Neurotoxin-Related Research: From the Laboratory to the Clinic L. IVERSEN, A. C. FOSTER, R. G. HILL, S. D. IVERSEN, J. A. KEMP, P. D. LEESON, N. M. J. RUPNIAK, K. SAYWF,LL, M. D. TRICKLEBANK, AND B. J. WILLIAMS M m k Sharp t 9Dohme Reseamh Ldmumia Neumcienu Ikseutzh Cenm Tnliqg~Park, Raarl Hiwhv, Essex, Eqghnd CM20 2QR Neurotoxins have provided valuable new insights into the molecular mechanisms underlying human neurodegenerative diseases, as evidenced by the previous papers in this symposium. In particular, the discovery that the synthetic chemical MPTP (l-methyl4phenyl-1,2,3,6-tetrahydropyridine)selectively targets dopaminergic neurons in human and animal brain represents a major advance in understandingthe possible biochemical basis of Parkinson’s disease. Similarly, the finding that L-glutamate and related excitatory amino acid derivatives are not only widely used as b t excitatory neurotransmitters in the CNS, but also are potential endogenous neurotoxic agents, represents another surprising insight. In this review we shall describe how both these discoveries can also be used to provide powerful new models fbr drug discovery research.

THE MPTP-TREATED PRIMATE AS A MODEL FOR ASSESSING NOVEL ANTIPARKINSONUN AGENTS

The MMP-treated squirrel monkey (Szimiti sciuffw) has been used as an experimental model in our laboratory, as first described by others.l.2 Animals were treated once weekly with a single dose of MPTP hydmchloride (2 mgkg, i.p.) and were monitored closely for signs of parkinsonism. Subjects were considered suitable for drugtesting when they had reached a stable parkinsonism score of 3 out of a total score of 4 on a scale that rated the cardinal symptoms: tremor, akinesia, bradykinesia, and rigidity. Once a stable score had been obtained fbr a 2-week period the subjects were used for drug testing. Although some monkeys remained stable fbr up to 14 months, most exhibited a gradual spontaneous recovery, and were periodically treated with further doses of MPTP. Stability was usually maintained fbr 1-2 months between MPTP treatments. The most prominent characteristic of the Mm-treated animals was the profbund reduction in motor activity (to around 25-3096 of that seen in normal monkeys), and 207

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we used this as a convenient measure which can be scored automatically by photocell activity devices. The model is, we believe, one of the most accurate animal representations of the cardinal neurologic features of Parkinson’s disease in man. The primate model has been used to test two types of novel antiparkinsonian agents: dopamine agonists and L-dopa p h g s (FIG.1).

N ~ o l h i t - D o p m i n eDZREceptm This naphthoxazine derivative, otherwise known as (+)PHNO (4propyl-9-hydroxynaphthoxazine) or MK458, was discovered in our laboratories as a potent and selective dopamine DZ receptor a g ~ n i s t . Naxagolide ~.~ is the most potent known dopamine agonist in PiPo and this, together with its physiochemical properties, suggested that it might be suitable fbr transdermal application in the treatment of parkinsonism. It was hoped that a sustained delivery system might lead to improved relief of clinical symptoms, avoiding the “on-off swings often associated with L-dopa therapy. These concepts were tested in the MPTP monkey model.5 It was first ascertained that naxagolide was able to reverse some of the features of the MPTP syndrome when administered as a single subcutaneous bolus. Treatment with single doses of 2.5-20.0 crglkg proved remarkably effective in increasing locomotor activity in MlTP-treated animals up to the levels observed in normal control

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FIGURE 1. Chemical structures of the Gdopa p&g NB-355 and the dopamine agonist (+)-PHNO.

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4-propyl-9- hydroxynaphthoxazine

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FIGURE 2. Spontaneous locomotor activity in untmted normal squirrel monkeys over a 2-day period compared with MPTP-treated animals bebre and after wearing a primate jacket alone (A) and before and after application of transdermal PHNO patches to cover a skin surface area of 2.39/cm2 (B)or 4.78 cm* (C). At the times indicated by pcrtiral amnw, the parkinsonian animals had patches applied. Each point is mean f SEM for number of photocell interruptions recorded during 3-h time bins for 4-7 animals. = p < 0.05 compared with control untreated animals; Student's t test. (FromRupniak ct a l . 5 Reprinted by permission.)

