The opioid receptors are a subfamily of the family A G protein-coupled opioid receptor superfamily and consist of mu (OPRM1), delta (OPRD1), and kappa (OPRK1), all of which activate inhibitory G proteins. The dynorphins act as endogenous agonists of OPRK to activate a variety of signaling transduction pathways including those involving mitogen activated protein kinases (MAPK). Activation of OPRK leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward. Hence, OPRK antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists are very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK- dynorphin system and as potential pharmacotherapies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports ML350 as a highly potent OPRK1 antagonist with an IC50 of 9-16 nM, with high selectivity (selectivities vs. OPRD1 and OPRM1 of 219-382–fold and 20-35–fold, respectively). ML350 was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that ML350 has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging in vivo pharmacokinetic profile in rats. ML350 was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, ML350 was shown to have a reversible analgesic effect when challenged with an OPRK agonist in a tail flick assay in mice. ML350 serves as a novel OPRK antagonist that can be developed as a therapeutic for the treatment of a variety of disorders involving the OPRK1-dynorphin system.

Probe Structure & Characteristics

Recommendations for scientific use of the probe

Prototypic OPRK antagonists are very long-acting, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. The phase I studies with PF-04455242 were terminated due to toxicity issues. Eli Lilly advanced LY2456302 to a phase I clinical trial study (oral treatment of alcohol dependence) to measure the occupancy of brain OPRK after single oral doses. The last update in ClinicalTrials.gov reported for this study is May 5, 2011; no information on any progress in further Phase I studies or progression to Phase II is available. Such a delay is unusual due to the costs involved. Hence, there is a need for additional drug-like highly potent and selective OPRK antagonists with a good safety profile and CNS exposure.

Selective OPRK antagonists are being explored for their effects in the treatment of a wide variety of areas including cocaine addiction [1], depression [2], and feeding behavior [3] and have been proposed as a treatment for psychosis and schizophrenia [4]. ML350 will be used in a stress-induced migraine model assay that measures cutaneous (tactile) allodynia in rats as a measurable translational endpoint for headache-related pain [5], an alcohol withdrawal assay that measures working memory performance and anxiety-like behavior in rats after withdrawal from alcohol [6], and a cocaine reward assay that measures the effect on drug-seeking behavior in a cocaine addiction model.

The probe will be of use to researchers interested in the role of the OPRK in neuropathic pain, drug addiction, or affective psychiatric disorders.

New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK-dynorphin system. Signaling through OPRK results in the activation of multiple signal transduction pathways including activation of PI3-kinase, PKCζ, and EERK-1 and ERK-2 [7]. In addition, signaling through OPRK results in activation of the p38 MAPK [8-15] and the JNK1 [16-17] signaling pathways, and the JAK2/STAT3 and IRF2 signaling cascade [18]. Different OPRK1 ligands are reported to activate distinct signal transduction pathways; this activation of multiple signal transduction pathways are thought to be due to ‘ligand-directed signal trafficking’ by OPRK1 [7-17, 19-21]. It is thought that ligands that are selective for one pathway over the other will help elucidate the role of each pathway in specific responses. This will help in the development of reagents and/or therapeutics that can target pathway responsible for the wanted effects versus unwanted side effects.

1. Introduction

Opioids are the most widely used class of analgesics [22]. The opioid receptors are a subfamily of the G protein-coupled opioid receptor (GPCR) superfamily and the three major types, mu (OPRM1), delta (OPRD1), and kappa (OPRK1) opioid receptors have been pharmacologically characterized and cloned [23-25]. All activate inhibitory G proteins. Signaling through OPRK results in the activation of a number of signal transduction pathways [7-21] and this, in turn, leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward [1-2, 4]. Hence, kappa opioid antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK system and as potential pharmacotherapies.

