Development of Novel, Highly Selective, Non-Peptide Opioid Delta Agonists and Antagonists.
The development of highly selective, systemically active and efficacious opioid delta agonists is a promising pathway for the identification of compounds of potential clinical importance. Such substances would provide effective medications for pain with decreased or no abuse liability. In an effort led by NIDA Grantee, Dr. Frank Porreca at the University of Arizona, a group of chemists and pharmacologists from the University of Arizona, University of Michigan, University of Texas and NIH has been actively exploring the development of systemically
available analgesics acting through opioid delta receptors. Based on a large amount of information gained with highly receptor-selective peptides, this group has now succeeded in identifying families of molecules which show a high degree of selectivity for delta receptors and which are systemically active in producing analgesic actions in a variety of animal models. This group (Drs. Kenner Rice and Silvia Calderon at NIH, Dr. Victor J. Hruby, University of Arizona) has succeeded in synthesizing several compounds with significant selectivity at opioid delta receptors. One of the lead compounds, SNC80 shows more than 2000 fold delta (compared to mu or kappa) opioid receptor selectivity and has recently been reported (Journal of Medicinal Chemistry, 37:2125-2128, 1994). Furthermore, the analgesic actions of this compound have been characterized and shown to be mediated via both supraspinal and spinal opioid delta receptors (findings which will soon appear in the Journal of Pharmacology and Experimental Therapeutics). This compound, and others in this series and other chemical series are being further characterized by Dr. James Woods at the University of Michigan and by Dr. Thomas Burks at the University of Texas. They have shown that SNC80 produces minimal disturbance of gastrointestinal functions as would be predicted by previous studies with highly selective peptidic delta agonists. Further characterization of the consequences of systemic activation of opioid delta receptors is underway in the laboratory of Dr. Porreca who is investigating the possibility of development of physical dependence, whether selective agonists at these receptors will produce a positive reward, and whether such compounds can be useful in the treatment of morphine resistant pain states such as those observed in neuropathies.
Two Novel Cannabinoid Receptor Antagonists.
In 1994, two successful efforts were made towards the discovery of reversible cannabinoid antagonists. One of these (SR 141716A) was discovered by the French pharmaceutical company, Sanofi, through the screening of large numbers of analogs (high throughput screening). The other (AM630) was developed by Dr. Makriyannis, a NIDA grantee (University of Connecticut) and this was developed by rational design. The pharmacological properties of AM630 were studied by Dr.R. Pertwee in Aberdeen, Scotland. Although the two antagonists have very different structures they showed similar potencies when tested in vitro. However the two analogs showed different degrees of selectivity towards the two cannabinoid receptors (central and peripheral). These receptor antagonists are very valuable as they will be: 1) New, experimental tools for studying the biochemical and physiological properties of the cannabinoid receptors; 2) new templates for ligand design; and 3) may give rise to new therapies.
A High Affinity Electrophilic Probe for the Cannabinoid Receptor with Long-Acting Analgesic Properties.
A number of analogs have been designed to elucidate the structural features of the active site of the receptor site. A family of photoaffinity probes have been designed by Dr. Markiyannis and his colleagues (University of Connecticut.) These probes were found to be very useful for in vitro experiments but could not be used for in vivo experiments. More recently, potentially useful analogs were obtained through the development of affinity probes. The most useful of these ligands, (-)-7'-isothiocyanato-11-hydroxy-delta-8-tetrahydrocannabinol, was shown to fully occupy the cannabinoid receptor at concentrations as low as 2 nM. This molecule was found to act as antagonist when tested for its effects on the calcium current in neuroblastoma cells. However, when tested in vivo in rats this molecule behaves as an irreversible agonist demonstrating analgesic properties over a long period of time thus being potentially useful as a potent, non-opioid, long acting analgesic.
Rewarding Properties of Cocaine Linked to D3 Receptor.
Dr. George Koob and his colleagues from the Scripps Research Institute have been using a variety of behavioral methods in laboratory animals that simulate the human drug-seeking and drug-taking behaviors. These researchers have documented the withdrawal phase of cocaine addiction in the rat. This is similar to that reported for human addicts, and is characterized by a lowering of mood, hypersomnia, hyperphagia, psychomotor retardation, anhedonia and an intense craving for the drug. In the rat, elevations in reward thresholds and psychomotor retardation have been demonstrated during withdrawal from a cocaine binge. Markou, A. and Koob, G. Postcocaine anhedonia: An animal model of cocaine withdrawal. Neuropsychopharmacology 4: 17-26, 1991.
