Gavril W. Pasternak - US grants

One of the most important concepts to emerge in recent years in opiate research is the concept of multiple opiate receptors. Originally proposed as "Receptor Dualism", this concept has now been expanded to a whole variety of pharmacologically-defined receptor classes, including mu, delta, kappa, sigman and epsilon. Most recently, two subtypes of mu receptors have been proposed. One, the mu2 site, corresponds to the morphine-selective mu receptor previously characterized. The other, the mu1 site, represents a site first recognized in homogenate binding studies which binds both opiates and opioid peptides with similar very high affinities. The selective mu1 antagonists naloxonazine and naloxazone have proven very helpful in defining the pharmacological role of mu1 sites, implicating them in supraspinal analgesia, for example, but not in respiratory depression or most signs of physical dependence. This application proposes three major aims. The first consists of the characterization of the various classes of opiate binding sites. It consists of the development of selective binding assays for mu1, mu2, delta and kappa sites, followed by their biochemical and pharmacological characterization. The second aim involves the localization of these binding sites using digital subtraction computerized autoradiography. These studies will attempt to localize mu1, mu2, delta and kappa sites using a sophisticated computer analysis to regions within the brain as well as their pre- versus post-synaptic distributions. The last major aim involves continuation of studies on the characterization of a number of irreversible opiates. These compounds will be characterized in binding studies and their selectivity correlated with their pharmacological actions. Selected compounds will be utilized as affinity labels for the receptor. Ultimately, studies such as these should lead to the development to a better understanding of the actions of opiates and their more effective use in the management of pain.

This is a request for a Research Scientist Award (KO5). Despite their importance in the management of pain, opiates are abused and this remains a major problem. By understanding the roles of the many opioid receptor subtypes in pain control and addictive behavior, it may be possible to develop analgesics without abuse potentia. Opiate receptor multiplicity has expanded to three major classes of receptors (mu, delta and kappa) with subtypes within each class. These receptor subtypes have been associated with specific pharmacological actions in vivo and have been studied in traditional binding assays. The recent cloning of delta, mu and kappa1 receptors has greatly facilitated the study of opioid receptor heterogeneity. For example, we have confirmed the role of spinal delta receptors in enkephalin analgesia using an antisense oligodeoxynucleotide to DOR-l and identified a new putative kappa3 receptor clone based upon sequence homologies. An antisense oligodeoxynucleotide based upon this clone prevents analgesia in vivo by a kappa3 ligand but not morphine. These investigations into opioid receptor heterogeneity will be continued, focusing upon mu and kappa3 receptors. They will include the binding, regulation and second messenger systems of mu and kappa3 receptors in cell lines using traditional biochemical and molecular biological approaches, as well as novel ligands synthesized by my laboratory. Unlike tissue culture, the brain is a highly complex network of neuronal systems. The descriptions of marked regional synergy among various brainstem nuclei and between the brain and the spinal cord underscore the need for in vivo studies. Our studies on synergy within the central nervous system will continue and the roles of the various receptor subtypes will be defined. Investigations into tolerance and the role of nitric oxide (NO) also will be continued while additional studies looking at an important morphine metabolite, morphine 6beta-glucuronide, will be expanded. Our recent identification of an antiopioid sigma1 system within the brain may help explain the wide variability of responses to opioids among strains of mice and possibly people. Additional studies exploring these sigma1/opioid interactions are planned. While understanding opioid pharmacology is crucial, it is important to disseminate this knowledge to the medical community and the general public. This can only lead to a more effective approach to the control of pain and a decrease in the abuse of these important drugs.

Our overall goal is to develop novel and useful drugs for the control of pain and drug abuse. We have three major aims. The first is a continuation of the original proposal and extends our studies exploring the structure- activity relationships for NaIBzoH (6-desoxy-6-benzoylhydrazido-N-allyl- 14-hydroxydihydronormorphinone), a unique kappa3 agonist/mu antagonist. We have synthesized over 40 analogs of NalBzoH and have observed marked changes in affinity and selectivity with substitutions on the benzoyl ring. Additional compounds are proposed to help understand the importance of the carbonyl and the hydrazone functions. Significant differences also are seen among the naloxone, naltrexone and oxymorphone series which need further study. We will extend these SAR studies to the pharmacological characterization of these agents. In addition to its unique pharmacological profile, NalBzoH has an extremely prolonged dissociation rate from mu receptors which does not correlate with its apparent affinity in equilibrium binding studies. The structural components important in this function also will be explored. The second aim addresses the importance of a previously unrecognized anti-opioid system mediated through sigma1 receptors. Sigma1 agonists block opioid analgesia while sigma antagonists markedly enhance the analgesic activity of all opioid analogs tested. The most profound effects are observed with kappa drugs. We will continue to characterize this system and its interactions with analgesia as well as other opioid functions, including tolerance, dependence, gastrointestinal motility and respiratory depression, and define its effects on the therapeutic index of opioids. The last aim addresses possible mechanisms of action to explain the very high potency of the active morphine metabolite, morphine 6beta-glucuronide (M6G). M6G is over 100-fold more potent than morphine when administered into the brain or spinal canal. Binding studies reveal little difference between morphine and M6G, implying that simple differences in affinity for the receptor are not responsible for the dramatically potency differences. We, will explore the possible role of pharmacokinetics, intrinsic activity and even novel binding sites in the mechanism of action of this agent.

