Ornithine decarboxylase as an enzyme target for therapy

Pharmac. Ther. Vol. 54, pp. 195-215, 1992 Printed in Great Britain. All rights reserved

Associate Editor: P. K.

0163-7258/92 $15.00 © 1992 Pergamon Press Ltd

CHIANG

ORNITHINE DECARBOXYLASE AS AN ENZYME TARGET FOR THERAPY PETER P. MCCANN*"~ a n d ANTHONY E. PEGG~

*Marion Merrell Dow Inc., Indianapolis, IN 46268-0470, U.S.A. ~Departments of Cellular and Molecular Physiology and of Pharmacology, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA, U,S.A. Abstract--Interest in ornithine decarboxylase (ODC) and the therapeutic effects of its inhibition with the consequent depletion of polyamine biosynthesis has been widespread since the late 1970s and 1980s. This review covers new information about the properties of ODC, recent findings with ODC inhibitors and a discussion of the mechanism of inactivation of ODC by eflornithine. Recent in vivo therapeutic approaches of ODC inhibition are also discussed including: cancer and cancer chemoprevention; autoimmune diseases; polyamines and the blood-brain barrier, ischemia and hyperplasia; the NMDA receptor and modulation by polyamines; hearing loss; African trypanosomiasis; Pneumocystis carinii pneumonia and Cryptosporidium in AIDS; and other infectious diseases/organisms.

CONTENTS 1. Introduction 2. Ornithine Decarboxylase (E.C.4.1.1.17): The Enzyme 2.1. Properties 2.2. Inhibitors of ODC 2.3. Mechanism of inactivation by eflornithine 3. Ornithine Decarboxylase Inhibition: In Vivo Therapeutic Approaches 3.1. ODC inhibition and cancer 3.1.1. Disease treatment 3.1.2. Chemoprevention 3.2. Autoimmune diseases 3.3. Polyamines and the blood-brain barrier, ischemia and hyperplasia 3.4. NMDA receptor and modulation by polyamines 3.5. ODC inhibition and hearing loss 3.6. African trypanosomiasis 3.7. Pneumocystis carinii pneumonia and Cryptosporidium in AIDS 3.8. Other infectious diseases/organisms 3.8.1. Malaria 3.8.2. Trypanosoma cruzi 3.8.3. Fungi Acknowledgements References

195 196 196 199 201 203 203 203 205 205 206 207 207 208 209 209 209 210 210 210 210

*Corresponding author. Abbreviations: DFMO, ~t-difluoromethylornithine; eflornithine, Ornidyl~; DFMP, ~t-difluoromethylputrescine; MAP, (R,R)-6-methyl-ct-acetylenicputrescine; MFMO, ~t-monofluoromethylornithine; A-MFMO, (E)
196

P.P. MCCANNand A. E. PEGG 1. I N T R O D U C T I O N

Numerous reviews and books have summarized the immense amount of work about ornithine decarboxylase (ODC), polyamine biosynthesis and the pharmacological and therapeutic effects of inhibition of ODC and consequent polyamine depletion which occurred in the late 1970s and 1980s. Detailed information on this early work and a fuller list of available reviews can be obtained from one of the following sources and references contained in them (Pegg and McCann, 1982; Tabor and Tabor, 1984; McCann et al., 1987; Morgan, 1987; Pegg, 1988; Hayashi, 1989; Bachrach and Heimer, 1989; Seller, 1990, 1991; J~inne et al., 1991). A detailed bibliography of the work with eflornithine (ct-difluoromethylornithine usually abbreviated to D F M O and also known as Ornidyl'~), which has been the most widely used ODC inhibitor, comprises 1556 papers published since the initial reports of this compound in 1978 (Metcalf et al., 1978; Mamont et al., 1978). The present review attempts to cover information about ODC that has emerged in the last few years and the status of the current in vivo therapeutic approaches of inhibition of this enzyme. Thus, on the whole, only papers published since 1989 are cited, while review articles are generally noted to give background information. The topic as stated in the title only covers mammalian disorders and infectious diseases related to polyamines and does not touch upon the significant literature related to polyamine biosynthesis in plants and plant pathogens. The therapeutic section of this review covers only pathologies where there is recent or new laboratory or clinical evidence to indicate the validity of the polyamine inhibition approach. Other possible therapeutic utilities for ODC inhibition discussed in earlier publications, such as psoriasis and contragestion, are not covered here since there has been no more recent clinical or other data to warrant their inclusion. However, the absence of such data should not be interpreted to mean that the validity of this approach has been disproven. For a discussion of these particular topics see an earlier review by Schechter et al. (1987). Other enzymes in the polyamine biosynthetic pathway may also be valuable targets for therapy, either alone or in combination with ODC inactivators in order to maximize the effects on cellular polyamines. The possibility of using specific inhibitors of S-adenosylmethionine decarboxylase in this way is described in a companion review.* 2. O R N I T H I N E D E C A R B O X Y L A S E (E.C.4.1.1.17): T H E E N Z Y M E 2.1. PROPERTIES ODC has been the subject of a vast amount of research since the discovery of the mammalian enzyme and its rapid turnover and response to growth promoting stimuli in 1968. Much of this work is summarized in the chapters of a recent book (Hayashi, 1989). ODC preparations have now been isolated from a considerable array of species and, in all cases, the enzyme has been found to be dependent on pyridoxal Y-phosphate (PLP) for activity, cDNA clones containing the entire coding region for ODC have been obtained from Escherichia coli (Barroso et al., 1990; Kashiwagi et al., 1991), various mammalian sources (Gupta and Cottino, 1985; Kahana and Nathans, 1985; Hickok et al., 1987; Wen et al., 1989; van Kranen et al., 1987; Grens et al., 1989), Xenopus laevis (Bassez et al., 1990), Trypanosoma brucei (Phillips et al., 1987), Saccharomyces cerevisiae (Fonzi and Sypherd, 1987), Leishmania donovani (Hanson et al., 1992) and Neurospora crassa (Williams et al., 1992). The subunit MW values from the amino acid sequences deduced from these sources are, in most cases, in good agreement with the values from the purified enzymes where these have been determined. Table 1 lists the sizes and probable subunit structure of these ODCs. In all cases, ODC consists of a single subunit type and the complete enzyme is a dimer or higher aggregate of these subunits. Although there is significant variation in the size of the subunits, comparisons of the eukaryotic ODC sequences show a remarkable similarity between ODCs from these sources with more than 90% identity between the mammalian proteins and an 81% (Xenopus), 69% (Trypanosoma), 42% (Neurospora), 40% (Leishmania) and 40% (Saccharomyces) identity between these proteins and the murine ODC over the common core region of the enzyme. *A. E. Pegg and P. P. McCann, S-Adenosylmethionine decarboxylase as a enzyme target for therapy. Manuscript in preparation.