Neuropathic pain: Studies have identified a role for dynorphin and OPRK in neuropathic pain [26]. The dynorphins act as endogenous agonists of OPRK1 [27]. The OPRK1-dynorphin system mediates astrocyte proliferation through the activation of p38 MAPK that is required for the effects of neuropathic pain on analgesic responses [15, 28]. Increased dynorphin expression in neuropathic pain leads to a sustained activation of OPRK [26, 29-30].

There is evidence that the endogenous dynorphin-derived opioids may produce either a sustained reduction or an increase in sensitivity to painful stimuli [15, 31]. Thus, dynorphin can elicit multiple effects to modulate analgesic responses. Antinociceptive effects of intrathecal and systemic administration of selective OPRK1 agonists have been documented [31-32]. It has also been reported that the OPRK1 antagonist norbinaltorphimine (nor-BNI) significantly lowers pain thresholds and increases pain sensation after sciatic nerve ligation [33]. Thus, the endogenous dynorphin-derived opioids may have both antinociceptive and pronociceptive actions. How sustained activation of opioid receptors by endogenous dynorphins contributes to the neuropathic pain state is not clear. One of the side effects of OPRK1-mediated analgesia is depression [30, 34]; the development of novel analgesics that bypass this side effect would be therapeutically beneficial.

Drug addiction: About 90 million people worldwide suffer from drug addiction [35]. A role for the OPRK1-dynorphin system in modulating drug addiction has been proposed; however its function appears to be diverse, and may modulate drug-seeking behavior depending on factors such as drug history, pattern of intake, and stress (for review see [36-37]). Multiple exposures to cocaine result in complex molecular changes in the brain, and, ultimately, in addiction [38]. Although a single exposure to cocaine in rats does not affect brain dynorphin levels, repeated exposures increase dynorphin concentrations in the striatum and substantia nigra [39].

The role of OPRK1 in cocaine addiction has been actively studied [40-50]. Results clearly demonstrate an activation of the OPRK1 system following chronic cocaine exposure. As OPRK1 stimulation in the brain produces aversive effects in animals and humans [51-53], it is likely that cocaine-induced up-regulation of OPRK1 in regions associated with reward might be part of a protective compensatory neuroadaptive mechanism to counteract the rewarding effect of cocaine and might contribute to the emergence of persistent dysphoria, an emotional state marked by anxiety, depression, and restlessness that is often reported in humans after the withdrawal of the drug [54]. Studies with OPRK1 knockout mice [55-62] suggest that the receptor antagonists might be useful in preventing stress-induced relapse in cocaine-dependent individuals and therefore help to prevent drug use relapse.

It has been reported that chronic nicotine exposure affects OPRKs modulation of neurotransmission, leading to enhanced negative affect and increased anxiogenic effects [63]. Furthermore, spontaneous nicotine withdrawal-induced anxiety-like behavior and somatic signs of withdrawal are blocked by pretreatment with the OPRK antagonists nor-BNI or JDTic [63].

Several studies have implicated the role of OPRK1 signaling in stress-induced reoccurrence of ethanol self-administration [64-65]. In addition, OPRK1-dynorphin systems have been shown to be altered by opiate treatment in reward-related neural circuits in animal models [64, 66-67]. Taken together, these studies suggest that antagonism of OPRK1 may mitigate negative reinforcing behavioral states associated with drug withdrawal.

Affective psychiatric disorders: Affective psychiatric disorders comprise a worldwide health challenge; about 120 and 25 million people suffer from depression and schizophrenia, respectively [35]. These afflictions are all characterized by changes in emotion, motivation, cognition, and stress reactivity. Imaging studies have consistently shown altered activity in the amygdala, hippocampus, basal ganglia, and prefrontal cortex of psychiatric patients [68]; areas involved in stress responsiveness, emotional reactivity, goal-directed behavior, motivation, and executive function. OPRK1 and the dynorphin peptides are enriched in these brain regions, where they play a role in modulating neurotransmission. This has been an increasingly active area of research in recent years, and resulting data suggest that dysregulation of this system may contribute to the development and maintenance of various affective psychiatric disorders [69]; for review, see [36, 70]. However, solid evidence from clinical studies is lacking.