DESCRIPTION (provided by applicant): Opiates have a unique place in medicine in the treatment of pain, although they also have problems due to the potential of abuse. Most clinically used opioids act through mu opioid receptors. Yet, the wide range of responses among patients and the demonstration of incomplete cross tolerance has raised questions regarding how these drugs could all be acting through a single mu receptor. Pharmacological studies going back over twenty years have suggested the existence of multiple subpopulations of mu receptors, a concept that has now been confirmed with the cloning of splice variants of the cloned mu receptor MOR-1 in mice, rats and humans. Understanding the role of these receptor variants in behavior is important for the optimal use of these drugs. In this application we propose to extend ongoing studies correlating the cloned MOR-1 variants with the functional roles of opioid receptors in vivo. The major focus is upon the mu receptors, although additional studies will examine delta and kappa receptors as well. Prior studies have shown that the traditional seven transmembrane domain MOR-1 variants all bind mu opioids selectively and with similar high affinities. Yet, recent studies indicate that the efficacy of these drugs for the variants varies widely despite their similarities in receptor binding assays. We believe that the effects of mu opioids in vivo reflect the summation of actions from a number of mu receptor variants and that differences among the mu drugs reflects their differing efficacies for these receptor populations. We propose to examine this hypothesis using both antisense mapping, knockout animals and traditional behavioral approaches. We also will map the expression of the variants in these models immunohistochemically. Additional studies will focus on topical mechanisms of opioid action as a model system to examine these variangs in a more defined system. We also will examine the role of genetic backgrounds in opioid pharmacology. Finally, we will expand upon our studies with the transporter P-glycoprotein. In addition to its well documented role in the blood brain barrier, evidence suggests it also is important in the production of opioid tolerance. Together, these studies should provide insights into the role of the MOR-1 splice variants on opioid action and a better understanding of the use of these drugs.

DESCRIPTION: The principal focus of this project is multiple opioid receptor binding sites. The principal investigator has a long-established interest in identifying multiple subtypes of opioid receptors, using a variety of techniques including receptor binding and analgesia studies. However, when the mu opioid receptor was cloned, it was clear that only one type of receptor was coded by the mu receptor gene, and that potential multiple receptor subtypes must arise from different mechanisms. During the previous funding period, the principal investigator has explored the concept that multiple mu receptors arise from alternative splice variants of the mu receptor (MOR-1) gene. These concepts arose originally from antisense studies of opioid analgesia, in which different antisense oligonucleotides representing different exons of the MOR-1 gene produced different effects on analgesia. The studies were furthered by sequencing of the MOR-1 gene encoding a number of introns and exons. Although their physiological significance is not yet clear, the mRNA coding for at least 12 variants of this receptor have been isolated. The proposed studies will further explore these potential receptor subtypes. The first specific aim will provide a detailed characterization of mu receptor splice variants, including pharmacological and anatomical studies and identification of new clones. The second aim will perform similar experiments on the ORL-1 receptor, identifying at least one novel splice variant of this receptor. The third aim will explore the possible existence of MOR-1/ORL-1 heterodimers, based upon the preliminary finding that co-expression of both of these receptors into cell lines produces binding sites which are different from either site alone. The fourth aim will compare the binding properties of several peptide ligands for the OPRL-1 receptor.