Ornithine decarboxylase

197

TABLE 1. Properties o f ODCs from Sources from which a cDNA has been Isolated and Sequenced

Subunit size Number of MW amino acids

Species Human Mouse Rat Hamster X. laevis S. cerevisiae N. crassa T. brucei L. donovani E. coli (biodegradative) E. coli (biosynthetic) Lactobacillus sp 30a

51,000 51,000 51,000 50,000 51,000 52,000 53,000 49,000 77,000 82,000 82,000 85,000

Subunits/molecule

461 461 461 455 460 466 484 445 707 732 731" "~

2 2 2 2 ? 2 2 2 ? 2 2 2 or 12

*The DNA derived sequence has 731 amino acids but it appears that the first 20 residues are removed to form the final enzyme (Kashiwagi et al., 1991; Barroso et al., 1990). tEnzyme has not been sequenced. Figure 1 shows the sequence of the murine ODC, which contains 461 amino acids and the residues that are invariant in all eukaryotic ODCs are underlined. There are 95 such amino acids and there are numerous other positions in which only highly conservative changes occur. The conserved amino acid nearest to the amino terminus is residue 40 and that nearest the carboxyl end is residue 410. These results are consistent with work showing that significant deletions of the protein at both ends do not abolish activity although, as discussed below, the turnover of the protein is affected. The eukaryotic O D C sequences show almost no similarity to the bacterial enzymes from E. coli and Lactobacillus sp. 30a O D C (Barroso et al., 1990; Kashiwagi et al., 1991; Guirard and Snell, 1980). Numerous pseudogenes of O D C are present in rodent tissues, but so far only one active O D C gene has been found in mouse and humans where it is located on chromosomes 12 (Berger, 1989) and 2 (Hickok et al., 1990), respectively. F r o m a structural point of view, the best studied O D C is the mouse enzyme but there are so few differences between this and the other mammalian ODCs including the human protein that it is highly likely that the catalytic properties are virtually identical. The mouse O D C consists of a

MSSFTKDEFDCHILDEGFTAKDILDQKINEVSSSDDKDAE-40 R2~FMO

YVA~L~DILKKHLR~LKAL[RVTEFYAVKCNDSRAIVSTL-80 AAIGTGFDCASKTEIQL~QGLGVPAERVIY~NPCKQVSQI-120 K Y A ~ S N G ~ Q M M ~ S E I~LM~VARAHEKAK L V L ~ D S - 160 KAVCRL~V~F~ATLKT SRLLLERAKELNIDVI GVSFHVGS-200 ~CTDP DTFVQAVSDARCI[EDMATEV~%FSMHLLD I GGGFP G-240 SEDTKLKFEEITSVINPALDKYFPSDSGVRIIA~YV-280 ASAFTL~VNII~KKTVWKEQPGSDDEDESNEQTFMY~V~-320 D~O

~V~GSFNC~LYD/IAHVKALLQKRPKPDEKYYSSSIWGPTC-360 ~GL~RIVERCNLPEMHV~D~MLFENM~VAAASTFNGF-400 QRPNIYYVM~RPMWQLMKQIQSHGFPPEVEEQDDGTLPMS-440 CAQESGMDRHPAACASARINV-461

FIG. 1. Amino acid sequence of mouse ODC. Residues that are conserved in all eukaryotic ODCs are underlined and the PLP binding site and the DFMO attachment positions are indicated. The amino acids shown in bold type are those known to play a major role in enzyme activity as a result of site specific mutagenesis.

198

P. P. MCCANNand A. E. PEGG PLP

N. c r a s s a T. b r u c e i

Mammalian X. l a e v i s S. c e r e v i s i a e L. d o n o v a n i

Leu Asn I Leu CyslLeu I Lys Ala Leu I Lys Ala Leu IL y s ~ Leu His GlulLeu

~

Pro Arg Pro Arg Pro Arg Pro Arg Pro Ar~ ProJMet

Vall LyslPro Phe Val Thr Pro Phe Val Thr Pro Phe Val Thr Pro Phe Ile Lys I Pro Phe VallArg ProlTyr

Tyr Ala Tyr Ala Tyr Ala Tyr Ala Tyr Ale PhelAla

Val Val Val Val Val Val

Lys CyslHis Pro m

Lys Cys Asn Lys Cys Asn Lys Cys Asn Lys~Asn LysISerlAsn

Aspl Asp Asp Pro Pro |

FIG. 2. Amino sequences of ODCs containing the PLP binding site. dimer of two identical subunits each having MW of ca. 51,000 (461 amino acids). It catalyzes the formation of putrescine from L-ornithine. The Km for L-ornithine is about 90 ~M and the kcat is about 40 sec -1 at 37°C. The enzyme will act on lysine but the Km is 100 times higher and this reaction is therefore of little physiological importance, occurring only under unusual circumstances where there is a large amount of enzyme and a very high lysine to ornithine ratio. Mammalian ODC has a striking requirement for sulfhydryl reducing agents such as dithiothreitol to maintain activity (Pegg and Williams-Ashman, 1981; Pegg, 1989a). The PLP binding site is at lysine-69 (Poulin et al., 1992). This lysine is present within a highly conserved region of eukaryotic ODCs (Fig. 2) but differs completely from that of the peptide forming the cofactor binding site of the E. coli ODCs which is -QSVHKQ-. The PLP-binding site of all eukaryotic ODCs except that from Leishmania contains the sequence -PYFAVKC- (Fig. 2). The presence of the cysteine residue in this sequence may contribute to the extreme dependency of ODC on sulfhydryl reducing agents. Another interesting feature of this sequence is that it does not conform to the consensus sequence of-SXHK- that contains the PLP binding lysine in many (but not all) other PLP-requiring enzymes including the E. coli ODC (Vaaler and Snell, 1989). It has been suggested that the histidine present in this sequence aids the binding of PLP to the enzyme by interacting with the phosphate group. Since mammalian ODC does not contain a basic residue in this location, this may account for the fact that this enzyme has a low affinity for PLP and is readily separated from the cofactor (Pegg, 1989a). In addition to the region around the PLP-binding site at position 69, there are several other highly conserved regions in the amino acid sequence of ODC including residues 164-171, 193-201 and 357-361 (Fig. 1). Mutation of residues within these regions namely lysine-69, lysine-169, histidine-197 and cysteine-360 to alanines abolishes or greatly reduces activity indicating that these sequences do indeed contribute towards the catalytic site (Lu et al., 1991; Pegg et al., 1992; Coleman and Pegg*). These are the only other point mutations that have been reported to abolish activity except for the change of glycine to aspartic acid at position 381 in the hamster ODC protein present in an ODC deficient mutant CHO cell line (Pilz et al., 1990). This position (residue 387 in the mouse sequence) is also in a conserved region, GAYT at amino acids 387-390 (Fig. 1). Changes to alanine that do not inactivate the enzyme include mutations of lysine-298, serine-303, glutamic acid-308 and lysine-349 (Lu et al., 1991; Rosenberg-Hasson et al., 1991b). The serine-303 to alanine change prevents phosphorylation at this site normally brought about in vivo by casein kinase II. Clearly, no functional significance can yet be assigned to this phosphorylation since the mutant protein is active and has a degradation rate similar to or only slightly less than the wild type enzyme. Another noteworthy conserved site in ODC is the region at residues 232-238 which contains the sequence -LDI(V)GGGF- (Fig. 1). Glycine-rich sequences of this type are found in a number of PLP dependent enzymes and have been postulated to form part of the cofactor binding region (Marceau et al., 1988). Since mouse ODC is normally a dimer containing two catalytic centers, it is possible that each active site involves residues from both subunits. A better understanding of the interaction of *C. S. Coleman and A. E. Pegg, Mutations affecting the active site of mouse ornithine decarboxylase. Manuscript in preparation.

