Inventors: Wen- Lung Ma, Yao-Chang Chiang, Ruey-Yun Wang, Chieh-Liang Huang, all of Taichung, TW

Applicant/ Assignee: China Medical University, No.91, Hsueh-Hsih Rd, Taichung 40402 TW Priority: Ser No. 62/502,682; Filed: May 07, 2017

[001] Introduction

[002] Opioid drugs are the most effective therapeutic analgesic for chronic and cancer pain[l]. Continual use of opioids, however, results in the development of tolerance and dependence[2,3]. Moreover, abuse of opioids (heroin and/or morphine) is a serious social problem worldwide (SAMHSA, 2014). Methadone, a synthetic mu- (μ-) opioid receptor (MOR) agonist, was developed and first introduced to replace morphine for pain treatment[4]. It's also considered as most effective alleviating agent for heroin-addiction withdrawal syndrome[5-7]. Although methadone has been shown to be effective in reducing withdrawal symptoms and impulsive injection of opioids[8], chronic use of methadone has also exhibited addictive liability and respiratory repression in some subjects[9].

[003] Methadone is major metabolized in the liver through CYP3A4, CYP2D6, CYP2B6, CYP2C19 and CYP1A2 (cytochrome p450s; xenobiotics oxidizing enzyme) through which converting methadone to inactive metabolites (EDDP; 2-ethylidene-l,5-dimethyl-3,3- diphenylpyrrolidine)[10,l l]. Oral taken methadone subjected to first-pass effect and could be detected in the plasma after 30 min of administration. And the methadone bioavailability range is widely from 41-76% to 85-95% in different subjects[10]. The discrepancies of bioavailability in patients reflect on pharmacokinetic data that a noticeable variation of plasma

EDDP/methadone concentration observed in same dosing patients[12]. CYP enzymes modulator has been demonstrated to alter plasma concentration of methadone[10]. For example, the off target effect of efavirenz could increases CYP3A4 activity, decrease 60% plasma concentration of methadone, therefore, an increase of 66-133% or 15 mg more methadone dosing is required to alleviate withdrawal symptoms[13,14]. It implicates a therapeutic benefit of CYP suppressor in MMT regimen.

[004] Gender in substances-abuse individuals have been reported in terms of severity, craving, medical conditions, and impairment in associated central nervous system (CNS) functional areas [15- 18]. Except for neuronal activity, it's been reported that sex hormones regulate differential panels of liver CYPs, namely feminine or masculine CYPs[19-21]. Estrogen (17β- estradiol, E2) is the active ligand for estrogen receptors (including ERa and ERfi) which expressions in liver throughout life[22,23]. Genome-wide association studies reported that the SNPs related to CYPs, opioid receptors, sex hormone producing enzymes can correlate to individualized methadone dosing[24]. Methadone metabolism is linked to MMT efficacy and also associated to genders. Different metabolic enzymes are involved in enantiomeric S- and R- methadone metabolism[10]. S-methadone can be catabolized to S-EDDP (2-ethylidene-l,5- dimethyl-3,3-diphenylpyrrolidine) by CYP2B6[25], where R-methadone can be catabolized to R-EDDP by CYP2C19([26]; Lu et al., Drug Metab Dispos. 2010 Aug; 38(8): 1308-1313), and methadone suppressed estrogen levels, possibly through CYP2C19 expression[27]. In addition, methadone clearance was increased during pregnancy [28]. A study of MMT patients suggested that males tend to use heroin as their opiate of choice, and are more likely to combine with cannabis, while females are more likely to select the street methadone with ketamine, benzodiazepines, and/or amphetamines [29].

[005] E2 has been reported to be involved in various neuronal activities, e.g. pain sensation, mood, seizures susceptibility, and stroke or Alzheimer's disease neuroprotection[47], and E2 can rapidly attenuate the ability of MOR to hyperpolarize hypothalamic (β-endorphin) neurons [48]. Opioid systems can influence the prolactin release through the estrogen regulation [49] or abolish estrogen-induced antinociception and hyperalgesia[50]. The sensitivity and the expressions of MOR reduced by E2 might decrease the responsiveness of MOR against its ligands (e.g., morphine or methadone) [48,51-53]. E2 can also regulate neurotransmitters release, e.g., β-endorphin, serotonin, choline and dopamine[54].

[006] E2 can also feedback through the Hypothalamus-Pituitary-Organs-circuit

(Gonadotropin-releasing hormone [GnRH] -Luteinizing hormone [LH])-E2 per se.); for example, CNS E2 level might influence the endocrine circuit via opioid system to regulate the plasma E2 level. Quesada and Micevych (2008) showed that E2 increased the nociceptin receptor (NOPR) number (Bmax) and maximal GTPyS binding (Emax) in the cell membrane of mediobasal hypothalamus. Besides, intracerebroventricular injected or microinjected nociceptin (N/OFQ) into the hypothalamus inhibited the release of GnRH from the hypothalamus and then to decrease plasma LH levels[55].