There is increasing evidence for a potential involvement of OPRK1-dynorphin in schizophrenia; OPRK1 agonists appear to induce symptoms in humans and animals that are present in schizophrenia [70-72]. The potent OPRK1 agonist Salvinorin A produces hallucinations in humans, supporting the idea of a OPRK1-dynorphin involvement in disorders characterized by disturbed perception [72]. Potential evidence for a role of dynorphin in psychotic disorders also comes from animal experiments. In rats, the selective OPRK1 agonist U- 50488H induced a dose-dependent reduction of pre-pulse inhibition [73], which is seen as a readout of sensorimotor gating and is impaired in schizophrenics. Pre-pulse inhibition was restored by the selective OPRK1 antagonist nor-BNI [73].

The role of OPRK1 in stress has been an active area of study, linking OPRK signaling and behavior [74-75]. Stress-induced opioid peptide release resulting in stress-induced analgesia through action at opioid receptors has been reported for all of the major opioid systems. It has been reported that OPRK1 activation after stress can also modulate numerous behaviors, including reward and depression [20, 76-77]. OPRK1 antagonist administration produces anxiolytic effects in several rat models of stress (elevated plus-maze, open-field, and fear potentiated startle paradigms) [78], suggesting a role for endogenous dynorphin release in the expression of anxiety-like behavior in these stress models. OPRK1 antagonists produce effects similar to that of traditional anti-depressants [2, 51, 79]. In an animal model widely used to model social defeat stress, wild-type mice treated with the selective OPRK1 antagonist Nor-BNI exhibit decreased social defeat postures [80].

In the forced swim test, a rodent model of depression and a procedure that identifies in rats treatments with antidepressant efficacy in humans [81], effects produced by OPRK1 agonists and antagonists have been interpreted as “prodepressive” and “anti-depressant”, respectively [2, 82]. The antidepressant-like effects of OPRK1 antagonists have also been observed in other studies [51, 83-84]. Interestingly, standard antidepressant drugs often cause anxiety [85-87]. The antidepressant and anxiety-relieving effects of OPRK1 antagonists is notable, and suggests that this class drug might be particularly efficacious for the treatment of concomitant depressive and anxiety disorders [88].

The physiological and pathophysiological mechanisms of OPRK1-dynorphin systems and their roles in neuropathic pain, drug addiction, and affective psychiatric disease in humans are active areas of study. The availability of new research tools such as potent and selective OPRK1 antagonists will facilitate understanding of the mechanisms involved in these processes and potentially have therapeutic value as novel therapies with an improved side effect profile to currently available drugs.

Several OPRK1 antagonists have been described in the literature. Norbinaltorphimine (nor-BNI) [89-90], 5′-guanidinonaltrindole (GNTI) [89, 91], and JDTic [89, 92] all exhibit a delay in the onset of action of approximately 24 hours [89, 93-95], and have very long lasting in vivo effects of up to 56 days [89, 95-96]. Due to their pharmacodynamics/pharmacokinetics and poor unbound brain/plasma ratio, morphine-like derivatives, nor-BNI and GNTI, are only used as pharmacological tools. The non-opioid compound JDTic exhibited a poor brain exposure but had an extraordinary persistence in brain (mean brain concentration declined by only 56% over 24 hours, and the drug was still detectable at 1 week in mice) despite its P-glycoprotein-mediated efflux, and its moderate lipophilicity and low affinity for cell homogenates. The presence of two basic nitrogens and entrapment in cellular compartments such as lysosomes have been proposed as a possible mechanism of this persistence [89, 97]. A Phase 1 clinical trial of JDTic for cocaine dependence was terminated in 2012 due to adverse events. Several short-acting compounds from distinct chemotypes have been developed with greater CNS exposure compared to the prototypic ligands (nor-BNI, GNTI, JDTic). AstraZeneca disclosed a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides. AZ-MTAB was efficacious in animal models of mood disorders, but was associated with a significant hERG liability (IC50 of 260 nM) [97-98]. Another OPRK antagonist PF-04455242 [99] was developed by Pfizer. However, on January 6, 2010 the phase 1 study for bipolar disorder and depression was terminated due to toxicology findings in animals exposed to PF-04455242 for three months. Eli Lilly identified a subnanomolar OPRK antagonist with selectivity of 21 and 135 over OPRM and OPRD respectively [100]. In 2010 LY2456302 was advanced to Phase I clinical trial for the oral treatment of alcohol dependence. The clinical study was to assess the brain OPRK1 occupancy after single oral doses of LY2456302 as measured by positron emission tomography with radioligand LY2879788 (¹¹C PKAB) in healthy subjects. The last update of the study was May 5, 2011, and no new studies for the compound are listed in ClinicalTrials.gov.

