Opiates are powerful analgesics (agents used for the treatment of pain), but their use is hampered by non-trivial side effects, tolerance to the analgesic effects, physical dependence resulting in withdrawal effects, and by concerns surrounding the possibility of addiction. By itself, enhanced analgesic efficacy of an opiate can result in opioid sparing, and therefore a reduction in opioid-related side effects. The side effects of opiates include nausea, vomiting, pruritus, insomnia, constipation, sedation and impaired physical function (Ballantyne et al., 2003 N Engl J Med 349:1943-1953).
In many cases, patients taking opioids balance side effects with analgesia, often choosing to tolerate a certain amount of pain so as to avoid side effects. The more severe side effect of respiratory depression can also limit the tolerated dose, and hence the effective analgesia in many patients.
One of the most problematic aspects of opioid therapy is analgesic tolerance with prolonged treatment. Tolerance can be defined as the need for progressively higher doses in order to maintain the same reduction in pain. Although opioid rotation is currently used to minimize tolerance, this approach requires close monitoring due to variable cross-tolerance and side effect profiles among different patients (Fine, 2004 J Pain Palliat Care Pharmacother 18:75-79).
In its most severe form, opioid tolerance can manifest as opioid-induced hyperalgesia; that is, the opiate no longer reduces pain but actually increases or induces pain (Arner et al., 1988 Acta Anaesthesiol Scan 32:253-259; Simonnet et al., 2003 Neuroreport 14:1-7; Fine, 2004 J Pain Palliat Care Pharmacother 18:75-79). This hyperalgesia is clinically similar to the hyperalgesia of neuropathic pain, and in vivo models show that brainstem descending pain facilitation pathways are activated in both syndromes (Vanderah et al., 2001 Pain 92:5-9). Like neuropathic pain, opioid-induced hyperalgesia is extremely difficult to treat and is often a physician's greatest fear in initiating opioid therapy.
Dependence and addiction are also among the greatest fears of pain patients surrounding the use of opiates. Dependence is characterized by physical or psychological withdrawal upon discontinuation of the opiate and can be independent of addiction, which itself is defined by repeated, often self-destructive behaviors focused on obtaining the drug, according to DSM-IV criteria (American Psychiatric Association, 2000).
However, it is still thought that physical dependence, or the desire to avoid withdrawal, contributes to opiate addiction, particularly at later stages of addiction; whereas, a craving for the euphoric effects of opiates can dominate in earlier stages (Koob et al., 1989 Neurosci Biobehav Rev 13:135-140). The somatic withdrawal signs that can occur when opioid therapy is abruptly stopped in physically dependent individuals include agitation, irritability, muscular jerks, abdominal pain, diarrhea, burning sensations, “gooseflesh” and itching (Miser et al., 1986 Am J Dis Child 140:603-604; Heit, 2003 J Pain Palliat Care Phamacother 17:15-29).
Abrupt cessation of opioid treatment can also cause a hyperalgesia, which has also been referred to as opioid-induced hyperalgesia (Li et al., 2001 Anesth Analg 93:204-209). Although patients receiving prolonged opioid analgesic therapy can or can not develop analgesic tolerance, they usually become physically dependent, requiring careful tapering off of the opiate in order to minimize withdrawal effects (Heit, 2003 J Pain Palliat Care Phamacother 17:15-29; Woolf et al., 2004 Curr Opin Investig Drugs 5:61-66).
Opiates produce analgesia by activation of opioid receptors that belong to the superfamily of G protein-coupled receptors (GPCRs). Opioid receptors are also involved in the development of the physical and psychological dependence that are important aspects of drug abuse and addiction.
Studies on GPCRs, including opioid receptors, have shown that the third cytoplasmic loop and the carboxyl-terminal tail are very important for signal transduction (Law et al., 2000 Annu Rev Pharmacol Toxicol 40:389-430), regulation (Law and Loh, 1999 J Pharmacol Exp Ther 289:607-624), and internalization of GPCRs (Trapaidze et al., 1996 J Biol Chem 271:29279-29285; Keith et al., 1998 Mol Pharmacol 53:377-384), and are frequently involved in the association of the receptors with other proteins. In addition to G proteins, examples of proteins known to interact with GPCRs are Gprotein-coupled receptor kinases (Pitcher et al., 1998 Annu Rev Biochem 67:653-692), β-arrestins (Lefkowitz, 1998 J Biol Chem 273:18677-18680), PDZ domain-containing adaptor molecules (Milligan and White, 2001 Trends Pharmacol Sci 22:513-518), and scaffolding proteins such as filamin A (Onoprishvilli et al., 2003 Molec Pharmacol 64:1092-1100).