[007] We hypothesized that sex hormones signals might be able to modulate motivation for opioid selection and preference. We disclose dissected estrogen signals in genetics,

serology/hormonal to the regulations of methadone metabolomics, including a preclinical trial targeting estrogen signals by selective estrogen receptor modulator (SERM) in MMT program, and show that we can decrease MMT dosing and reduce the opioid liability. [008] Summary of the Invention

[009] The invention provides methods and compositions for treating withdrawal syndromes and symptoms by using methadone and a selective estrogen receptor modulator (SERM). The invention provides reduced dosing and liability in methadone maintenance treatment (MMT) for opiate addiction by inhibiting estrogen receptor signaling.

[010] In an aspect the invention provides a pharmaceutical composition comprising methadone and a SERM.

[011] In embodiments:

[012] - the SERM is clomifene, ormeloxifene, raloxifene, tamoxifen, toremifene, lasofoxifene or ospemifene;

[013] - the SERM is tamoxifen;

[014] - the methadone and SERM are each in separate or combined unit dosage form;

[015] - the methadone and SERM are combined in unit dosage form;

[016] - the methadone and SERM are copackaged;

[017] - the methadone and SERM are copackaged and each is in separate unit dosage form;

[018] - the methadone and SERM are coformulated;

[019] - the methadone and SERM are coformulated and in unit dosage form;

[020] - the composition comprises 1-100 or 2-50 or 5-40mg methadone and 0.01-50 or 0.05-

20 or 0.1-10 mg SERM; and/or

[021] - the weight ratio of the methadone and the estrogen receptors inhibitor is 1:2 to 1:1000 or 1: 10 to 1:1000.

[022] The SERM preferably inhibits effects of estradiol receptors, and the methadone can comprise S- and R-enantiomers, or can be a substantially pure form of either the S- or R- enantiomer. Any compatible dosage form may be used, including pills (e.g. tablet or capsule syrups), specialty tablet like buccal, sub-lingual, thin film, liquid solution or suspension (e.g., drink or syrup), powder or liquid or solid crystals, pastes, drops, cream, liquid solution, aerosol, inhaler, nebulizer, vaporizer, intradermal, subcutaneous (SC), intramuscular (IM), intraosseous (IO), intraperitoneal (IP), and intravenous(IV), and any other pharmaceutically acceptable dosage forms and method.

[023] In an aspect the invention provides use of the disclosed compositions in the manufacture of a medicament for treating pain or opioid dependence (e.g. maintenance therapy or detoxification in opioid dependence), such as manifested as heroin or morphine withdrawal symptoms.

[024] In an aspect the invention provides a method of treating pain or opioid dependence comprising administering to a person in need thereof a disclosed composition, such as wherein the methadone and SERM are administered simultaneously or within 24, 12, 3, or 1 hour of each other.

[025] The invention encompasses all combination of the particular embodiments recited herein, as if each combination had been laboriously recited.

[026] Brief Description of the Figure

[027] Changes in methadone liability and craving during MMT are shown in three different treatment approaches.

[028] Description of Particular Embodiments of the Invention

[029] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms "a" and "an" mean one or more, the term "or" means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.

[030] The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[031] Embodiment 1: Estrogen signal increases MMT dosing. In order to examine the association of estrogen-CYP-MMT dosing in human subjects, we recruited first cohort-based study to associate estrogen-related CYPs gene polymorphism to maximal methadone dose (Max) in MMT patients. We've evaluated the association of 10 putative ERE-SNP sites and Max MMT in the cohort. We found the Max MMT dosing in ERE-SNP (rs 16974799; C:T) CYP2B6 is significantly higher (p=0.0441) in CC genotype compared to TT and CT genotypes. We then performed association on ERE-SNPs genotypes to Max MMT dosing, and found dominant model (p=0.0147) of rsl6974799 in patients. Patients carried TT genotype (48.2%) taking lower dose among three MMT dosing groups. Next, we conducted second cohort study to observe the association of sexes and female hormones to MMT dosing (pMax: maximal MMT dosing before enter the trial; SS: steady-state MMT dosing in the trial; Max: maximal MMT dosing during the trial). In our validation cohort study, we found either pMax, SS, and Max MMT dosing are gender-related. Females were significantly correlated with higher MMT dosing compared to males. While associate female hormones (estradiol, E2 and progesterone, P4) to MMT dosing, it's E2 (p=0.0472) but not P4 (P=0.4576) is associated with Max MMT in the trial. This result implicating estrogen is the confounder in the sexual dimorphic MMT dosing. [032] We next sought to determine if estrogen participates in methadone metabolism and MMT efficacy in 2nd cohort patients. The correlation analysis found E2 levels significantly correlated with S-form (r=0.25822, P=0.0146), R-form (r=0.23588, P=0.0261), and total (r=0.26566, P=0.0119) EDDP/methadone ratio. Literature reported that R-form methadone exerts opioid function, which related to alleviating withdrawal syndrome[41]. On the contrary, S-form is associated with the electropathological cardiac QT-interval prolongation