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subjects, with a minimum effective dose of approximately 2.5 pgkg. Doses of naxagolide in this range did not have any significant effects on locomotor activity in control animals. Naxagolide was then applied to MPTP-treated animals by means of a transdermal patch device. This consisted of a thin adhesive plaster comprising an outer covering membrane, a drug reservoir of naxagolide as free base, a rate-controlling membrane, and an adhesive contact surfice. The patches were circular and covered a skin surface area of 2.39 cm2. The release rate of drug in vim was 2.6 pg/cm2/h. Various doses of drug were delivered by applying between 1 and 8 patches to the shaved skin on the chest of the monkeys. During the experiments the monkeys wore a lightweight nylon jacket to prevent removal of the patches. Applying a single patch had little effect, but application of 2 patches (covering a skin area of 4.78 cm2) markedly increased locomotor activity in the MM'P animals to control levels (FIG.2 ) .There was a delay of about 9 hr after applying the patches before locomotor activity was fully restored and, thereafter, it reflected the diurnal pattern observed in normal animals over the remainder of the 2 4 h observation period, in con-

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tmt to the akinetic state characteristic of MPTP-treated animals. Dyskinesias, in mild

form,were observed in only one animal, and did not interfere with locomotion. Thus our findings demonstrate the fcasibility of sustained delivery of a dopamine agonist by means of a transdermaldevice to provide a prolonged relief of the neurologic symptoms in an animal model of Parkinson's disease. Unfortunately, although naxagolide was shown to have clinical efficacy when administered by the conventional oral route,6 the transdermal delivery system was not tested extensively clinically, as further development of the compound was terminated at that stage. L-Dopa

Ptv-drnfl: NB-355

The MPTP monkey was also used to assess a different approach to the development of novel antiparkinsonian therapy. The compound NB-355 [c3-(3-hydroxy+pivaloyloxyphenyl)alanine] (FIG.1)was discovered by our colleagues in the Banyu Research Laboratories in Japan. It acts as a prodrug for Ldopa, providing, in animal models, a prolonged plasma level of cdopa. We found that NB-355 was superior to L-dopa in the MMl' monkey model, providing a significantly more prolonged reversal of the locomotor deficit in the treated animals, with less risk of dyskinesias.'

EXCI'IWIDXIC MODELS FOR NEUROPRUXTXXIVE COMPOUNDS

It is now widely believed that the neurotoxic effects of L-glutamate and related excitotoxins which activate glutamate receptors in the CNS play a role in the neurodegenerative changes seen in human brain afier a variety of insults that lead to excessive glutamate leas^. These include stroke, perinad ischemia of various types, head injury, and global cerebral ischemia (as after cardiac arrest or drowning) (for rWiews Re&. 8-10). Consequently there has been an upsurge of interest in the possibility that drug that block the excitatory effects ofbglutamate at CNS receptors may act as novel neuroprotective writs, with particular attention being paid to antagonists acting at the Nmethyl-D-aspartate receptor subtype (NMDA receptor).9J0 Models fbr drug discovery in this new area of research into two categories. The first employ Gglutamate or other excitotoxins to cause neural damage, which can then be reversed by glutamate antagonists. In studies of NMDA antagonists, agonists with selectivity for this receptor subtype have been widely used. These include NMDA itself, ibotenic acid, and the potent excitotoxin quinolinic acid. These agonists are applied either in piho (brain slice, isolated retina, tissue culture mode1s)ll or by local infusion to particular brain areas in v b .l2 A second type of model seeks to identlfy experimental conditions in which neural damage is caused principally as a consequence of increased glutamate release. These models include brief exposure of neuronal cultures to hypoxia, or brief periods of global cerebral ischemia in the whole animal (e.g., reversible occlusion of carotid arteries in rodents). More realistic models of human stroke are those in which fbcal ischemia is induced in animal brain by surgical occlusion of the middle cerebral artery (a vessel commonly involved in human stroke).13

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m s ofMK-801 and Gmptitiaw N W A Antagmrkts