Ornithine decarboxylase

199

residues making up the active site requires knowledge of the three dimensional structure and attempts to obtain this by crystallography are under way. Deletion of all or part of the 36 residues at the carboxyl terminus of mouse ODC does not affect the enzyme activity but has a profound effect on the stability of the protein preventing its rapid turnover (Ghoda et al., 1989; Rosenberg-Hasson et al., 1991a; Lu et al., 1991). Furthermore, addition of the 37 carboxyl terminal residues from ODC to dihydrofolate reductase increased the degradation of this protein in reticulocyte lysates or Xenopus extracts (Loetscher et al. 1991). This suggests that this section of the mammalian ODC protein contains a recognition signal for rapid degradation and the region does include the critical part of residues 423-449, a PEST, i.e. proline-glutamate-serine-threonine, region that was postulated to be such a recognition signal (Rogers et al., 1986). These results are entirely consistent with comparison with the Trypanosoma ODC that lacks this carboxyl sequence and is not rapidly degraded (Ghoda et al., 1990). However, the exact role of the PEST recognition sequence is unclear. Partial deletions of the carboxyl region, not all of which alter the PEST amino acids, also stabilize mouse ODC; and the Xenopus ODC, which also turns over rapidly, has significant differences with the mammalian enzyme in the carboxyl region and lacks a clear PEST region in this location (Osborne et al., 1991). Mouse ODC contains a second PEST region (residues 298-333) that may also have an effect on protein turnover. Deletions or alterations of these amino acids stabilized the protein under some circumstances but to a lesser extent that the truncation of the carboxyl terminal (Rosenberg-Hasson et al., 1991a; Lu et al., 1991; Pegg et al., 1992).

2.2. INHIBITORSOF ODC A myriad of ODC inhibitors have been described and their properties and potential have been discussed in several recent reviews (Sjoerdsma and Schechter, 1984; Bey et al., 1987; Danzin and Mamont, 1987; Pegg, 1989b; J/inne et al., 1991). The only compounds having sufficient potency and specificity to have pharmaceutical potential are the enzyme-activated irreversible inhibitors of which eflornithine is by far the best known and most widely studied and applied. Eflornithine (Fig. 3) is accepted as a substrate at the active site by the enzyme. It is then converted into a reactive intermediate that forms a covalent bond with ODC. As shown in Table 2, the Ki towards the mammalian enzyme is about 40 #M and inactivation proceeds with a half life of 3.1 min at saturating levels of the drug. A considerable number of compounds that act in a similar way have now been synthesized. A detailed comparison of these compounds is given by Bey et al. (1987) and the properties of some of the compounds of particular interest are shown in Table 2. Their structures are given in Fig. 3. These compounds have certain differences from eflornithine that may be exploitable to provide therapeutic benefits. For, example, A-MFMO has a lower Ki of only 2.7 #M and its methyl and ethyl esters were designed to facilitate uptake into the cell where it is converted to the active inhibitor by non-specific and widely distributed esterases (Sjoerdsma and Schechter, 1984; Danzin and Mamont, 1987; Claverie and Mamont, 1989). Irreversible inhibitors of ODC can be designed using derivatives of putrescine, the product of the reaction, as a basis rather than the ornithine substrate. Because of the microreversibility of the protonation step in the enzymatic decarboxylation, putrescine analogs having a latent reactive group attached to the ~tposition are also strong irreversible inhibitors ofODC (Metcalf et al., 1978; Bey et al., 1987). As shown in Table 2, MFMP and DFMP are comparable to MFMO and eflornithine towards the mammalian enzyme although the rate of reaction is somewhat slower. However, MAP in which the ~t-fluoromethyl group is replaced by an ~-acetylenic (ethynyl) moiety is a very effective inhibitor having both a lower K~ and a more rapid rate of inactivation (t~/2 of 1.8 min) than eflornithine. (The 6-methyl group is not needed for inactivation of ODC but was added to prevent the metabolism of this putrescine derivative by diamine oxidase.) MAP is very well taken up by cells and produces inhibition of polyamine synthesis at concentrations much less than eflornithine (Sjoerdsma and Schechter, 1984; Danzin and Mamont, 1987; Claverie and Mamont, 1989). In several animal models, it is a more potent antitumor agent. Whether this can

200

P. P. MCCANNand A. E. PEGG .COOH

H2N"~~,,,CHF2 NH2

H,N~,,'~cHOO2H NH2

ct-Difluoromethylornithine(DFMO;eflomithine)

H2N~ " ' C H 2 F

.COOH

a-Monofluoromcthylomithine(MFMO)

H2N*~~~,CcO~H2 CH3

NH2

NH2

(E)-o~-Monofluoromethyldchydroomithine (A-MFMO)

H 2 N ~

H

CHF2 H2N" / ~ ~ , ~ , C H 2 F NH2 NH2

cc-Difluoromethylputrescine(DFMP)

H...

A - M F M O methylester

a-Monofluorornethylputrescine (lVIHC~P)

NH2 H

CHa (R)-~-Ethynyl-(R)-8-rnethylputrescine(MAP*)

FIG. 3. Structures of some important inhibitors of ODC. More information on the properties of these inhibitors is given in Table 2. It should be noted that MAP is also known as (2R,5R)-6-heptyne-2,5-diamine and as (R,R)-6-methyl-~-acetylenicputrescine.

be transferred to therapeutic benefit remains to be determined since MAP appears to be more toxic than eflornithine (Cornbleet et al., 1986). There are some interesting species differences in the interaction of these inhibitors with ODCs. As shown in Table 2, although eflornithine inactivates all of the eukaryote ODCs, it produces no time-dependent inactivation of either of the E. coli enzymes (Kallio and McCann, 1981; Bey et al., 1987). However, M F M O and A-MFMO are inactivators of the E. coli ODCs (Table 2). The difference between the mono- and di-substituted fluoro-derivatives is maintained when the putrescine based inhibitors are considered since M F M P inactivates the E. coli enzymes and D F M P does not (Kallio et al., 1982). The explanation for the difference between the response of the prokaryote and eukaryote enzyme undoubtedly lies in the very different amino acid sequences of the prokaryotic ODCs that lack the sequence containing a cysteine residue which, as described in Section 2.3. is the major site of binding of eflornithine. However, the difference in response to the mono- and di-substituted fluoro-compounds is puzzling since the additional fluorine atom makes little difference to the size of the inhibitor and only one fluorine is needed to form a reactive intermediate. Recent work on the structure of the adduct formed by eflornithine in mammalian ODC does allow a tentative hypothesis to explain this difference (see below). Although the growth of Trypanosoma brueei is very sensitive to eflornithine and this parasite is effectively treated by the drug, the K~for the trypanosomal ODC is actually higher at 130/~M than the K~ for the mammalian enzyme (Bitonti et al., 1985; Table 2). It has been suggested that the long half life of the parasite ODC renders it less able to restore ODC activity (Ghoda et al., 1990) but many other factors are likely to contribute to the therapeutic response of sleeping sickness to eflornithine. A-MFMO methyl ester (but not the ethyl ester) is also a good antitrypanosomal agent but MAP, which is a potent inactivator, is not (Sjoerdsma and Schechter, 1984; Bitonti et al., 1985). These examples emphasize that toxicity, bioavailability and other pharmacokinetic considerations are equally or even more important than the biochemical parameters for the ODC inhibitors

201

Ornithine decarboxylase TAaLE2. Selected ODC Inhibitors Enzyme source

Compound Eflornithine (DFMO, Ornidyl®, ~t-Difluoromethylornihine)

Rat

Eflornithine (DFMO, Ornidyl~) Eflornithine (DFMO, Ornidyl~)

T. brucei E. coli

MFMO (ct-Monofluoromethylornithine)

Rat

MFMO

E. coli

A-MFMO [(E)-~t-Monofluoromethyldehydroornithine]

Rat

A-MFMO

T. brucei

A-MFMO

E. coli

A-MFMO methyl ester

tt/2

Ki (pM)

at saturation (min)

39

3.1

130 1 No irreversible inactivation at 5 mM 75 360 2.7

1.6 12 2.6

14

1.1

1700

3.7

Inhibitory only after action of esterases to generate A-MFMO

DFMP (ct-Difluoromethylputrescine)

Rat

DFMP

E. coli

MFMP (~-Monofluoromethylputrescine)

Rat

MFMP

E. coli

MAP [(R)-ct-Ethynyl-(R)-f-methylputrescine or (R,R)-fi-methyl-aactylenicputrescine] MAP

30

7.4

No irreversible inactivation at 5 mr,i 56

4.4

110

2.1

Rat

3

1.7

T. brucei

5

1.0

Data from Bitonti et aL (1985) and Bey et al. (1987).