Dr. Koob and his colleagues have hypothesized that activation of dopamine transmission within specific structures of the basal forebrain is a major determinant for cocaine self-administration in laboratory animals, while decreased dopamine function may be responsible for some of the aspects of the withdrawal phase.
In a number of recent articles, Dr. Koob classifies potential treatment drugs for cocaine addiction in man. While there have been many clinical trials looking at the dopamine D2 agonists and antagonists, none have been very successful. However, increasing knowledge of a role for D1 and D3 receptors in cocaine dependence points to new targets for pharmacotherapeutics. Perhaps even more interesting, as Dr. Koob relates, the concept of partial dopamine agonists is gaining favor. These experimental compounds are not stimulatory or self-administered by naive animals, as cocaine is, yet they appear to attenuate the cocaine self-administration behaviors in the animals. The author concludes that "there is enough evidence to indicate that compounds with partial agonist activity at dopamine receptors may in the future represent novel psychoactive agents with important advantages compared to previously used antipsychotic drugs". Pulvirenti, L. and Koob, G. Dopamine Receptor Agonists, Partial Agonists and Psychostimulant Addiction. Trends in Pharmacological Sciences 15 (10): 374-379, 1994; Caine, B. and Koob. G. Modulation of Cocaine Self-Administration in the rat through D-3 Dopamine Receptors. Science 260: 1814-1816, 1993.
Cocaine During Pregnancy Disrupts Structure of the Cerebral Cortex.
Dr. Michael Lidow (Yale University) is studying the effects of orally administered cocaine to pregnant rhesus monkeys on the development of the cerebral cortex in the offspring. In his research presented at the Society for Neuroscience Meeting in November, Dr. Lidow discussed findings that cocaine virtually destroys the laminations of the cortex of the developing fetus so completely that no individual cortical layers could be discerned. Furthermore, there was an almost complete absence of glial fibers which are normally present in the upper layers of the cerebral cortex. In these studies, monkeys received 20 mg/kg/day from Day 40 to Da 102 of pregnancy, and were allowed to deliver at term (approximately Day 165). The offspring were allowed to develop normally for two months, at which time the observations were made. This study demonstrates that cocaine, administered daily during pregnancy, may significantly affect the development of the primate cerebral cortex, and that the rhesus monkey is a valuable and potentially predictive model for studies on the effects and mechanisms of action of cocaine during development of the cerebral cortex in embryos of human mothers addicted to this drug.
Potential Analgesic for Use in AIDS Patients.
Dr. Charles France (Louisiana State University, New Orleans) has been studying the properties of fentanyl derivatives. As part of his progress, he has examined a novel fentanyl derivative OHM3295 and found that it enhances natural killer cell (NK) activity in mice; all other mu opioids have been reported to decrease significantly NK activity. That OHM 3295 enhances NK activity while also producing opioid receptor-mediated antinociceptive effects suggests this compound might provide an important lead in the discovery of better treatments for pain in patients with compromised immune function.
Therapy of Memory Deficit Due to Drug Abuse and Other Causes.
Acquisition of eyeblink conditioning in rabbits is greatly delayed by PCP or MK-801 (dizocilpine) given daily, probably due to their blockade of NMDA receptors in the hippocampus. This is the primary model employed by Dr. John Disterhoft and his group at Northwestern University Medical School. Human PCP abusers show similar cognitive deficit. They had previously demonstrated that daily D-cycloserine, an agonist at the glycine site on the NMDA receptor (a site which can facilitate NMDA neurotransmission), enhances acquisition of trace eyeblink conditioning in young rabbits. They recently demonstrated the same effect, but more robust, in aged rabbits. They further showed that the most effective doses of cycloserine did not increase sensory sensitivity of the rabbits to the tone (conditioned stimulus) or airpuff (unconditioned stimulus). These studies have important implications for treatment of the cognitive consequences of drug abuse. It is possible that cycloserine, or other compounds acting as agonists at the glycine site on the NMDA receptor, can compensate for dysfunction in NMDA-mediated neural transmission as a result of a history of PCP abuse, and possibly other causes such as Alzheimer's disease.
Drug Discovery and Chemical Libraries.