Even though a few drug-like OPRK antagonists with more favorable pharmacokinetics than nor-BNI, GNTI, JDTic have been developed, new OPRK antagonists possessing novel scaffolds and improved selectivity are needed both as pharmacological tools to better understand the OPRK1-dynorphin system and as potential pharmacotherapies.

2. Materials and Methods

2.2. Probe Chemical Characterization

The probe structure was verified by ¹H and ¹³C NMR (see Section 2.3) and high resolution LC-MS (Figure 1). Purity was assessed to be greater than 95% by LC-MS.

Figure 1

LC/MS analysis of ML350.

Solubility (at room temperature) for ML350 in PBS was determined to be 557 μM. Solubility in water and saline was determined to be 1.1 mM. ML335 has a half-life of >48 hours in PBS at room temperature (79% compound remaining at 48 hours) (Figure 2).

Figure 2

Stability of ML350 (CYM50202) in PBS.

No Michael acceptor adducts were observed when a sample of the probe was incubated with 50 μM glutathione and analyzed by LC-MS.

The following compounds have been submitted to the SMR collection (Table 1).

Table 1

Compounds submitted to the SMR collection (2-21-2013).

2.3. Probe Preparation

Figure 3Synthetic scheme for ML350 (CYM50202)

To a stirred solution of trans-rac-N-Boc-4amino-3-hydroxy piperidine I and cyclohexanone in DCE were added NaBH(OAc)₃ and AcOH. The reaction mixture was stirred overnight at room temperature. The mixture was diluted with ethyl acetate and washed with brine (2X). The organic phase was concentrated, and the product II purified by column chromatography using CH₂Cl₂/MeOH (9:1).

To a solution of II in CH₂Cl₂ was added TFA and the reaction mixture was stirred for 30 minutes at room temperature. The mixture was concentrated under reduced pressure. The residue was dissolved in ethanol followed by the addition of DIPEA and pyridine III. The reaction mixture was heated at 145°C for 35 minutes under microwave irradiation. The crude was concentrated under reduced pressure and the product purified by HPLC furnishing the pure compound ML350 (CYM50202; CID 60156214).

¹H NMR and ¹³C NMR of methyl 5-bromo-2-(4-(cyclohexylamino)-3-hydroxypiperidin-1-yl)nicotinate are as follows: ¹H NMR (600 MHz, CDCl₃): δ 8.20 (s, 1H), 8.07 (dd, J = 9.5, 2.4 Hz, 1H), 3.95-3.90 (m, 2H), 3.83 (s, 3H), 3.79 (d, J = 13.3 Hz, 1H), 3.15 (bs, 2H), 2.89 (t, J = 12.4 Hz, 1H), 2.81 (t, J = 12.1 Hz, 1H), 2.07-2.02 (m, 3H), 1.90-1.82 (m, 3H), 1.67 (d, J = 12.4 Hz, 1H), 1.51 (q, J = 11.5 Hz, 1H), 11.4 (q, J = 11.4 Hz, 1H), 1.29-1.16 (m, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 166.63, 157.96, 152.16, 143.80, 115.87, 110.44, 68.27, 59.70, 56.08, 55.39, 53.34, 48.62, 30.61, 29.16, 27.24, 25.67, 25.47, 25.31. MS (EI) m/z: 412, 414 (M+H). The purity was assessed to be greater than 95% by LC-MS.