SO long as the compound is capable of bringing about a complete and rapid reduction in ODC activity. 2.3. MECHANISMOF INACTIVATIONBY EFLORNITHINE The original mechanistic reasoning that led to the design and synthesis of ct-methyl substituted ornithine derivatives such as eflornitbine was that it was thought that the incorporation of a leaving group in this position would, after decarboxylation by the enzyme, generate a conjugated imine that would then alkylate the apoprotein (Metcalf et al., 1978; Bey et al., 1987). Studies of the kinetics of inactivation and of the fate of radioactively labeled eflornithine during the inactivation were in full agreement with this hypothesis. The fact that the inactivation was active-site directed was confirmed by showing that the presence of L-ornithine or putrescine prevented the inactivation (Metcalf et al., 1978). Furthermore, when [5-14C]eflornithine was used, it was found that inactivation was accompanied by the binding of a stoichiometric amount of eflornithine to the ODC subunit and that this binding was prevented by L-ornithine (Seely et al., 1982; Pegg et al., 1987). When [l~4C]eflornithine was incubated with ODC, there was a release of ~4CO2 and the partition ratio of decarboxylation to binding was about 3.3 (Pegg et al., 1987). This indicates that decarboxylation of eflornithine by ODC does indeed occur and generates a highly reactive intermediate at the active site. Furthermore, this intermediate very frequently (slightly

202

P.P. MCCANNand A. E. PEt_a3 +

0

~/co~

H3N""'-~

11( ~ .

NH2 I~'--CHF2 ~ , , ~ ~

H I~3

(•)--Cys•O-S"

002 . J4~F_= H3N ~

N CHFT~

J

/----~CHF

'~

N~

Py s-c,,~ H3N~'~C" H NH2~

H

.,t~__/'~c... N~

3

PY

..Cs_c,,.,0e 'a"H,

l

. H3+N--J~

S--Cys3e°~) I ~CH~j ~,,~ "F N

rL.?"

Py=

H FIG. 4. Inactivation of ODC by eflornithine. less than one time in three) binds to a residue in the ODC molecule and brings about the inactivation. The actual sites of eflornithine binding have been identified recently using the recombinant mouse ODC (Poulin et al., 1992). Expression of this protein in E. coli was used to obtain large quantities of this normally non-abundant protein and, after reaction with [5-14C]eflornithine, the labeled protein was analyzed by protease digestion followed by HPLC separation of the labeled peptides and protein sequencing. The major site of binding accounting for about 90% of the total was the cysteine residue at position 360. The actual structure of the adduct at cysteine-360 was shown by FAB-MS (fast action bombardment mass spectrometry) to be the cyclic imine, S-((2-(1-pyrroline))methyl)-cysteine (Poulin et al., 1992). This adduct is very readily oxidized to the S-((2pyrrole)methyl)cysteine unless stabilized by reduction with NaBH 4 to S-((2pyrrolidine)methyl)cysteine. The mechanism of the formation and structure of this adduct is shown in Fig. 4. Eflornithine is accepted by the active site of ODC where it forms a Schiff base with PLP. Subsequent decarboxylation of eflornithine followed by elimination of a fluoride anion generates a conjugated imine. This reactive electrophilic imine is capable of alkylating the nucleophilic thiol group of cysteine-360. Subsequent elimination of another fluoride anion yields a second conjugated imine, which then undergoes a transaldimination reaction with the amino group of lysine-69. The corresponding enamine formed by this reaction may then cyclize internally with a concomitant loss of ammonia to give a cyclic imine, S-((2-(1-pyrroline))methyl)cysteine. This mechanism is entirely consistent with the postulated mechanism that was the basis for the synthesis of eflornithine (Metcalf et al., 1978). There is also a minor site of binding of eflornithine to ODC accounting for about 10% of the total at lysine-69, which, as described above, is the site of PLP attachment (Poulin et al., 1992). The structure of the lysine-69 adduct has not yet been determined but it could be formed in a similar reaction to that described above in which lysine is alkylated by the imine or via the pathway described by Likos et al. (1982) for the inactivation of PLP-dependent enzymes by suicide substrates. (In this pathway, the activated form of the inhibitor engages in a nucleophilic attack on the aldimine carbon of the cofactor to initiate the normal transaldimination process leading to

Ornithine decarboxylase

203

DFMO

11

N. crassa T. brucei

Mammalian X. laevis S. cerevisiae L. donovani

Ser Ile Trp Gly Ser[Val]Trp Gly Ser Ile Trp Gly Ser Ile Trp Gly Ser I l e ~ G l y Thrl IlelPhelGly

Pro Pro Pro Pro Pro Pro

Thr Thr Thr Thr Thr Thr

Cys Cys Cys Cys Cys Cys

Asp G l y [ I l e [ A s p ~ Ile[! Asp Gly Leu Asp Ile Asp Gly Leu Asp Arg Ile Asp Gly Leu A s p ~ Ile[ Asp Gly Leu Asp[Cys[Ile[ Asp lSer MetlAsplCys

FIG. 5. Amino sequences of ODCs containing the major eflornithine (DFMO) binding site.

product release and reformation of the lysine-PLP Schiff base. The liberated reactive enamine intermediate can then react with the reformed internal aldimine.) In any case, it is clear that either adduct inactivates ODC since inactivation occurs with a stoichiometry of 1 molecule of eflornithine bound per subunit (Seely et al., 1982; Poulin et al., 1992). The presence of two different sites that can be attacked including the PLP-binding lysine, which is an essential residue for activity, renders it most unlikely that mutant forms of eukaryotic ODC resistant to eflornithine will occur readily. Recent studies in which cysteine-360 has been converted to an alanine by site-specific mutagenesis are in agreement with this.* The C360A mutant ODC has a greatly reduced activity but retains about 1.5% of the activity of the wild type enzyme. However, it is still sensitive to inhibition by eflornithine, albeit with different kinetics. The eflornithine-binding site at cysteine-360 is contained in a highly conserved region of ODC in the pentapeptide -GPTCD- that is present in all known eukaryotic ODC sequences (Fig. 5). Such conservation also indicates that this residue is likely to be located close to the active site of the enzyme. Mutation of cysteine-360 to serine completely abolished ODC activity* but the small activity of the C360A mutant described above renders it unlikely that the cysteine acts as an essential proton donor needed for the decarboxylation mechanism. Such as proton donor could be lysine-169 or histidine-197 since the K169A and H197A mutants are inactive (Lu et al., 1991). The general features of the inactivation of ODC by other enzyme-activated irreversible inhibitors are likely to be similar to the scheme shown in Fig. 4 for eflornithine. However, compounds such as MFMO, A-MFMO and MFMP, which have only a single fluorine substituent, could not undergo the second elimination of a fluoride ion and thus the nature of the adduct would be different. The fact that the adduct produced by eflornithine does undergo a second reaction after binding to the enzyme may be relevant to the lack of irreversible inactivation of the E. coli ODC described above (Section 2. 2.). It is conceivable that an adduct is formed with the E. coli enzyme by both eflornithine and M F M O but that the further reaction of the eflornithine-derived adduct leads to its instability and the regeneration of the active enzyme.