DESCRIPTION (provided by applicant): The primary goal of this project is to develop novel and useful drugs for the control of pain and drug abuse. Recent years have seen a resurgence of opioid abuse, as well as the need for additional analgesics with limited abuse potential. Our objectives are to identify new lead drug candidates for further development and to identify and characterize modulatory systems that influence opioid action. The project contains three major aims. The first aim addresses the role of the anti-opioid sigma1 receptor system in the modulation of opioid analgesia. Sigma1 agonists potently reverse the analgesic activity of a variety of classes of opioid analgesics while sigma1 antagonists potentiate opioid analgesia. The sigma1 receptor has recently been cloned and will serve as a basis for examining this system at the molecular level. The cloned receptor also can serve as a screening system for potential new drugs. The second aim involves the role of ATP-binding cassette (ABC) transporters in opioid pharmacology. The most prominent member of this category, P-glycoprotein (i.e. mdr) has been implicated in the blood-brain barrier, as well as providing a system for transporting endogenous neuropeptides/transmitters from the brain to the peripheral circulation. It also plays a major role in morphine tolerance, with chronic morphine upregulating its expression. In addition to Pgp, MRP1 also has been implicated in opioid action. The last aim involves further studies with a series of opioid analogs synthesized in the laboratory during the previous granting period. By understanding how opioids and their modulatory systems interact, we hope to develop better approaches towards the management of pain and the discovery of new agents.

[unreadable] DESCRIPTION (provided by applicant): Opiates play a major role in the management of pain. Most opioids used clinically act through the mu opioid receptor, a G-protein-coupled receptor encoded by the Oprm gene. Despite a common receptor class through which they act, the pharmacology of mu opioids vary widely among patients. Twenty-five years ago we proposed multiple mu opioid receptors based upon a series of receptor binding and traditional pharmacological studies. The mu opioid receptor has been cloned and termed MOR-1. We have now identified 25 different splice variants of MOR-1 in the mouse, 10 in humans and 8 in rats, confirming our earlier suggestion of multiple mu opioid receptors. In the mouse, the splicing is complex due to the presence of two independent promoters located over 10 kb apart. The exon 1 promoter generates 16 different variants while the exon 11 promoter is responsible for 9. In addition to traditional, full length 7 transmembrane receptors, a number of truncated forms have been identified. The objective of this proposal is to continue our studies exploring MOR-1 and its actions, trying to understand the roles of these new splice variants. This will include receptor binding and functional assays. Studies on the truncated variants will be undertaken in an effort to assess their potential significance and function. A major component of this work will address the issue of the proteins associated with the receptors in the membrane. This receptor complex, which likely includes G-proteins and other regulatory proteins, may play a major role in understanding the functional differences observed among the full length variants and possibly even the activity of the truncated forms. We also will explore potential new splice variants. ORL1 is a member of the opioid family and is selective for its endogenous ligand OFQ/N. Expressed alone, ORL1 binds OFQ/N with very high affinity and is insensitive to traditional opioids. However, ORL1 dimerizes with MOR-1 to form a complex with unique pharmacological properties. Specifically, co-expression with MOR-1 renders ORL1 binding sensitive to a range of mu opioids. We will continue our studies of OFQ/N binding sites identified in brain and explore putative receptors for the OFQ/N-related peptides. Together, these studies should give a better understanding of the actions of opioids and their pharmacology. [unreadable] [unreadable] [unreadable]

The primary goal of this project is to identify and characterize new targets for medication development in the area of pain control and drug abuse. Recent years have seen a resurgence of opioid abuse, as well as the need for additional analgesics with limited abuse potential. Our objectives are to identify new lead drug candidates for further development and to identify and characterize modulatory systems that influence opioid action. The project contains three major aims. The first aim addresses molecular mechanisms of the anti-opioid sigma1 receptor system, looking for additional splice variants of the recently cloned sigma1 receptor and mechanisms through which these proteins modulate the functional aspects of G-protein coupled receptors. By isolating and cloning additional splice variants, it may be possible to identify novel targets for drug development. Sigma1 agonists potently reverse the analgesic activity of a variety of classes of opioid analgesics while sigma1 antagonists potentiate opioid analgesia. The second aim will explore the ability of sigma1 systems to selectively enhance opioid analgesia and not other opioid actions, as well as the role of sigma1 receptors in influencing the expression of ATP-binding cassette (ABC) transporters. The most prominent member of this family, P-glycoprotein (i.e. mdr) has been implicated in the blood-brain barrier, as well as providing a system for transporting endogenous neuropeptides/transmitters from the brain to the peripheral circulation. It also plays a major role in morphine tolerance, with chronic morphine upregulating its expression. In addition to Pgp, MRP1 also has been implicated in opioid action. The last aim involves further studies with a series of opioid analogs synthesized in the laboratory during the previous granting period and the synthesis of a series of new tools to assist in the study of opioid systems. By understanding how opioids and their modulatory systems interact, we hope to develop better approaches towards the management of pain and the discovery of new agents. This proposal explores new potential targets for medication development. Through a better understanding of sigma1 receptors it is hoped to maintain pain control while minimizing side-effects and possibly abuse liability while other aspects of the project explore novel opioid analgesics and antagonists with unique pharmacological profiles.

