Inhibitors of nitric oxide synthase as potential treatments for opioid withdrawal

165 INHIB1TORS OF NITRIC OXIDE SYNTHASE AS POTENTIAL TREATMENTS FOR OPIOID WITHDRAWAL E.D. London, D.B. Vaupel, and A.S. Kimes. Neuroimaging and Drug Action Section, Intramural Research Program, National Institute on Drug Abuse, P.O. Box 5180, Baltimore, MD USA.

Nitric oxide is an intercellular mediator that is produced in brain in a reaction catalyzed by the enzyme nitric oxide synthase (NOS). The enzyme exists as isoforms found primarily in vascular endothelial cells and in brain. Prior to the introduction of inhibitors specific to the respective isoforms, we showed that two nonselective inhibitors of NOS, L-NG-nitroarginine(L-NNA) and L-NG-nitroargininemethyl ester (L-NAME), reduced some signs of naloxone-precipitated morphine withdrawal in rats (1), rendering NOS inhibition as a new potential target for ameliorating the opioid abstinence syndrome. The present work was an extension of these studies to include inhibitors specific for cerebral NOS (7nitroindazole; 7-NI) and highly potent for endothelial NOS (N(5)-(1-iminoethyl)-L-ornithine;L-NIO). Clonidine, an alpha2 adrenoceptor agonist, was included as a pharmacological standard because it effectively reduces behavioral and physiological signs of morphine withdrawal in rats and is used clinically to treat opioid withdrawal. Effects of the NOS inhibitors were assessed in rats that were made morphine-dependentby subcutaneous (s.c.) implantation of pellets. Each rat received one 75-mg morphine pellet on Day 1 of the experiment, and withdrawal was precipitated by naloxone (0.5 mg, s.c.) on Day 4. NOS inhibitors, clonidine, or vehicle were administered intraperitoneally I h before the naloxone challenge. Signs and symptoms of opioid withdrawal were scored over a 15-min period beginning 1 min after the administration of naloxone. 7-NI (1.8 - 100 mg/kg) and L-NIO (1 - 300 mg/kg) produced dose-dependent decreases in weight loss, diarrhea, wet dog shakes and grooming, as did L-NAME (1-56 mg/kg) and L-NNA (1-30 mg/kg). In addition, 7-NI reduced salivation and masticatory movements, and tended to decrease genital effects (penis licks, ejaculations). In contrast, L-NNA, L-NAME and L-NIO all increased these genital effects significantly. Neither 7-NI nor L-NIO produced overt toxicity when the rats were observed for 3 days after testing; however, some deaths occurred during the 3 days following treatment with L-NAME and L-NNA. Clonidine (0.01-0.1 mg/kg) reduced many of the signs that were attenuated by 7-NI, but some signs were enhanced. For example, 7-NI, L-NIO, and clonidine all increased exploratory activity and escape jumps. These results are summarized in Table 1. In a second study, unanesthetized morphine-naive and morphine-dependent rats were prepared with left femoral arterial catheters, and the blood pressure effects of the highest doses of NOS inhibitors used in the withdrawal studies were evaluated. Among the four NOS inhibitors, 7-NI had no effect on blood pressure in either condition; whereas the other NOS inhibitors significantly increased mean arterial pressure by approximately 40 mm Hg, at 1 h after treatment (Table 2). With the exception of 7-NI in morphine-naive rats, all of the treatments with inhibitors of NOS also reduced heart rate (Table 2). In conclusion, the ability of 7-NI to affect more signs of opioid withdrawal than L-NNA, L-NAME or L-NIO may reflect the selectivity of 7-NI for cerebral NOS (2). It is noteworthy that in the rat model of naloxone-precipitated opioid withdrawal, clonidine and 7-NI, which act by different pharmacological mechanisms, produced similar profiles of activity. The efficacy of 7-NI in reducing signs of opioid withdrawal without the complicating effect of hypertension may make this compound more suitable for development as a therapeutic agent than NOS inhibitors that are not selective for the cerebral enzyme.

166 Table 1. Effects of NOS Inhibitors and Clonidine on Naloxone-precipitated Opioid Withdrawal 7-NI Weight Loss Diarrhea Wet Dog Shakes Grooming Mastication Salivation Penis Licks/Ejaculations Exploratory Activity Escape Jumps Abnormal Posturing Ptosis

L-NNA

Clonidine

$ $ $ $ $ ,1, $ Q t t

L-NIO

$ $ $ $ o t 1" t 1"

L-NAME

$ $ $ $ o o $NR ~ o

$ $ $ $ o

$ $ $

@

@

@

@

1" NR t @

? NR

$ @

1" Q t t $ $ NR

The symbols $ and 1" indicate a significant decrease or increase, respectively, as determined by analysis of variance (ANOVA) and log-linear regression. @ = no effect. NR = no significant regression, but significant treatment effect by ANOVA. Q = a qualitative effect (i.e. treatment effect did not attain statistical significance).

Table 2. Cardiovascular Effects of NOS Inhibitors and Clonidine

MAP, Naive MAP, Dependent Heart Rate, Naive Heart Rate, Dependent

7-NI

L-NIO

L-NAME

L-NNA

Q

1"

1"

1"

o

1"

1"

t

Clonidine $ H

$ H

®

$

MAP = Mean arterial pressure. The designations "naive" and "dependent" refer to morphine-naive and morphine-dependent rats, respectively. @ = no effect. ,1, or 1"indicate a significant treatment effect. H = decreased blood pressure also observed in human studies.

REFERENCES 1. A.S. Kimes, D.B. Vaupel and E.D. London (1993) Psychopharmacology 112:521-524 2. P.K. Moore, R.C. Babbedge, P. Wallace, Z.A. Gaffen and S.L. Hart (1993) British Journal of Pharmacology 108:269-297