3. O R N I T H I N E D E C A R B O X Y L A S E INHIBITION: I N V I V O T H E R A P E U T I C APPROACHES 3.1. O D E INHIBITIONAND CANCER 3.1.1. Disease Treatment

Eflornithine has had significant efficacy in slowing the growth of tumor cells in vitro and in many animal models in vivo both alone (see Sunkara et al., 1987) and in combination with several cytotoxic agents (see Porter and J/inne, 1987). However, when used as a single agent in more than 500 patients with a variety of malignancies, eflornithine did not seem to significantly *C. S. Coleman and A. E. Pegg, Mutations affecting the active site of mouse ornithine decarboxylase. Manuscript in preparation.

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affect tumor growth or progression of disease (reviewed by Schechter et al., 1987). Some studies had suggested that a combination of eflornithine with interferon elicited a response in some patients with malignant melanoma (Meyskens et al., 1986). However, subsequent phase II studies indicated this combination showed little promise as therapy (Creagan et al., 1990a,b). Other combinations that had been tried in patients with recurrent brain tumors were eflornithine with methylgloxal bis(guanylhydrazone (Levin et al., 1987 and submitted manuscript) and eflornithine with BCNU (1,3bis(2-chloroethyl)-l-nitrosourea) (Prados et al., 1989) both of which showed promise and indicated some sustained response or stable disease in an unexpected number of patients. However, the most interesting finding was when eflornithine alone was tried against recurrent gliomas and 46% of the evaluable patients benefited from the monotherapy.* This quite striking result was significant enough to warrant a second study in similar patients which if, when repeated, showed similar data, would allow filing of an eflornithine New Drug Application with the FDA for a cancer indication. Another interesting use of eflornithine was initiated in a phase I study, again against metastatic melanoma, wherein it was combined with systemic hyperthermia. The rationale for this was that polyamine depletion caused by eflornithine in several tumor cell and animal models sensitized the cancer cells to the cytotoxic effects of hyperthermia (Harari et al., 1990). No clinical results are known as yet. Several other recent clinical trials with eflornithine have been done wherein colon or rectal carcinoma patients were treated with continuous-infusion of a median dose of 8g/m2/day. There seemed to be a correlation between the steady-state plasma levels of eflornithine and lowering of platelets (Ajani et al., 1989) as well as reduction of the gastrointestinal toxicity generally noted in oral doses of eflornithine (Ajani et al., 1990). However, there did not seem to be any significant activity against tumors in either study. Although numerous animal studies on the efficacy of eflornithine in dozens of tumor models have published in the past ten years, there are at least two recent ones worth mentioning here. Evers et al. (1991a,b) have shown that a human carcinoid tumor, a endocrine neoplasm derived from neuroectodermal cells, could be transplanted to nude mice and its growth could be suppressed by eflornithine as a single agent. Treatment of carcinoid tumors has been hampered by both a lack of effective drugs and an animal model and now eflornithine may prove to be useful in treating patients with this tumor (Evers et al., 1991a,b). Other studies were also revealing in the context that they compared four different, but very specific, potent irreversible inhibitors of ODC: eflornithine, MAP and A-MFMO methyl and ethyl esters. As described above, although widely used, eflornithine is not as potent biochemically as the latter inhibitors, and, because of supply limitations, direct biological comparison of these inhibitors has been quite limited. A-MFMO methyl and ethyl esters were considerably more potent than eflornithine against L1210 leukemia in mice giving equal effects at one fifth the dose. MAP was even more effective on this basis being at least 4 times more active than the esters (Claverie and Mamont, 1989). Against Lewis lung carcinoma, the ethyl ester of A-MFMO was more active than the other compounds and, once again, the esters of A-MFMO and MAP were more potent than eflornithine. In a B16F1 mouse melanoma model, A-MFMO methyl ester was significantly more effective in inhibiting tumor growth (95%) than MAP (79%) or eflornithine (87%) used at a 4 times higher concentration (Bowlin et al., 1990). Another interesting facet of this study was that eflornithine and A-MFMO methyl ester augmented mouse macrophage tumoricidal activity against the B16F1 target cells, independent of their direct effects on the actual growth of the tumor cells; MAP did not augment macrophage activity (Bowlin et al., 1990). (Another immunopotentiating activity of eflornithine will also be discussed below in Autoimmune Diseases.) In any case, the advantages of A-MFMO esters over eflornithine are a reminder that other inhibitors of ODC may ultimately

*V. A. Levin, M. D. Prados, W. K. A. Yung, M. J. Gleason, S. Ictech and M. Malec, Treatment of recurrent gliomas with eflornithine. J. natn Cancer Inst., submitted.

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prove to be more clinically useful than eflornithine itself, although given the current development status of eflornithine, this possibility seems a long way off. Another interesting possibility utilizing eflornithine or other ODC inhibitors for the treatment of neoplastic disease arises from the discovery by Persson et al. (1988) that a mutant line of L1210 leukemia cells deficient in polyamine transport was significantly more sensitive to the therapeutic effects of eflornithine in inoculated mice. There was an 87% increase in median survival time of the mice and these results were directly attributable to an inability of the mutant L1210 cells to 'scavenge' polyamines and partially overcome the polyamine depletion caused by eflornithine. Subsequent experiments using normal L1210 cells or Lewis lung carcinoma cells in mice indicated that the gastrointestinal tract was a source of polyamines for animals treated with eflornithine and greater efficacy could be achieved by treatment of animals with eflornithine plus a polyamine oxidase inhibitor. Furthermore, a polyamine deficient diet along with antibiotics to decontaminate microorganisms in the gut would further reduce the availability of gastrointestinal polyamines and again increase efficiency of eflornithine treatment of the above tumors (Sarhan et al., 1989). Later studies were done on rats with intracranial glioblastomas with similar results (Sarhan et al., 1991). Overall, the potential of the use of a polyamine deficient diet, combined with gut decontamination, may significantly increase the utility of eflornithine in cancer. 3.1.2. Chemoprevention