The Merck compound dizocilpine (MK-801) was the first selective NMDA antagonist that exhibited unimpeded CNS penetration and high systemic potency. 14.15 Numerous studies have been performed with this and other NMDA antagonists during the past five years to assess the neuroprotective potential of these new agents. The results have been positive in the large majority of cases and have been reviewed in detail elsewhere.1G18In more than 50 publications, mainly on dimilpine, NMDA antagonists have been found to offer complete protection against the excitotoxic damage elicited by NMDA agonists both in pino and in vbu. Furthermore, in several models the neuroprotective effects were observed even when the NMDA antagonist was administered some hours after the excitotoxic insult. Most importantly, the NMDA antagonists were also effective in models in which ischemia, rather than an excitotoxin, was used to provoke neural damage. The NMDA antagonists were fdly effective in vim.l1 In vim the efficacy of the compounds depends on what type of model was employed and when the NMDA antagonist was given. The most consistent neuroprotective effects were seen in models of bcal ischemia, although some laboratories have reported positive effects also in global ischemia models. In the latter the severity of the ischemic insult appears to be of critical importance. In fbcal ischemia models the NMDA antagonists were most effective when given at the time of the ischemic insult, or within 1-2 hours thereafter, with 3-4 hours apparently representing the maximal “time window” available. Some competitive antagonists, because of their highly polar character, do not penetrate rapidly into the brain. Such compounds (e.g., d-CPPene) may have no neuroprotective efficacy when given at any time after the start of an ischemic insult because they cannot enter the CNS quickly enough.s In focal ischemia models in a variety of species (mouse, rat, rabbit, cat, primate) dizocilpine has consistently proved effective in reducing the volume of the cerebral cortical damage by 50-60%, and it was equally effective in models in which outcome was assessed at a short time interval after the ischemic insult (3-4 hours) or in long-term survival models (1-7 days).8J6 Despite the remarkable neuroprotective efficacy ofsuch NMDA antagonists as dim cilpine, the clinical development of these compounds has not proved easy. Dizocilpine, and other compounds in this class, exhibit a wide variety of other CNS effects, some of which are undesirable. Thus NMDA antagonists show some similarities, in their pharmacological profile, to the intravenous anesthetic and psychostimulant drug phencylidine (PCP), which also acts as a noncompetitive NMDA antagonist. NMDA antagonists of both noncompetitive and competitive classes will cross-generalize to PCP in animals trained to “recognize” PCP as a discriminative stimulus19 At low dose levels dizocilpine and related compounds cause a syndrome of behavioral activation, described originally as ‘csympathomimetic.”20This includes increased motor activity, stereotyped behavior, increased autonomic outflow (leading to sustained elevation in blood pressure), and increased glucose metabolism as measured by the 2deoxyglucose technique, particularly in limbic brebrain areas (for a review see Ref. 21). In these same areas of brain (e.g., cingulate cortex, entorhinal cortex, hippocampus) the activation is accompanied by a reversible histolop change in many large neurons, which become swollen and exhibit prominent fluid-filled vacuoles in their cytoplasm. This response was first observed with PCP, dimilpine, and other noncompetitive NMDA antapnists, but is now known to occur also after administration of large doses of competitive

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NMDA antagonists.22The effect reaches a maximum 4-8 hr after a single dose of dimcilpine and diminishes thereafter, with no residual effects at 24 hr. Animals subjected to repeated doses of dizocilpine rapidly developed tolerance, and vacuolization was no longer seen after the first dose.22 In our own studies we observed that rats treated for 14 days with a maximum tolerated dose of dizocilpine maleate (1mg/kg/&y i. p.) showed no evidence of vacuolization or other neurodegenerative histologic changes in cingulate cortex neurons, nor any evidence of cell loss.23 Only in animals treated with a very high single dose of dizocilpine (5 mgkg, i.p.) did we observe any evidence of cellular necrotic changes 48 hr later, and these were seen only in a small number of cingulate neurons (approximately 0.3% of the total population).% Although none of these undesirable side effects of dizocilpine was considered sufficiently serious alone to prevent the clinical development of the compound, the overall profile was considered sufficiently problematic to prevent further clinical studies in stroke. Furthermore, as outlined below, a second generation of neuroprotective NMDA antagonists acting by a different receptor mechanism appears to offer an alternative approach and to yield compounds with more benign side-effect profiles.

G l y c k S i & - W N m A Antagmists The discovery some fbur years agu of a glycine-sensitive modulatory site on the NMDA receptor suggested an alternative approach to the development of neuroprotective agents acting at this receptor25326 (FIG.3). Glycine in submicromolar concenGiycine

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V t w p k q i a t i o n Site

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FIGURE 3. Diagram illustrating the various drug-recognition sites on the NMDA receptor complex (drawn by A. Foster).

