arnold Brossi of National Institute of Health, Bethesda, MD, for helpful discussion and providing reference standards.

REFERENCES ACTON, N. and KLAYMAN,D. L. (1985) Artemisitene, a new sesquiterpene tactone endoperoxide from Artemisia annua. Planta Med. 441-442. BAKER, J. K., YARBER, R. H., HUFFORD, C. D., LEE, I.-S., ELSOHLY, H. N. and McCHESNEY, J. D. (1989) Thermospray mass spectroscopy/high performance liquid chromatographic identification of the metabolites formed from artcether using a rat liver microsome preparation. Biomed. Environ. Mass Spectrom. 18: 337-351. BROSSl,A., VENUGOPALAN,B., GERPE, L. D., YEH, H. J. C., FLIPPEN-ANDERSON, J. L., BUCHS, P., LUO, L. D., MILHOUS, W. and PETERS,W. (1988) Arteether, a new antimalarial drug: synthesis and antimalarial properties. J. med. Chem. 31: 645-650. CAO, M., Hu, S., LI, M. and ZHANG, S. (1982) Synthesis of carboxylic esters of dihydroartemisinin. Nanjing Yaoxueyuan Xuebao 1: 53-54. CHINA COOPERATIVERESEARCHGROUPON QINGHAOSUAND ITSDERIVATIVESASANTIMALARIALS(1982a) The chemistry and synthesis of qinghaosu derivatives. J. trad. Chin. Med. 2(I): 9-16, CHINA COOPERATIVERESEARCHGROUPON QINGHAOSUAND ITS DERIVATIVESAS ANTIMALARIALS(1982b) Antimalarial efficacy and mode of action of qinghaosu and its derivatives in experimental models. J. trad. Chin. Med. 2(1): 17-24. CHINA COOPERATIVERESEARCHGROUPON QINGHAOSUAND ITS DERIVATIVESAS ANTIMALARIALS(1982C) Metabolism and pharmacokinetics of qinghaosu and its derivatives. J. trad. Chin Med. 2(1): 25-30. CLARK, A. M., HUFFORD, C. D. and McCHESNEY, J. D. (1981) Primaquine metabolism by microorganisms and ~3C nuclear magnetic resonance assignments. Antimicrob. Ag. Chemother. 19: 337-341. CLARK, A. M., MCCHESNEY, J. D. and HUFFORD, C. D. (1985) The use of microorganisms for the study of drug metabolism. Med. Res. Rev. 5: 231-253, COUTTS, R. T. and JONES, G. R. (1980) Significance of Analytical Techniques in Drug Metabolism Studies. In: Concepts in Drug Metabolism, Part A, pp. 1-51, JENNER, P. and TESTA,B. (eds) Marcel Dekker, New York. DUTTA, G. P., BAJPAI,R. and VISHWAKARMA,R. A. (1989) Comparison of antimalarial efficacy of artemisinin (qinghaosu) and arteether against Plasmodium cynomolgi B infection in monkeys. Trans. R. Soc. trop. Med. Hyg. 83: 56-57. ELMARAKBY, S. A., EL-FERALY, F. S., ELSOHLY, H. N., CROOM, E. M. and HUFFORD, C. D. (1987) Microbial transformation studies on arteannuin B. J. Nat. Prod. 50: 903-909. FERR1S,J. P., FASCO,M. J., STYLIANOPOULOU,F. L., JERINA, D. M., DALI, J. W. and JEFFREY,A. M. (1973) Monooxygenase activity in Cunninghamella bainieri: evidence for a fungal system similar to liver microsomes. Archs Biochem. Biophys. 156: 97-103. GOODHUE, C. T. (1982) The Methodology of Microbial Transformation of Organic Compounds. In: Microbial Transformations o f Bioactive Compounds, Vol. 1, Chap. 2, pp. 9-44, ROSAZZA,J. P. (ed.) CRC Press, Boca Raton, Florida.