High throughput screening of natural products and fermentation broths has resulted in the discovery of several new drugs. At present, generation and screening of chemical diversity is being utilized extensively as a major technique for the discovery of lead compounds, and this is certainly a major fundamental advance in the area of drug discovery. Peptide libraries present potential for producing millions of new peptide compounds for screening at a reasonable cost thus eliminating the time consuming, laborious and expensive procedures involved in the isolation, characterization, and synthesis of peptides obtained from natural sources (fermentation products) or conventional synthetic methods. It is not unusual to generate and screen tens of millions of peptides from a "peptide library". One of the approaches was earlier reported by a NIDA grantee, Dr. Richard Houghten. This approach consists of synthesis of non-support bound or soluble peptides (SPCLs= synthetic peptide combinatorial libraries) and these peptides can be used in all types of bioassays and screening methods. This method permits incorporation of D-amino acid or unnatural amino acid residues as well as specific secondary structures. Recently, an all-D-amino acid SPCL was successfully utilized to identify an all D-amino acid peptide ligand. Dooley et al. (in press), reported the identification of Ac-D-Arg-D-Phe-D-Trp-D-Ile-D-Asn-D-Lys-NH2 (Ac-rfwink-NH2), a potent mu agonist. This peptide bears no resemblance to any known opioid peptide and still, it produced an analgesic effect in mice. This effect, in comparison with morphine, was more potent and longer lasting. The fact that this highly stable peptide also induced analgesia after systemic administration demonstrates its ability to cross the blood-brain-barrier. Molecular dynamics calculations revealed similarity in the conformational characteristics of this peptide with PL 107, another potent opioid peptide. This result is of great fundamental importance because for the first time screening of an SPCL produced an agonist rather than an antagonist. These results also confirm the power of the SPLC approach for the discovery of novel analgesic drugs.
Cardiovascular Effects of Voltalized Cocaine Free Base.
The illicit use of cocaine is associated with a large array of mild stimulatory to deleterious effects to the cardiovascular system including cardiac arrhythmia, depressed contractility, myocardial infarction and sudden death. In particular, the increased popularity of smoking cocaine free base is associated with concomitant increase in its cardiotoxicity. The effects of cocaine are dependent, in part, upon prior and present history, dose, and route of administration. Dr. Billy Martin and his colleagues compared the cardiovascular effects elicited by voltalized cocaine to i.v. administration (in rats) and their findings indicate that inhalation of cocaine free base can produce cardiotoxic events with rapid onset that differ from i.v. drug administration. Acute exposure to voltalized cocaine produced a dramatic increase in blood pressure, a severe bradycardia, and heart blocks. In contrast, an i.v. injection of 1.5 mg/kg cocaine, a dose which has been estimated to be achieved after 5 min of inhalation exposure failed to elicit the profound bradycardia or heart blocks, but did increase mean arterial blood pressure (MAP). In addition, the cardiovascular effects that occurred during cocaine exposure underwent a rapid tolerance.
Diet and Taste Preferences May Predict Drug Use.
Dr. Blake Gosnell at the University of Wisconsin (Madison) has shown that preferences for fat and sweet tastes in rats predict a high preference for alcohol and/or morphine. Diet and taste preferences may be vulnerability factors for drug abuse, and raise the possibility that alteration of such preferences can be used in prevention and treatment efforts.
Other evidence that points to a role for diet and taste factors in the development of drug use is: (a) a high comorbidity between alcohol and drug abuse and eating disorders, (b) sweet cravings and high sweet intake in opiate addicts; and, (c) self-administration of a wide variety of drugs enhanced by food deprivation. Further research into the relationship between feeding motivation and drug motivation could lead to the development of important drug abuse prevention and treatment interventions.
Morphologic and Electrophysiologic Effects of Cocaine.
Dr. David Anderson at the Minneapolis Medical Research Foundation has shown that chronic cocaine abuse has definite morphologic and electrophysiologic effects on the brain. The studies are testing whether the long-term abuse of cocaine induces cerebral atrophy resulting in cognitive abnormalities, and whether these adverse consequences are partially reversible with abstinence from cocaine. Evidence for an ischemic mechanism for irreversible brain damage may suggest important approaches to treatment and prevention efforts.