A second major interest in the use of eflornithine in cancer has been as a chemopreventive agent. Ideas that low doses of eflornithine could be used clinically in prophylactic situations have been proposed for some time. Luk and Baylin (1984) raised the possibility for the treatment of familial polyposis where dysplastic polyps and ultimately colon cancer might be prevented by the use of eflornithine in specific prone populations. A number of animal models have shown that various types of cancers, e.g. skin, breast, colon, urinary bladder and intestinal, can be inhibited, dramatically in some cases, by eflornithine (see Thompson et al., 1986; Verma and Boutwell, 1987; Verma, 1989). Recently, it was demonstrated that doses of eflornithine (0.5-3g/m2/day), many times lower than the maximally tolerated doses (ca. 9g/mZ/day) used in cancer treatment (see above), can inhibit a tumor promoter-mediated induction of ODC in human skin (Loprinzi et al., 1989). Other studies have also shown significant effects of eflornithine on methylnitrosourea induced tracheal carcinoma in hamsters (Ratko et al., 1990) and significant additive effects of piroxicam (a nonsteroidal anti-inflammatory) plus eflornithine in the chemoprevention of colon carcinogenesis in rats (Reddy et al., 1990; Rao et al., 1991). Furthermore, exposure of the offspring to eflornithine in the drinking water exerted a slight but significant inhibitory effect on the incidence of tumors in the offspring of rats treated transplacentally with ethylnitrosourea (Alexandrov et al., 1991). This is a remarkable result in view of the potency of tumor initiation in this model. All of the above mentioned studies have led the Chemoprevention Branch of the National Cancer Institute (see Boone et al., 1990) to designate eflornithine as a high priority compound to evaluate against several types of human cancers, including familial polyposis. Another clinical trial has shown that low doses of eflornithine (0.5g/m2/day) can significantly lower polyamines in G.I. mucosa of patients with Barrett's Esophagus, a premalignant precursor lesion for adenocarcinoma of the esophagus (Garewal et al., 1991). Additional strategies are planned to look at the actual lesion response itself. Overall, the potential use of low doses of eflornithine for chemoprevention of cancer seems likely to be one of the most promising avenues for the future success of ODC inhibitors in the treatment of malignancies.

3.2 AUTOIMMUNE DISEASES

A question often raised is, as eflornithine will inhibit red and white cell and platelet production in man, why is there no apparent effect on the immune system? There is still no simple, single response to this, but recent evidence has demonstrated some inhibition of skin graft rejection in mice by eflornithine (Campbell et al., 1991) indicating a general suppressive effect on the mouse immune system. Curiously, another study has shown that eflornithine would not only inhibit skin JPT 54/2--F

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carcinogenesis induced by ultraviolet B (UVB) irradiation in mice (the first report of its efficacy against cancer caused by a physical carcinogen), but that the drug actually blocks the immunosuppression also induced by UVB irradiation (Gensler, 1991). The latter result was ascribed to the inhibition of local immunomodulation which in turn would increase the host capacity to respond immunologically to UVB-induced antigenic skin tumors. Thus, one has to be cautious in defining whether or not polyamine depletion effected by ODC inhibition has 'macro' effects on immune function. Recently, it was demonstrated, as well, that eflornithine was a potent enhancer of interleukin 2 production by rheumatoid arthritis cells and suggested that in the absence of eflornithine, increased polyamine levels suppressed interleukin 2 by a mechanism related to the products of spermidine oxidation. Furthermore, these immunosuppressive products related to polyamines and their oxidation may play a role in the T cell hyporesponse of rheumatoid arthritis patients (Flescher et al., 1989), thus indicating yet another potential clinical utility for eflornithine. Other immune diseases have also been suggested as targets for ODC-inhibition therapy, particularly in the treatment of lupus, which has been linked with production of anti-DNA antibodies. Originally, it was shown that when MAP was administered to MRL-lpr/lpr mice, a strain genetically predetermined to develop an autoimmune disease similar to lupus erythematosus, it inhibited the characteristic splenomegaly, retarded the development of lymphadenopathy and prolonged survival (Claverie et al., 1988). Although eflornithine was found to be inactive in this study, when used in the same model and administered in a different regimen and at a higher dose by Thomas and Messner (1989), the survival time of female MBL-lpr/lpr mice was indeed increased and onset of lymphadenopathy was delayed by five weeks as well. Furthermore, the sera of the eflornithine treated mice contained a significantly lower concentration of anti-DNA antibodies compared with untreated mice (Thomas and Messner, 1989). Subsequent studies also showed a major beneficial effect on lupus nephritis as well and found that there was a 79% increase in the median life span with an optimal dose of eftornithine (Gunnia et al., 1991). In another lupus model, NZB/W female mice treated with eflornithine, there was an 80% reduction of anti-DNA antibodies as well as significant effects on other biochemical parameters associated with the clinical manifestation of lupus (Thomas and Messner, 1991). All these studies thereby suggest that polyamine biosynthesis inhibition by eflornithine may provide a new approach to the treatment of lupus, now an essentially untreatable disease. A related study was also done with MAP wherein its effects on collagen-induced arthritis in mice were examined. Development of arthritis was inhibited only in mice receiving 0.5% MAP. Furthermore, when control mice were developing arthritis, serum anti-collagen antibodies were substantially lower (70%) in the drug treated group (Wolos et al., 1990). The results indicated that ODC inhibition and polyamine depletion could prevent development of collagen-induced arthritis by inhibiting the autoimmune response and again suggested that drugs such as MAP and eflornithine deserve clinical investigation as immunosuppressive drugs targeted to specific diseases. 3.3. POLYAMINESAND THE BLOOD--BRAINBARRIER, ISCHEM1AAND HYPERPLASIA The polyamines have been implicated as possible mediators and even being obligatory for several pathophysiological brain responses related to blood-brain barrier breakdown. An early study showed that cold induced blood-brain barrier breakdown and consequent edema involved accumulation of polyamines in rats. Eflornithine suppressed the cold-induced injury by 70% in these animals (Koenig et al., 1983). Later work has shown similar results in a rat spinal cord model of late-delayed radiation injury wherein rat spinal cord polyamines and water content could be significantly reduced by treatment with eflornithine after irradiation (Gutin et al., 1990). These data strongly suggest that the use of eflornithine to inhibit polyamine synthesis could be clinically useful in treatment of edema caused by radiation injury. Another exciting recent finding was in a dog model of J25I-radiation induced brain injury. The volumes of edema, necrosis and brain tissue showing evidence of blood-brain barrier breakdown was significantly altered by eflornithine treatment of the dogs compared to controls (Gobbel et al., 1991). This again indicates that the effect of ODC inhibition on restricting edema is mediated by a specific action preventing blood-brain barrier breakdown. What is still unclear in all these studies is the mechanism by which polyamines affect the actual breakdown of the blood-brain barrier and why a specific blockage of new