Gu, H. M., Lu, B. F. and Qu, Z. X. (1980) Activities of 25 derivatives of artemisinine against chloroquine-resistant Plasmodium berghei. Acta pharmac, sin. 1: 48-50. HUFFORD, C. D. and ELSOHLY, H. N. (1987) ~H and t3C NMR assignments of arteethers. Spectrosc. Lett. 20: 439--444. HUFFORD, C. D., BAKER, J. K. and CLARK, A. M. (1981 a) Metabolism of phencyclidine by microorganisms. J. Pharm. Sci. 70: 155-158. HUFFORD, C. D., CAPITON, G. A., CLARK, A. M. and BAKER,J. K. (1981 b) Metabolism of imipramine by microorganisms. J. Pharm. Sci. 70: 151-155. HUFFORD, C. D., LEE I.-S., ELSOHLY, H. N., CHI, H. T. and BAKER, J. K. (1990) Structure elucidation and thermospray HPLC/MS of the microbial and mammalian metabolites of the antimalarial arteether. Pharm. Res. 7: 923-967. IDOWU, O. R., EDWARDS,G., WARD, S. A., ORME, M. L'E. and BRECKENRIDGE,A. M. (1989a) Determination of arteether in blood plasma by high-performance liquid chromatography with ultraviolet detection after hydrolysis with acid. J. Chromat. 493: 125-136. IDOWU, O. R., WARD, S. A. and EDWARDS, G. (1989b) Determination of artelinic acid in blood plasma by highperformance liquid chromatography. J. Chromat. 495: 167-177. IMAKURA,Y., YOKOl,T., YAMAGISHI,T., KOYAMA,J., HU, H., MCPHAIL, D. R., McPHAIL, A. and LEE, K.-H. (1988) Synthesis of desethanoqinghaosu, a novel analogue of the antimalarial qinghaosu. J. chem. Soc., chem. Commun. 372-374. JENNER,P. and TESTA,B. (1981) Concepts in Drug Metabolism, Part B, Marcel Dekker, New York. JEREMIC, D., JOKIC, A., BEHBUD,A. and STEFANOVIC,M. (1973) A new type of sesquiterpene lactone isolated from Artemisia annua L. Tet. Lett. 32: 3039-3042. JIANG,J., YAN, H., ZHUANG,Y., XU, G., ZENG,Y. and DING, S. (1983) Absorption, distribution, and excretion of artemether in mice and rats. Zhongguo Yaoli Xuebao 4(3): 197-201. JIANG,J.-R., ZOU, C.-D., SHU, H.-L. and ZENG,Y.-L. (1989) Assessment of absorption and distribution of artemether in rats using a thin layer chromatography scanning technique. Acta pharmac, sin. 10(5): 431-434. JUNG, M., LI, X., BUSTOS,D. A., ELSOHLY,H. N. and McCHESNEY,J. D. (1989) A short and stereospecific synthesis of (+)-deoxoartemisinin and (-)-deoxodesoxyartemisinin. Tet. Lett. 30: 5973-5976. KAR, K., NATH, A., BAJIPAI, R., DUTTA, G. P. and VISHWAKARMA, R. A. (1989) Pharmacology of ct/fl arteether--A potential antimalarial drug. J. Ethnopharmac. 27: 297-305. KIESLICH, K. (1976) Microbial Transformations o f Nonsteroid Cyclic Compounds, Wiley-Georg Thieme, Stuttgart. KLAYMAN,D. L. (1985) Qinghaosu (artemisinin): an antimalarial drug from China. Science 228: 1049-1055. LEE, I.-S., ELSOHLY, H. N., CROOM, E. M. and HUFFORD, C. D. (1989) Microbial metabolism studies of the antimalarial sesquiterpene artemisinin. J. Nat. Prod. 52" 337-341. LEE, I.-S., ELSOHLY, H. N. and HUEFORD,C. D. (1990) Microbial metabolism studies of the antimalarial drug arteether. Pharm. Res. 7: 199-203. LI, G. Q., Guo, X. B., JIN, R., WANG, Z. C., JIAN, H. X. and Ll, Z. Y. (1982) Clinical studies on treatment of cerebral malaria with qinghaosu and its derivatives. 3". trad. Chin. Med. 2: 125-130. Ll, Z. L., Gu, H. M., WARHURST,D. C. and PETERS,W. (1983) Effects of qinghaosu and related compounds on incorporation of [G-3H] hypoxanthine by Plasmodium falciparum in vitro. Trans. R. Soc. trop. Med. Hyg. 77: 522-523.