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trations has a powerful modulatory effect on the NMDA receptor. In the absence of glycine or in the presence of an antagonist acting at the glycine recognition site glutamate responses are almost completely suppressed. We argued, nevertheless, that NMDA antagonistsacting at this site might have a different overall profile from channelblocking (dizocilpine-like) or competitive antagonists. This has proved to be the case. Although glycine site-directed NMDA antagonists are noncompetitive blockers, the antagonism they cause is not “use-dependent,” as with the dizocilpine channel blocking group, and we believe that this may be an important h t conkrring ~ ~ their improved side-effect profile. In a search fbr NMDA antagonists that act at the glycine modulatory site we first synthesized a series of analogues of kynurenic acid since weak glycine site affinity existed in this compound. The analogue 7-chloro-kynurenic acid (743-KYN) (FIG.4) proved to be a potent displacer of [jH]glycine binding (I& = 0.56pM) with a similar potency in blocking glycine-induced enhancement of NMDA responses in neurophysiological experiments using rat brain slices or cortical neurons in tissue cult~re.2~ 7-Cl-KYN was also fir more selective than the parent compound, having only low affinity for the glutamate recognition site of non-NMDA receptors (quisqualate and kainate). It was possible to demonstrate, by using the model described by Choi,” that 7-CL KYN was able to offer complete neuroprotection against a hypoxic insult in primary cultures of cortical neurons.28 In intact brain, the compound was also able to protect against neuronal damage in rat striatum elicited by the excitotoxin quinolinate (200 nmol) administered intrastriatally. Because 7x1-KYN does not penetrate readily into brain it was necessary to deliver the compound also by intrastriatal injection, at doses of 50 nmol given 1 hr after the excitotoxin administration. Under these conditions 7-CI-KYN provided complete protection against the loss of neurotransmitter marker enzymes (glutamate decarboxylase, choline acetyltmskrase) measured in striatum 7 days later.29

7-Chlorokynurenlc acid

(+) HA-966

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FIGURE 4. Chemical strumm ofglycinc-site dkctcd NMDA antagonists refmd to in t a t .

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The disubstituted kynurenic acid analogue 5-iodo-7-chloro-kynurenic acid (FIG.4) proved to be an even more potent glycine site-directed antagonist (Ic50 against [AH] glycine = 0.032 pM).30>31 We have also pursued other leads, notably the compound HA-966 (FIG. 4), first described more than 30 years 9 . 3 2 HA-966 has been recognized for some time as an NMDA antagonist with unusual pharmacologic features. It proved to be a moderately potent glycine site-directed antagonist or weak partial agonist (I& against [3H]glycine binding = 27 pM).j3 O n resolution of the racemic compound, the (+) enantiomer w a s fbund to account for most of the glycine-site activity (Ic50 fbr (+) HA-966 = 12.5 pM). (+)HA-966 possessed in PiPo activity as an anticonvulsant, protecting mice against NMDA-induced or sound-induced convulsions when administered systemically (i.p.).%The (-) enantiomer of HA-966, although only weakly active as a glycine-site NMDA antagonist, proved to have its own pharmacology, being more sedative and ataxic than the (+) enantiomer-apparently acting in a gammabutryolactone-like manner34 (FIG. 5). (+)HA-966 and the more potent analogue (+)-ck-fl-methyCHA-966 (M87,

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Webster mice (upppancl) and DEW2 mice (kpanc Mice l). were trained to remain on the revolving rotarod for 2 min. Four doses of each test compound were administered i.v. (SwissWebster) or i.p. (DBA/2) 15 minutes before testing to determine the latency to Fall off the rotarod. Results m means f SEM for at least 8 mice per group. solid rpum = (*)HA-%; mhif thgh = (-)HA-%; and mhifcirclcr = (+)HA-%. (From Sigh ctuf.31 Reprinted by permission.)

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Neurotoxin-related research: from the laboratory to the clinic.

Neurotoxin-Related Research: From the Laboratory to the Clinic L. IVERSEN, A. C. FOSTER, R. G. HILL, S. D. IVERSEN, J. A. KEMP, P. D. LEESON, N. M. J...
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