Opiates have a unique place in medicine in the treatment of pain, although they also have problems due to the potential of abuse. Most clinically used opioids act through mu opioid receptors. Yet, the wide range of responses among patients to individual drugs and the demonstration of incomplete cross tolerance among mu opioids has raised questions regarding how these drugs could all be acting through a single mu receptor. Pharmacological studies going back over twenty years have suggested the existence of multiple subpopulations of mu receptors, a concept that has now been confirmed with the cloning of splice variants of the cloned mu receptor MOR-1 in mice, rats and humans. Understanding the role of these receptor variants in behavior is important for the optimal use of these drugs. In this application we propose to extend ongoing studies correlating the cloned MOR-1 variants with their functions in vivo. We believe that the effects of mu opioids in vivo reflect the summation of actions from a number of mu receptor variants and that differences among the mu drugs reflects their differing efficacies for these receptor populations. We propose to examine this hypothesis using both antisense mapping, knockout animals and traditional behavioral approaches. We also will map the expression of the variants in these models immunohistochemically. Additional studies will focus on topical mechanisms of opioid action as a model system to examine these variants in a more defined system. Finally, we will expand upon the role of transporters in opioid tolerance. Together, these studies should provide insights into the role of the MOR-1 splice variants on opioid action and a better understanding of the use of these drugs.

Opiates remain among the most useful and important class of drugs in medicine, but not without problems. This submission proposes to examine two clinically relevant issues in opioid use ? safety and tolerance. Most opioids used clinically act through the mu opioid receptor. The mu opioid receptor gene Oprm1 undergoes alternative splicing to generate a family of opioid receptors that can be categorized into three classes based upon their structure, each of which contain a number of variants. Knockout mouse models that selectively remove different sets of Oprm1 variants suggest that morphine acts through only one of these sets of variants while drugs acting through different sets of mu receptors lack respiratory depression, physical dependence and reward while maintaining their analgesic activity, thus enhancing their safety. The focus of this application is to understand the role and significance of the sets of mu opioid receptor splice variants in opioid analgesia, side-effects and tolerance. The current application will explore this concept by generating a mouse model in which selected opioid receptor splice variants can be expressed under native control of the Oprm1 gene, thereby permitting the exploration of their actions in vivo. The second aspect of this application involves tolerance. Preclinicla models reveal that short-term opioid administration leads to progressive tolerance. Yet, cancer patients can be maintained on fixed opioid doses without dose escalation to relieve their pain for many months. In a recent study using an extended chronic administration paradigm we reconciled these observations, showing a progressively increasing tolerance to morphine for up to three weeks that then stabilized with no further increases for as long as 6 weeks. Furthermore, this stabilization was associated with changes of select Oprm1 splice variants in specific brain regions of as much as 400-fold. The second component of this application will explore the stabilization of opioid tolerance with extended administration and potential mechanisms.

The expansion of opioid prescribing in recent years to better treat pain has markedly increased their usage and availability and fueled an epidemic of abuse. Estimates of up to 80% of addicts reported initiating their habit through prescriptions drugs. Decreasing opioid prescriptions would lower opioid exposure with fewer people receiving the drugs and less drug available for diversion. We have identified a novel target in brain distinct from any of the traditional opioid receptors capable of mediating potent analgesia without the reward behavior and side-effects seen with traditional opioids. We have targeted this site with a series of arylepoxamides and have identified a clinical candidate (MP1000) and backup compound. MP1000 is a potent analgesic in a range of thermal, inflammatory and neuropathic analgesic assays. It fails to show reward behavior and does not produce respiratory depression at doses 5-fold greater than its analgesic ED50. Chronic administration does not produce physical dependence or withdrawal when challenged with an antagonist. It shows no cross tolerance to morphine and can be co- administered to subjects already on opioids for pain to lower their opioid usage (i.e. ?opioid sparing), facilitating the eventual discontinuation of the opioid. Preliminary safety and toxicology studies are encouraging, and, based upon these results we are proposing to carry out IND- enabling studies and a Phase 1 clinical trial.