Metabolism of antimalarial sesquiterpene lactones LIANG) X.-T. (1985) The chemistry of peroxidic antimalarials. Adv. Chin. Med. Mater. Res. 427--432. LIANG,X.-T,, YU, D.-Q., WE, W.-L. and DENG,H.-C. (1979) The structure of Yingzhaosu A. Acta chim. sin. 37: 215-230. LIN, A. J., KLAYMAN,D. L., HOCH, J. M., SILVERTON,J. V. and GEORGE,C. F. (1985) Thermal rearrangement and decomposition products of artemisinin (qinghaosu). J. org. Chem. 50: 4504-4508. LIN, A. J., KLAYMAN,D. L. and M1LHOUS,W. K. (1987) Antimalarial activity of new water-soluble dihydroartemisinin derivatives. J. reed. Chem. 30: 2147-2150. LIN, A. J., LEE,M. and KLAYMAN,n. L. (1989) Antimalarial activity of new water-soluble dihydroartemisinin derivatives. 2. Stereospecificity of the ether side chain. J. reed. Chem. 32: 1249-1252. LIE, J. M., NI, M. Y., FAN, J. F., TU, Y. Y., Wu, Z. H,, Wu, Y. L. and CHOU, W. S. (1979) The structure and reactions of qinghaosu. Aeta chim. sin. 37: 129-143. LuG, X.-D. and SHEN, C,-C. (1987) The chemistry, pharmacology, and clinical applications of qinghaosu (artemisinin) and its derivatives. Med. Res. Rev. 7: 29-52. LUG, x.-n., YEH, H. J. C. and BROSSl,A. (1984) Detection of metabolites of qinghaosu in nanomolar quantities. DADF-esters of dihydroqinghaosu and its epoxy analog. The chemistry of drugs, V. Heterocycles 22: 2559-2562. LuG, X.-D., YEH, H. J. C., BROSSI,A., FLIPPEN-ANDERSON, J. L. and GILARDI,R. (1985) The chemistry of drugs, VI. Thermal decomposition of qinghaosu. Heterocycles 23: 881-887. LtJo, X.-D., XIE, M. and Zou, A.-Q. (1987) Subnanogram detection of dihydroartemisinin after chemical derivatization with diacetyldihydrofluorescein followed by highperformance liquid chromatography and uv absorption. Chromatography 23:112-114. NIu, X., HO, L., PEN, Z. and SONG,Z. (1985) Metabolic fate of qinghaosu in rats: a new tic densitometric method for its determination in biological material. Eur. J. Drug Metab. Pharmacokinet. 10: 55-59. ORRENIUS, S. and ERNESTER,L. (1974) Microsomal Cytochrome P-450-1inked Monooxygenase Systems in Mammalian Tissues. In: Molecular Mechanisms of Oxygen Activation, pp. 216-218, HAYAISHIO. (ed.) Academic Press, New York.

355

REPORT OF THE STEERING COMMITTEEOF THE SCIENTIFIC WORKING GROUP ON THE CHEMOTHERAPYOF MALARIA (1986) tdr/chemal/art/86.3, p. 19. WHO, Geneva, Switzerland. Rivcrtm, J. M. (1975) The Aliphatic Alcohols. In: Pharmacological Basis of Therapeutics, pp. 146-148 (5th ed.), GOODMAN, L. S. and GILMANA. (eds) Macmillan, New York. SMITH, R. V. and ROSAZZA,J. P. (1974) Microbial models of mammalian metabolism. Aromatic hydroxylation. Archs Biochem. Biophys. 161: 551-558. SMITH,R. V. and ROSAZZA,J. P. (1975) Microbial models of mammalian metabolism. J. Pharm. Sci. 64: 1737-1759. TANI, S., FUKAMIYA,N., KIYOKAWA,H., MUSALLAM,H. A., PICK, R. O, and LEE,K.-H. (1985) Antimalarial Agents. 1. ~-Santonin-derived cyclic peroxide as potential antimalarial agent. J. reed. Chem. 28: 1743-1744. TESTA,B. and JENNER,P. (1976) Drug Metabolism: Chemical and Biochemical Aspects, Marcel Dekker, New York. THAITHONG, S. and BEALE,G. H. (1985) Susceptibility of Thai isolates of Plasmodium falciparum to artemisinine (qinghaosu) and artemether, Bulletin of the Worm Health Organization 63(3): 617M19. THEOHARIDES, A. D., SMYTH, M. H., ASHMORE, R. W., HALVERSON,J. M., ZHOU, Z. M., RIDDER,W. E. and LIN, A. J. (1988) Determination of dihydroqinghaosu in blood by pyrolysis gas chromatography/mass spectrometry. Analyt. Chem. 60: 115-120. WARHURST,D. C. (1986) Antimalarial drugs: mode of action and resistance. J. antimicrob. Chemother. 18 (Suppl. B): 51-59. ZHAO, S.-S. and ZENG, M.-Y. (1985) Spektrometrische Hochdruck-Fliissigkeits-Chromatographische (HPLC) Untersuchungen zur Analytik yon Qinghaosu. Planta Med. 233-237. ZHU, D. Y., HUANG,B. S., CHEN,Z. L. and YIN, M. L. (1980) Studies on the physiological disposition of qing-hao-sou, I. Biotransformation of qinghaosou after oral medication in human, Yaoxue Xuebao 15: 509-512. ZHU, O. Y., HUANG, B. S., CHEN, Z. L., YIN, M. L., YANG, Y. M., DAI, M. L., WANG, B. D. and HUANG, Z. H. (1983) Isolation and identification of the metabolite of artemisinin in human. Zhongguo Yaoli Xuebao 4(3): 194--197.