3. Results

3.1. Summary of Screening Results

Prior to implementing the OPRM1-OPRD1 agonist HTS screen of the MLPCN library, a series of pilot screens of the Maybridge Library was done at the SRIMSC Screening Center (Figure 4). Screens for each opioid receptor were run in agonist and antagonist mode. An OPRK1 antagonist screen with an activity cutoff of >50% inhibition identified 72 hits out of 16,000 compounds screened (1X%INH; AID 652031). A confirmation OPRK1 antagonist screen (3X%INH; AID 652082) and an S1P1 counterscreen (3X%INH; AID 652087) identified 11 compounds that confimed OPRK1 antagonism and were inactive at S1P1; these were repurchased as powders. These 11 compounds were screened in four dose response assays: OPRK1 antagonist (AID 652077), OPRM1 antagonist counterscreen (AID 652081), OPRD1 antagonist counterscreen (AID 652080), and S1P1 antagonist counterscreen (AID 652079). From these screens a hit compound that appeared to exhibit potency and selectivity as an OPRK antagonist was identified (CID 2796048) (Figure 5). Structural integrity and potency of CID 2796048 were verified by independent synthesis. Chemical archeology was performed on and around the structure. Medicinal chemistry optimization by SAR by purchase and synthesis was begun.

Figure 4

Flow chart describing Maybridge screening results.

Figure 5

Compound identified from Maybridge library; CID 2796048.

3.2. Dose Response Curve for Probe

Figure 6Dose response curve for ML350

3.4. SAR Table

The original screening hit CID 2796048 can be conceptualized as consisting of three regions A, B, and C. Table 2 shows structures of compounds used for probe optimization.

Table 2

SAR Table for optimization of antagonist probe ML350 for OPRK.

The HTS hit compound (entry 1) was purchased (SID 26534319) with confirmed IC50s of 410 nM at OPRK1, 4.59 μM at OPRM1 and no demonstrated OPRD1 activity at concentrations up to 50 μM. Compound 1 does not contain reactive functional groups, is chemically amiable and is moderately and highly selective against the OPRM1 and OPRD1, thus making this compound suitable for medicinal chemistry optimization.

Pyridine region (A): We started our SAR studies by modifying the two metabolic soft spots (ester groups) in region A using the piperidine-4-yl and cyclohexylamine as regions B and C. 4-cyclohexylamine piperidine was the constant moiety to simplify the synthetic work and allow rapid screening of region A. First, we sought to minimize the structure of the hit (Andrews' analysis) by reducing the number of substituents on the pyridine ring. Interestingly, the 5-acetamide (entry 2) was found less that 2-fold less potent than compound 1. Changing the acetamide group for halogens led to an interesting series of compounds. The increase in potency was inversely related to the electronegativity of the halogen atoms: the fluoride (entry 6) was ∼16-fold less potent than compound 1, while the iodine (entry 3), bromide (entry 4) and chloride (entry 5) were ∼5-, ∼3-, ∼2.5-fold more potent, respectively. Conversely, the selectivity against the OPRM1 increased directly proportionally to their electronegativity: iodide, bromide and chloride were ∼6-, 7.5- and 12.5-fold selective against the OPRM. Interestingly the selectivity against the OPRD1 was higher for the bromide (143-fold) followed by the iodide (32-fold) and chloride (25-fold). Installing a phenyl ring (entry 7) on the 5-position led to complete loss of potency. Replacing the methyl ester of compound 4 for an isopropyl ester furnished compound 8 with ∼3-fold loss in potency at the OPRK1. A decrease in selectivity against the OPRD (7.5-fold) and an increase in selectivity against the OPRM1 (50-fold) were observed, indicating that steric factors at the 3-position of ring A are important for modulating potency and selectivity. A 39-fold loss in potency was observed for the carboxylic acid (entry 9) compared to compound 4.