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polyamine synthesis will, in fact, preserve the barrier and restrict or block edema formation after radiation injury. Despite this, the potential clinical implications of the use of eflornithine in radiation treatment of brain tumors could ultimately be as useful as direct eflornithine treatment of brain tumors themselves. Experiments looking at prevention of cell death following ischemia in rat skin flaps, also demonstrated that eflornithine increased survival of the flaps, suggesting that clinical utility could be to increase the viability of such flaps and salvage compromised skin flaps. Furthermore, a general protection of ischemic tissue by eflornithine could be of even greater clinical importance in protecting tissues such as heart and brain (Perona et al., 1990). Again, the direct mechanism of why ODC inhibition protects non-cerebral ischemic tissue is not clear, although a number of hypotheses have been proposed in the case of ischemic brain damage (see Section 3.4 below). Intimal hyperplasia, a significant cause of restenosis seen in carotid arteries after surgery, has long been suggested as a target for the restriction of cell growth by use of an ODC inhibitor to deplete polyamines. This possibility has now been confirmed by an in vivo study done in rabbits that demonstrated reduced development of intimal hyperplasia in rabbit arteries after both two (83% reduction) and four weeks (74% reduction) of eflornithine treatment (Endean et al., 1991). Although more confirmatory experiments need to be done, particularly to define the duration of eflornithine treatment needed to sustain the restriction of intimal hyperplasia, clearly this could have significant therapeutic potential. 3.4. NMDA RECEPTORAND MODULATIONBY POLYAMINES A specific recognition site for polyamines has been shown to exist as a part of the N-methyl-Daspartate (NMDA) receptor complex in membranes from rat brain. Polyamines are essentially agonists at this recognition site and may actually modulate exitatory synaptic transmission by binding at this site of the NMDA receptor (see Williams et al., 1991). Many studies have been done looking at the effects of the natural polyamines, i.e. spermidine and spermine and novel synthetic polyamines on the binding of molecules such as MR-801, an open-channel blocker and on NMDA-elicited currents in neurons. The results have found various compounds to be partial agonists, antagonists and inverse agonists at the polyamine recognition site (Williams et al., 1990, 1991; Romano et al., 1991). Thus, the polyamine site may prove to be important as a therapeutic target for neurodegenerative diseases and other neurological pathologies such as Alzheimer's disease, epilepsy and ischemic brain damage (Williams et al., 1991). In fact, evidence indicates that stimulation of the NMDA receptor is implicated in neuronal damage produced by a number of environmental and chemical mediators. Thus, if polyamines are agonists for this receptor, specific inhibition of ODC and consequently polyamines could be of some pharmacological value in reducing neurological damage caused by stimulation of the NMDA receptor. Two recent studies, although preliminary, seem to indicate that eflornithine could protect against the neurotoxic effects of exogenously administered NMDA. Treatment of rats with eflornithine partially blocked the toxic effects of intrastriatally administration of NMDA (measured after a ten day period) (Porcella et al., 1991). An analogous experiment was done in rat pups where eflornithine was administered either by i.p. injection or through the milk of the mother. The brain damage produced by intrastriatal NMDA was significantly reduced by 48% and 62%, respectively (Kish et al., 1991). Although neither study demonstrated the specificity of the eflornithine effect, i.e. co-administration of polyamines, measurement of levels of polyamines in the brain tissues, etc., clearly the results were interesting enough to warrant further experimentation. 3.5. ODC INHIBITIONAND HEARINGLOSS Although the reversible loss of hearing encountered in a number of patients treated with eflornithine over a period of time can in no way be considered a therapeutic benefit, it is a common side effect of eflornithine treatment and one which has attracted much attention in terms of curiosity about the mechanism of action. More knowledge of the mechanism by which this change is brought about may enable regimes that minimize its occurrence to be designed. A recent study in 58 melanoma patients found that hearing loss was dose related and that minimal ototoxicity

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( < 10% incidence) was observed in the majority of patients who received a cumulative dose of eflornithine less than 150 g/m 2. Conversely, up to 75% of the patients who received more than 250 g/m 2 developed a clinically demonstrable but reversible hearing loss (Croghan et al., 1991). Several animal models have been utilized to investigate the mechanism of eflornithine related loss of hearing. The pioneering immunochemical localization studies of Persson et al. (1983) first demonstrated the presence of ODC in the cochlear spiral and spiral ganglion of the guinea pig ear. This was confirmed by Mfiller et al. (1988) as well as by Schweitzer et al. (1986) who showed the presence of polyamines in the cochlea of the rat. Only recently was an animal model developed in guinea pigs that could duplicate the hearing loss caused by eflornithine seen in the clinic. Jansen et al. (1989) found that after 6-8 weeks of treatment with eflornithine, guinea pigs developed significant hearing loss that was at least partially reversible on discontinuation of the drug. Histologic and electrophysiological data suggested that the cochlea again was the anatomic site of the eflornithine-induced hearing loss. Subsequent studies indicated that eflornithine caused cochlear damage and increased the auditory threshold in guinea pigs (Salzer et al., 1990) as well as significantly inhibiting ODC and depleting polyamines in the entire inner ear (Marks et al., 1991). Thus, the data are consistent with the hypothesis that eflornithine induced hearing loss is related to inhibition of polyamine biosynthesis in the inner ear. However, the specific mechanism of the role of polyamines in the normal functioning of the cochlea remains to be elucidated. 3.6. AFRICANTRYPANOSOMIASIS Ever since 1980 when Bacchi and colleagues reported that eflornithine would totally cure acute infections of the African trypanosome T. b. brucei in mice, there has been a tremendous interest in the use of ODC inhibitors for this and other parasitic diseases (see Bacchi and McCann, 1987). These initial dramatic results led to the remarkably rapid use of eflornithine in what would have been fatal cases of drug-resistant late-stage sleeping sickness in Africa (see Schechter et al., 1987; Schechter and Sjoerdsma, 1989). The initial finding of the effects of eflornithine against African trypanosomes had led to an explosion of work demonstrating the role of polyamines in trypanosome growth and metabolism (see Bacchi and McCann, 1987). A great deal of interest has also focused on the role of the synthesis of trypanothione (N~,NS-bis(L-7-glutamyl-L-hemicystinyl-glycyl)spermidine) a novel spermidine-containing cofactor, obligatory for glutathione reductase activity in trypanosomitids. Not only does eflornithine reduce general polyamine levels in trypanosomes (Bacchi and McCann, 1987) but it also decreases the levels of trypanothione as a consequence of spermidine depletion (Fairlamb et al., 1987). The recent great interest in the role and function of trypanothione has led to a concerted effort to understand its pharmacological importance. It was reported that trypanothione was the primary target for the arsenical drugs that have been widely used to treat late-stage sleeping sickness, and, in fact, trypanothione actually forms a stable adduct with melarsen oxide itself (Fairlamb et al., 1989). This would also explain the pronounced synergism noted between the arsenical drug and eflornithine (McCann et al., 1983). A recent study with trypanosomes has also shown a potentiation of eflornithine with antimonial compounds, even though these traditional anti-leishmanial drugs had no trypanocidal effect given as monotherapy (Jennings, 1991). It is, however, completely unclear whether the antimonials directly bind to trypanothione as was shown for the arsenicals. Although both the arsenicals and eflornithine may have at least one site of action in common, the use of the most common of the arsenicals, melarsoprol, results in a > 5% mortality rate in all patients treated, caused by a specific reactive encephalopathy. Eflornithine has few adverse effects, particularly when given intravenously (Schechter et al., 1987). Recent clinical studies directly comparing the two drugs indicated a strong preference for the use of eflornithine in late-stage sleeping sickness cases for the above reasons (Hamon et al., 1990). A major milestone in the clinical use of eflornithine came in November of 1990 when the FDA approved it as the first new drug in forty years for the treatment of sleeping sickness (Nightingale, 1991). The World Health Organization has also approved the use of eflornithine, citing its efficacy as a 'resurrection drug' in more than 600 patients, most of whom had failed melarsoprol therapy