NIDA-supported research at Columbia University is investigating whether children exposed to cocaine in utero are at increased risk of neurodevelopmental and behavioral impairments at 6-7 years of age. Dr. Margaret Heagarty has described the spectrum of neurodevelopmental and behavioral disabilities associated with maternal cocaine use during pregnancy. Importantly, she is distinguishing between neurodevelopmental effects directly attributable to intrauterine cocaine exposure and those caused by postnatal factors associated with maternal drug use, maternal education and IQ, and family environment.
Chimeras of Dopamine (DA) and Norepinephrine (NE) Transporters.
The dopamine (DA) and norepinephrine (NE) transporters share similar primary sequences and predicted topology, yet demonstrate important differences in their selectivity for ligands. To delineate discrete structural domains contributing to pharmacologic and kinetic differences between these transporters, a series of recombinant chimeras was generated by Susan Amara and her coworkers at Vollum Institute using a novel restriction site-independent method, and expressed in mammalian cells. Functional analyses of the chimeras delineate two discrete regions spanning the first through the third transmembrane domains (TM1-3) and TM10-11 that contribute to differences in their apparent affinities for DA, NE or MPP+(1-methyl-4-phenylpyridinium). These results also suggest that TM2-3 of the DA transporter have a role in selectively increasing the rate of DA uptake as compared to NE. TM4-8 of the DA transporter may influence the relative rate with which MPP+ is taken up into cells and could contribute to its selective toxicity in neurons expressing the DA transporter. These studies provide a framework for identifying the specific structural or regulatory determinants contributing to substrate recognition and translocation by the DA and NE transporters.
New Inhibitor of the Prohormone Convertase PC2.
Recently Dr. Iris Lindberg and her coworkers at Louisiana State University Medical Center discovered for the first time the properties of an endogenous inhibitor of PC2, the enzyme responsible for the synthesis of bioactive enkephalins and endorphins in brain and neural tissue. Deficiencies in the biosynthetic capacity for opioid peptides may be responsible for the addictive properties of opiate drugs in certain individuals. This discovery should greatly enhance our understanding of regulatory mechanisms in endorphin biosynthesis. A thorough understanding of regulatory mechanisms in opioid peptide synthesis might one day lead to enzyme-based drugs serving as therapeutic agents in opiate addiction.
Marijuana and Genetic Mutations.
Researchers at the University of Texas Medical Branch, Galveston, demonstrated a more than three-fold and more than two-fold increase over non-smoking pregnant women in mutations of the hypoxanthine phosphoribosyl transferase (hprt) gene among pregnant women who smoked marijuana and cigarettes, respectively, prior to and early in their pregnancies. Expansion of these preliminary studies should further establish the ability of marijuana to cause genetic mutations in women and the developing fetus (Ammenheuser, MM, Batinson, AB, Babiak, AE, et al. Elevated frequencies of hprt mutant lymphocytes in pregnant women who smoked marijuana, Environmental & Molecular Mutagenesis, 1995, in press).
In a twin study of drug abuse, 4,000 pairs of twins, monozygotic [MZ] and dizygotic [DZ], have been assessed for drug abuse and dependence. Research shows that there is a strong tendency that to a great extent, the abuse of some drugs (cocaine and marijuana) is affected by genetic factors. For marijuana, the common or family environment also made a significant contribution. Initiation of marijuana and cocaine use was influenced by characteristics of the environment (drug availability, peer groups) and the characteristics of the individual (personality). Data suggest that for the continuation of drug use, other individual characteristics, such as physiological and subjective reactions to the drugs, may also be important. Further, among the marijuana users, suspiciousness and agitation appeared to be genetically related, while the pleasant psychological effects appeared to be mediated by the environment shared by twins, and not by genes. Using this twin model, additional studies are underway to examine medical and health, including psychiatric, consequences of drug abuse, and genetic influences on drug use/abuse and associated conduct disorders and antisocial behaviors in childhood and adults. (Tsuang, MT., Llyons, M., Isen, S., Goldberg, J., & True, W. Heritability of initiation and continuation of drug use. Psychiatric Genetics, 1993, 3(3):141; Tsuang, MT., Llyons, M., Goldberg, J., True, W., Meyer J., & Eaves, L. Genetic influences on abuse of illicit drugs: A study of 3,297 twin pairs, submitted to JAMA; Llyons, M., Tooney, R., Green, A., Isen, S., Goldberg, J., True, W., Meyer J., & Tsuang, MT. How do genes influence marijuana use? The role of subjective effects. Submitted to Am. J. Psychiatry.)