Cyclohexylamine region (C): The cyclohexylamine analog of compound 1 (entry 10) was ∼6-fold less potent at OPRK1. To continue our SAR studies we selected the methyl 5-bromopyridine-3-carboxylate moiety from compound 4 due to its potency (19.5-fold more potent than compound 10) and selectivity profile. The 1,2-trans-aminoalcohol (entry 11), 1,2-cis-aminoalcohol (entry 12) and diastereomeric 1,3-aminoalcohol (entry 13) were ∼2-, ∼4-, and ∼5-fold less potent than the un-substituted compound 4, respectively. The selectivity against the OPRM1 of trans- and cis-aminoalchol were 12- and 33-fold, respectively, while the selectivity of the 1,3-aminoalcohol was 25-fold. Drastic loss of potency resulted from the noncyclic 1,2-aminoalcohol (entry 14). The diasteromeric 2-methoxycycloxylamine (entry 15) was nearly equipotent to the trans-1,2-aminoalcohol (entry 11), but the selectivity against the OPRM1 was 3.7-fold bigger. Introducing a methyl group at the 4-position of the cyclohexyl group (entry 16) led to 4-fold loss of potency at the OPRK, 2-fold increase in selectivity against OPRM1 and ∼4-fold decrease in selectivity against the OPRD. Introducing an oxygen atom into the cyclohexyl ring (entry 17) led to 4.5-fold loss in potency at the OPRK1, but the selectivity against OPRM1 was ∼11-fold higher than for compound 4. Increasing (cyloheptyl, entry 18) or decreasing (cyclopentyl, entry 19) the ring size did not affect the potency at the OPRK1, whereas it increased the selectivity against the OPRM (4-5-fold) and decreased the selectivity against the OPRD1 (1.2-2.4-fold). Moving the cyclohexyl ring (entry 20) or the cyclohexylamine (entry 21) one methylene further from ring B led to 51-and 26-fold loss in potency, respectively. Methylation of the amino group (entry 22) led to 20-fold loss in potency. When the cyclohexyl group was removed (entry 23), the basicity of the amine reduced (entry 24) or the nitrogen replaced by oxygen (entry 25), the potency was completely lost. Taken together, it can be concluded that the basicity of the amine and its position in relation to the pyridine ring are fundamental for the activity at the OPRK1, while the installation of polar groups decreases the potency for the OPRK1, has small impact on the selectivity against OPRD1 but a greater impact against the OPRM1. A small lipophilic portion is important for the activity at the OPRK, however long lipophilic moieties lead to a decrease in potency on both OPRK1 and OPRM1.

Piperidine region (B): When the cyclohexylamine was moved from 4- to 3-position (entry 26) within the piperidine ring a complete loss of potency was observed. Interestingly, when a pyrrolidine system (entry 27) was introduced the potency slightly decreased. Adding a methyl group on position 3 (entry 28) led to small loss in potency. Interestingly, installing a hydroxyl group oriented in trans to the amino group (entry 29) led to a substantial increase of potency at OPRK (8-14.5-fold) and the selectivity against the OPRM1 and OPRD1 were 20-35- and 219-382-fold, respectively.

Complementary SAR on compound 29: Removing the cyclohexyl group (entry 30) led to a 368-644–fold loss in potency, as similarly observed for compound 23. Removing the bromine from position 5 of the pyridine ring led to 137-240-fold loss in potency.

3.6. Profiling Assays

ML350 was submitted to CEREP for broad panel screening of 52 protein targets (AID 652083), including hERG (AID 662075). The purpose of this panel of binding assays was to identify potential receptors, transporters, or ion channels for which ML350 displays affinity. ML350 exhibits 67% and 71% inhibition of the Na⁺ channel (site 2) and the NOP receptor, respectively (Table 3). The IC50s were determined to be 1.8 μM and 3 μM for these two targets, respectively. Thus, ML350 is >100-fold selective for OPRK1 over the Na+ channel (site 2) and approximately 200-fold selective over the NOP receptor. These data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects.