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and were near death (Lancet Editorial, 1991). Further approval for its use in sleeping sickness came in March 1991 from the European Community (E.C.) regulatory authority. 3.7. PNEUMOCYSTIS CARINII PNEUMONIAAND CR YPTOSPORIDIUM IN AIDS A second major clinical indication for the use of eflornithine has been against the pneumonia caused by the parasitic microorganism Pneumocystis carinii, thought traditionally to be a protozoan but now described as being more related to the fungi (Davey and Masur, 1990). Compassionate clinical trials in patients with Pneumocystis carinii pneumonia (PCP) were initiated in 1983 in individuals who had failed standard treatment and were based on the known activity of eflornithine against several classes of protozoa (Schechter et al., 1987). Almost all of the several hundred patients who have been treated were either refractory to or intolerant of drugs such as trimethoprim-sulfamethoxazole or pentamidine. Overall, the general positive response to eflornithine has been 35% of AIDS patients treated with it because they were intolerant of other drugs or had PCP unresponsive to them (Sahai and Berry, 1989). In one recent study, 31 patients with PCP who had failed either trimethoprim-sulfamethoxazole or pentamidine received eflornithine therapy and of these, 21 survived to the time of discharge (Smith et al., 1990). Another study indicated a clinical response in 15 of 33 episodes of PCP treated with eflornithine, again in intolerant or unresponsive patients (Paulson et al., 1992). In a randomized comparative study where eflornithine and trimethoprim-sulfamethoxazole were each given as primary therapy for the first episode of PCP, 20 of 51 patients (39%) successfully completed treatment with eflornithine and 19 of 47 patients (40%) successfully completed treatment with trimethoprim-sulfamethoxazole, indicating no difference in PCP mortality between treatments. However, significantly more patients were withdrawn from eflornithine because of treatment failure (49% versus 21%) while significantly more patients were withdrawn from trimethoprim-sulfamethoxazole because of serious drug-related side effects (38% versus 12%)*. These results would seem to indicate that while eflornithine may not be indicated as primary therapy, it would probably continue to have a role as salvage therapy in patients who have failed other treatments. While clinical studies were continuing, several laboratory findings have confirmed that eflornithine does have significant in vivo activity against Pneumocystis carinii in a rat model (Clarkson et al., 1990). Furthermore, although an earlier study had not been able to detect ODC activity in Pneumocystis carinii from rats, subsequent experiments demonstrated first, the presence of polyamine metabolism in this organism, the sensitivity of this metabolism to D F M O and A-MFMO methyl ester and ultimately, detection of ODC activity itself (Clarkson et al., 1990; Lipschik et al., 1991). Although clinical infections of Cryptosporidium, a coccidian protozoan parasite which causes a severe opportunistic gastrointestinal disease in AIDS patients, had been sporadically treated with eflornithine, the results were largely inconclusive (Schechter et al., 1987). A more definitive prospective study was done wherein 22 AIDS patients with severe debilitating diarrhea caused by Cryptosporidium were treated with eflornithine. Of 17 evaluable patients, only 7 did not respond to eflornithine at all, while 10 had complete resolution of the disease or significant partial response (Rolston et al., 1989). Thus, while cryptosporidiosis is not as common or as life threatening in AIDS patients as PCP, there is no effective therapy to treat it and eflornithine may very well be one of the few utilizable possibilities. 3.8. OTHER INFECTIOUSDISEASES/ORGANISMS 3.8.1. Malaria Eflornithine had been shown to inhibit human malaria (Plasmodium falciparum) parasites in vitro, cure exoerythrocytic infections of Plasmodium berghei in mice and appreciably reduce erythrocytic parasitemia of P. berghei in mice as well (see Bacchi and McCann, 1987). Based on *D. E. Smith, S. Davies, J. Smithson, I. Harding and B. Gazzard, Eflornithine vs co-trimoxazole in the treatment of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. J. Antimicrobial Chemotherapy, submitted.

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these results, a pilot trial was devized to observe the effect of eflornithine on P. falciparum asymptomatic parasitemia in several patients whose infections had resisted standard initial chloroquine treatment. After a three day treatment with eflornithine, parasitemia reduction ranged between 42-70% in the four patients, but no patient showed complete clearing of parasitemia. The clinical observations did confirm that eflornithine can exert an antimalarial activity in man against chloroquine resistant disease, but further studies need to be done.* 3.8.2. Trypanosoma cruzi It has been shown that T. cruzi, the American trypanosome and cause of Chagas' disease, is not sensitive to inhibition of ODC by eflornithine, but rather responds to high concentrations of ~-difluoromethylarginine, a specific irreversible inhibitor of arginine decarboxylase. This enzyme converts arginine to agmatine (which then serves as a precursor to putrescine) and is normally only found in bacteria and plants (see Bacchi and McCann, 1987; Pegg et al., 1989). Studies to prove the existence of arginine decarboxylase in T. cruzi have so far been unsuccessful. One recent approach to investigate how ~-difluoromethylarginine may act against T. cruzi was to use a non-pathogenic trypanosome, Crithidia fasciculata, as a model as it is easy to culture in a fully defined media. Although both eflornithine and ~-difluoromethylarginine inhibited the growth of C. fasciculata and reduced polyamine levels, it was determined that ~-difluoromethylarginine functioned via its enzymatic conversion to eflornithine and the subsequent inhibition of ODC (Hunter et al., 1991). Thus, the elusive search for arginine decarboxylase in T. cruzi continues. 3.8.3. Fungi Although no clinical work has yet been attempted, evidence has shown that a number of zoophilic yeast and fungi and also phytopathogenic fungi are sensitive to the effects of eflornithine (see Pegg et al., 1989). Recently Cryptococcus neoformans, the cause of severe fungal infections in immunosuppressed patients, was shown to be partially inhibited by eflornithine (Pfaller et al., 1990). Furthermore, ten species of human dermatophytic fungi were also shown to be inhibited by eflornithine (Gruhn and Boyle, 1991). Although these studies were limited in vitro results showing only partial efficacy in inhibiting growth, the potential use of ODC inhibitors as therapy for some almost untreatable fungal diseases remains an option that should be further investigated. Acknowledgments--Work on polyamine metabolism in AEP's laboratory is supported by grants CA-I 8138, GM-26290 and CA-37606.

REFERENCES AJANi, J. A., OTA, D. M., GROSSIE,B., LEVIN,B. and NISHIOKA,K. (1989) Alterations in polyamine metabolism during continuous intravenous infusion of ~t-difluoromethylornithine showing correlation of thrombocytopenia with ct-difluoromethylornithine plasma levels. Cancer Res. 49: 5761-5765. AJANI, J. A., OTA, D. M., GROSSIE,J. V. B., ABBRUZZESE,J. L., FAINTUCH,J. S., PATT, Y. Z., JACKSON,D. E., LEVIN,B. and NISHIOKA,K. (1990) Evaluation of continuous-infusion alpha-difluoromethylornithine therapy for colorectal carcinoma. Cancer Chemother. Pharmac. 26: 223-226. ALEXANDROV,V. A., BESPALOV,V. G., BOONE,C. W., KELLOFF,G. J. and MALONE,W. F. (1991) Study of postnatal effects of chemopreventive agents on offspring of ethylnitrosourea-induced transplacental carcinogenesis in rats. I. Influence of retinol acetate, ~t-tocopherol acetate, thiamine chloride, sodium selenite and ct-difluoromethylornithine. Cancer Letts 60: 177-184. BACCHI, C. J. and MCCANN, P. P. (1987) Parasitic protozoa and polyamines. In: Inhibition of Polyamine Metabolism, pp. 317-344, MCCANN, P. P., PEGG, A. E. and SJOERDSMA,A. (eds) Academic Press, Inc., Orlando. BACHRACH,U. and HEIMER,Y. M. (eds) (1989) The Physiology of Polyamines, CRC Press, Boca Raton. BARROSO, L., MOORE, R., WRIGHT,J., PATEL,T. and BOYLE,S. M. (1990) Analysis and sequence of the speC (ornithine decarboxylase) gene of Escherichia coli. GenBank/EMBL Data Bank Accession Number M33766. *P. L. Mulamba, G. Roscigno, A. J. Bitonti, A. Sjoerdsma, P. P. McCann and P. J. Lewis, Eflornithine reduces chloroquine-resistant Plasmodium faliparum parasitemia in man. Trans. Royal Soc. Trop. Med. Hygiene, submitted.

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