More specifically, opiates produce analgesia by activation of mu (μ) opioid receptor-linked inhibitory G protein signaling cascades and related ion channel interactions that suppress cellular activities by hyperpolarization. The μ opioid receptor (MOR) preferentially couples to pertussis toxin-sensitive G proteins, Gαi/o (inhibitory/other), and inhibits the adenylyl cyclase/cAMP pathway (Laugwitz et al., 1993 Neuron 10:233-242; Connor et al., 1999 Clin Exp Pharmacol Physiol 26:493-499). The analgesic effects of MOR activation have been predominantly attributed to the Gβγ dimer released from the Gαi/o protein, which activates G protein activated inwardly rectifying potassium (GIRK) channels (Ikeda et al., 2000 Neurosci Res 38:113-116) and inhibits voltage-dependent calcium channels (VDCCs) (Saegusa et al., 2000 Proc Natl Acad Sci USA 97:6132-6137), thereby suppressing cellular activities by hyperpolarization.
Adenylyl cyclase inhibition can also contribute to opioid analgesia, or its activation can contribute to analgesic tolerance. This inhibition is due to overexpression of adenylyl cyclase type 7 in the CNS of mice that leads to more rapid tolerance to morphine (Yoshimura et al., 2000 Mol Pharmacol 58:1011-1016). Additionally, adenylyl cyclase activation has been suggested to elicit analgesic tolerance or tolerance-associated hyperalgesia (Wang et al., 1997 J Neurochem 68:248-254). Although the superactivation of adenylyl cyclase after chronic opioid administration is more often viewed as a hallmark of opioid dependence than as a mediator of tolerance (Nestler, 2001 Am J Addict 10:201-217), both are consequences of chronic opioid administration, and tolerance often worsens dependence. Chronic pain patients who have escalated their opioid dose over time often experience more withdrawal than patients on a constant dose.
An important but underemphasized cellular consequence of chronic opioid treatment is excitatory signaling by opioid receptors in place of the usual inhibitory signaling (Crain et al., 1992 Brain Res 575:13-24; Crain et al., 2000 Pain 84:121-131; Gintzler et al., 2001 Mol Neurobiol 21:21-33; Wang et al., 2005 Neuroscience 135:247-261), possibly as a result of the decreased efficiency of coupling to the native G proteins; that decrease in efficiency being the index of desensitization (Sim et al., 1996 J Neurosci 16:2684-2692). Although the cellular effects of opiates are normally inhibitory, several in vitro studies have demonstrated that opiates can elicit excitatory effects either at low doses (Shen et al., 1989 Brain Res 491:227-242; Crain et al., 1990 Trands Pharmaol Sci 11:77-81) or after chronic exposure (Crain et al., 1992 Brain Res 575:13-24).
In vivo, opiates can cause “paradoxical hyperalgesia” at low doses (Kayser et al., 1987 Brain Res 414:155-157; Kiyatkin, 1989 Int J Neurosci 45:231-246; Crain et al., 2001 Brain Res 888:75-82), or after chronic administration, opioid-induced hyperalgesia (Arner et al., 1988 Acta Anaesthesiol Scan 32:253-259). Although descending facilitation of spinal cord dorsal horn neurons has been implicated in tolerance-associated hyperalgesia (Vanderah et al., 2001 Pain 92:5-9), alterations in opioid receptor signaling also occur with chronic opioid treatment (Shen et al., 1989 Brain Res 491:227-242; Crain et al., 1990 Trends Pharmacol Sci 11:77-81; Crain et al., 1992 Brain Res 575:13-24; Gintzler et al., 2001 Mol Neurobiol 21:21-33) and can contribute to the enhanced firing of descending brainstem projections.