Table 3

Targets that exhibit ≥ 50% inhibition by ML350 in CEREP screen.

3.7. Pharmacokinetic Assays

Permeability: ML350 has high passive membrane permeability as measured in a PAMPA assay (AID 652113) as compared to standard compounds (Figure 7).

Figure 7

ML350 has high passive membrane permeability, similar to positive controls propanolol and antipirine.

Brain penetration: ML350 exhibited good brain penetration when administered by intraperitoneal (IP) injection in mice (AID 662085). Thus, dosed at 10 mg/kg in mice, ML350 (1 mg/mL in 10/10/80 DMSO/Tween/Water), reached brain at levels 3 times higher than plasma (Table 4).

Table 4

Levels of ML350 in plasma and brain of mice 30 and 120 minutes after IP administration.

Cytochrome P450 inhibition: ML350 had no significant activity at 3 of 4 human cytochrome P450 subtypes (Table 5, AID 652076). ML350 inhibited CYP2D6 by 89% at 10 µM.

Table 5

Inhibition of human cytochrome P450 subtypes 1A2, 2C9, 2D6, and 3A4 by ML350. Furafylline, sulfaphenazole, quinidine, and ketoconazone were the positive controls used for CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively.

Plasma protein binding: ML350 (1uM) had modest plasma protein binding in human (73%), but was higher in rodent, 96% in mouse and 99% in rat (AID 652078, Table 6).

Table 6

Binding of ML350 to human, mouse, and rat plasma proteins.

Hepatic microsome stability: In vitro hepatic microsomal stability of ML350 was measured (AID 652086, Table 7). The half life in 0.2 mg/mL hepatic microsomes was >120, 20, 5, 13 and >120 minutes in human, rat, mouse, monkey and dog hepatic microsomes, respectively.

Table 7

Hepatic microsome stability of ML350.

Rat pharmacokinetics: ML350 has an encouraging in vivo pharmacokinetic profile in rats (Table 8). The oral results are particularly exciting with, concentrations in plasma 10 times the cell-based IC50 after the relatively modest dose of 2 mg/kg.

Table 8

In vivo pharmacokinetic profile of ML350 in rats. ML350 was administered by IV at 1 mg/kg, or by mouth at 2 mg/kg

3.8. Reversibility of Analgesic Effects in Mice

In order to assess the reversibility of analgesic effects of ML350 in mice, a tail flick assay was used (AID 652108, Figure 8). This is a pain receptive assay in which a mouse is placed within a restraining tube with its tail protruding. The tail is placed on a level surface, radiant heat is applied to the tail and the latency of the mouse to remove its tail from the heat is recorded. This latency is used as a measure to indicate neurological pathology. In this assay, the mice are administered ML350, vehicle, or the OPRK1 antagonist Nor-BNI, and subsequently challenged with OPRK agonist U-69,593 at one hour, 24 hours, and 1 week post pre-treatment. The ability of ML350 to block the analgesic effect of the agonist compound is measured. After each agonist challenge, each moue was tested by application of a heat source and the latency time of the mouse to remove its tail from the heat is measured and reported in seconds. The ability of ML350 to block the analgesic effect of the agonist is gone after 24 hours, whereas Nor-BNI is still efficacious after 1 week.

Figure 8

Results of Tail Flick assay to determine the reversibility of the analgesic effect of ML350. Mice received ML350 (5 mg/kg) administered IP, Nor-BNI (10 mg/kg) administered SC, or vehicle/. Mice were then challenged with agonist U-69,593 (2mg/kg) administered (more...)

4. Discussion

ML350 was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that ML350 has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging in vivo pharmacokinetic profile in rats. ML350 was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, ML350 was shown to have a reversible analgesic effect when challenged with an OPRK1 agonist in a tail flick assay in mice.

4.1. Comparison to existing art and how the new probe is an improvement

Several OPRK1 antagonists have been described in the literature. Norbinaltorphimine (nor-BNI) [89-90], 5′-guanidinonaltrindole (GNTI) [89, 91], and JDTic [89, 92] all exhibit a delay in the onset of action of approximately 24 hours [89, 93-95], and have very long lasting in vivo effects of up to 56 days [89, 95-96]. Due to their pharmacodynamics/pharmacokinetics and poor unbound brain/plasma ratio, morphine-like derivatives, nor-BNI and GNTI, are only used as pharmacological tools. The non-opioid compound JDTic exhibited a poor brain exposure but had an extraordinary persistence in brain (mean brain concentration declined by only 56% over 24 hours, and the drug was still detectable at 1 week in mice) despite its P-glycoprotein-mediated efflux, and its moderate lipophilicity and low affinity for cell homogenates. The presence of two basic nitrogens and entrapment in cellular compartments such as lysosomes have been proposed as a possible mechanism of this persistence [89, 97]. A Phase 1 clinical trial of JDTic for cocaine dependence was terminated in 2012 due to adverse events. Several short-acting compounds from distinct chemotypes have been developed with greater CNS exposure compared to the prototypic ligands (nor-BNI, GNTI, JDTic). AstraZeneca disclosed a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides. AZ-MTAB was efficacious in animal models of mood disorders, but was associated with a significant hERG liability (IC50 of 260 nM) [97-98]. Another OPRK1 antagonist PF-04455242 [99] was developed by Pfizer. However, on January 6, 2010 the phase 1 study for dipolar disorder and depression was terminated due to toxicology findings in animals exposed to PF-04455242 for three months. Eli Lilly identified a subnanomolar OPRK1 antagonist with selectivity of 21 and 135 over OPRM1 and OPRD1 respectively [100]. In 2010 LY2456302 was advanced to phase I clinical trial for the oral treatment of alcohol dependence. The clinical study was to assess the brain OPRK1 occupancy after single oral doses of LY2456302 as measured by positron emission tomography with radioligand LY2879788 (¹¹C PKAB) in healthy subjects. The last update of the study was May 5, 2011, and no new studies for the compound are listed in ClinicalTrials.gov.

Even though a few drug-like OPRK1 antagonists with more favorable pharmacokinetics than nor-BNI, GNTI, JDTic have been developed, new OPRK1 antagonists possessing novel scaffolds and improved selectivity are still needed both as pharmacological tools to better understand the OPRK1-dynorphin system and as potential pharmacotherapies.

Table 9Prior Art OPRK Antagonist Compounds

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Compound Name	Structure	OPRK IC50 (nM)	Selectivity (nM)	B/P*	Ref(s)
Norbinaltorphimine (nor-BNI)		0.07 ± 0.03	OPRM: IC50 15.8 ± 5.7; OPRD: IC50 12.1 ± 3.1	< 0.05	[92, 97]
5′-guanidinonaltrindole (GNTI)		0.04	OPRM: IC50 3.2; OPRD: IC50 15.5	< 0.0007	[97, 102]
JDTic		0.006 ± 0.001	OPRM: IC50 3.42 ± 0.83; OPRD: IC50 > 100	< 0.05	[92, 97]
AZ-MTAB		20 ± 3	OPRM: IC50 722 ± 47; OPRD: IC50 8306 ± 1635; (hERG: IC50 260 nM)	∼ 1	[97-98]
PF-04455242		1.23	OPRM: IC50 10 nM; OPRD: Ki > 4000	∼ 1	[99, 103]
LY2456302		0.813 ± 0.285	OPRM: IC50 17.4 ± 6.33; OPRD: IC50 110 ± 33.6	∼ 1	[97, 100]

*

Unbound brain/plasma ratio

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