Chronic opioid treatment causes excitatory signaling of opioid receptors via a switch in their G protein coupling from Gi/o to Gs proteins (Wang et al 2005 Neuroscience 135:247-261; Chakrabarti et al., 2005 Mol Brain Res 135:217-224) and by stimulation of adenylyl cyclase II and IV by mu opioid receptor-associated Gβγ dimers (Chakrabarti et al., 1998 Mol Pharmacol 54:655-662; Wang et al., 2005 Neuroscience 135:247-261). The interaction of the Gβγ dimer with adenylyl cyclase had previously been postulated to be the sole signaling change underlying the excitatory effects of opiates (Gintzler et al., 2001 Mol Neurobiol 21:21-33). It has further been shown that the Gβγ that interacts with adenylyl cyclases originates from the Gs protein coupling to MOR and not from the Gi/o proteins native to MOR (Wang et al., 2006 J Neurobiol 66:1302-1310). Importantly, the switch in G protein coupling by MOR and the interaction of the Gβγ dimer with adenylyl cyclase II and IV, are both signaling alterations attenuated by co-treatment of ultra-low-dose opioid antagonists, such as naloxone (NLX) or naltrexone (NTX), with opioid agonists (Wang et al., 2005 Neuroscience 135:247-261).
Ultra-low-dose opioid antagonists have been shown to enhance opioid analgesia, minimize opioid tolerance and dependence (Crain et al., 1995 Proc Natl Acad Sci USA 92:10540-10544; Powell et al. 2002. JPET 300:588-596), and attenuate the addictive properties of opioids (Leri et al., 2005 Pharmacol Biochem. Behav 82:252-262; Olmstead et al., 2005 Psychopharmacology 181:576-581). An ultra-low dose of opioid antagonist was an amount initially based on in vitro studies of nociceptive dorsal root ganglion neurons and on in vivo mouse studies, wherein the amount of the excitatory opioid receptor antagonist administered is about 1000- to about 10,000,000-fold less, preferably about 10,000- to about 1,000,000-fold less than the amount of opioid agonist administered. It has long been hypothesized that ultra-low-dose opioid antagonists enhance analgesia and alleviate tolerance/dependence by blocking the excitatory signaling opioid receptors that underlie opioid tolerance and hyperalgesia (Crain et al., 2000 Pain 84:121-131).
The attenuation of analgesic tolerance by administration of ultra-low doses, defined herein after, of NLX has been demonstrated in rat studies, where rats treated with morphine and low doses of NLX showed no antinociceptive tolerance (or tolerance to reducing sensitivity to painful stimuli) when compared to rats treated with morphine alone. Signs of physical dependence were also markedly reduced when morphine was administered with ultra-low dosages of NLX. Antinociception (reducing sensitivity to painful stimuli) was not observed in rats administered NLX alone. Further, co-administration of morphine and NLX resulted in a marked reduction in MOR-Gs coupling associated with analgesic tolerance and dependence. The interaction of Gβγ with adenylyl cyclase II or IV was also markedly attenuated or abolished when rats were co-treated with morphine+NLX. These findings suggest that ultra-low-dose NLX reduces antinociceptive tolerance and dependence by preventing the mu opioid receptor-Gs coupling that results from the chronic opiate administration.
The development of novel therapeutics that combine ultra-low-dose opioid antagonists with opiates is currently under development in products such as Oxytrex™ (oxycodone plus ultra-low-dose NLX) from Pain Therapeutics, Inc (San Mateo, Calif.). The combination of ultra-low-dose opioid antagonists with opioid agonists formulated together in one medication has been shown to alleviate many of these undesirable aspects of opioid therapy (Burns, 2005 Recent Developments in Pain Research 115-136, ISBN:81-308-0012-8). This approach shows promise for an improvement in analgesic efficacy, and animal data suggests reduced addictive potential.
The need still remains for a deeper understanding of the cellular mechanism of action of ultra-low-dose opioid antagonists. Specifically, the identification of the cellular target of ultra-low-dose opioid antagonists in their inhibition of mu opioid receptor-Gs coupling can permit development of assays to screen as well as against this target to create a new generation of pain therapeutics that can provide long-lasting analgesia with minimal tolerance, dependence and addictive properties. Importantly, a non-opioid cellular target of ultra-low-dose NLX or NTX would provide potential for developing either a therapeutic combination of which one component is not required to be ultra-low-dose, or a single-entity novel analgesic.
The present invention identifies the precise target for such screening assays, and describes one such screening assay as well as the characteristics of potential single-entity drug candidates that provide strong opioid-like analgesia, while minimizing tolerance, dependence and addictive properties.