complications of methadone [42]. In order to associate methadone metabolism to MMT therapeutic efficacy, we introduced the questionnaire[30] by which measuring the heroin using and craving after receiving MMT. The questionnaire HUC 1-6 represents the extent of mind using heroin and whether one can shift attention from heroin use, where HUC 7-14 was used to investigate the daily or weekly frequency of heroin usage, life and work disturbed, prong to anxiety emotion, desiring to use heroin, and the ability to overcome heroin use[30]. We scored and calculated the result in each patient to evaluate the association with methadone metabolism data. Our data showed that S-form, R-form, and total- EDDP/methadone ratio is significantly correlated with estradiol (r=0.25822, 0.23588, 0.26566; and P=0.0146, 0.0261, 0.0119, respectively) but not associated with progesterone. In addition, the results demonstrated that estradiol affect both S-form or R-form methadone metabolism. Furthermore, the study revealed that S-form EDDP/methadone ratio is negatively associated with medication-related HUC 7-14 score (r=-0.24246, p=0.0213), but not HUC 1-6. It indicates that the higher S-EDDP/methadone ratio, the lower score, and the less cravings or less dependent for heroin use. In other words, the higher S-form methadone metabolite, the lower is heroin craving compulsive behavior.

[033] Furthermore, insignificance association result of HUC 1-6 scores indicated the heroin obsessive craving, assessing the mind using of heroin is independent to methadone metabolite. Together, our results indicate that estrogen signal can contribute to MMT sex biased dosing, alter methadone pharmacology actions, through which abate methadone efficacy in patients.

[034] Embodiment 2: Estrogen mitigates methadone antinociception and rewarding behavior in mice. We determined the E2 effect on the methadone antinociception in mice by removing endogenous female hormones (OVX), or exogenous injection of E2 in male. OVX female mice exhibited lowered 17 -estradiol level and prolonged methadone-induced antinociceptive effects (tail flick test). Meanwhile, the serum EDDP/methadone ratio is also significantly decreased in the OVX mice. On the contrary, E2 supplement leads to elevated 17β- estradiol and ablated antinociceptive response with raised EDDP/methadone ratio in male mice.

[035] In order to delineate the relation of estrogen on methadone metabolism and CYPs expressions, we measured the related liver enzymes mRNA expressions. The liver CYPs mRNA expression (CYPla2: an ortholog of human CYP1A2; CYP2d22: an ortholog of human CYP2D6) were significantly decreased in OVX female group as compared with sham control. On the contrary, the CYPla2, CYP2d22, and CYP2c37 (an ortholog of human CYP2C19) and CYP2blO (an ortholog of human CYP2B6) were increased in E2 supplemented male mice.

[036] The conditioned place preference (CPP) test was also used to test the estrogen effect on methadone-induced rewarding behavior. Our results demonstrated that chronic methadone injection prolonged CPP duration, while OVX further enhances this effect. Restoration of E2 in OVX mice could reverse OVX-mediated CPP duration in female mice. On the other hand, chronic methadone administration could also prolong CPP duration in male mice, while coadministration of E2 could diminish methadone effect. In sum we demonstrated that estrogen can upregulate liver CYPs to facilitate methadone metabolism, therefore, ablate its

antinociception and rewarding effect in mice.

[037] Embodiment 3. Differential regulation of estrogen on ERE-SNPs. Since we demonstrated E2-CYP-MMT dosing axis, we further examined direct regulation of E2/ER signals on CYP expression. We compared two ERE-SNPs site, where one of them is significantly associated with Max dose of MMT (rsl6974799), and the other is not (rs3760657). We calculated the putative ERE sequence alignment score by BEST software with input of the known ERE as reference[42]. The ERE-SNP rs 16974799 (patient #348 : C allele vs. #898 : T allele) C allele gained higher ERE alignment score than that of T allele (0.85 vs. 0.65). On the other hand, the ERE-SNP rs3760657 (patient #348 : A allele vs. #928 : G allele) A and G alleles exhibited fewer contrast on alignment score (0.79 vs. 0.73). That implicated CC genotype of rs 16974799 exerts higher estrogen/ER transactivation function but not on rs3760657 SNP locus. Furthermore, we constructed ERE-SNPs into pGL3-basic luciferase reporter plasmid to test the levels of ERE-SNP genotype ER transactivation function in hepatoma cells. The CC genotype of rs 16974799 demonstrated significant higher activity than TT genotype, while comparable luciferase activity in AA and GG genotype of rs3790957.

[038] Embodiment 4. Targeting estrogen signal via SERM in MMT program. For heroin/morphine addiction patients entering MMT program, it is generally necessary to titrate methadone doses to tolerance phase, then move on to tapping and abstinence phases.

Conceptually, at the beginning of methadone tolerance phase, the dosing goal is to reduce withdrawal syndrome and avoid drug-seeking behavior. After tolerance phase, the patient can be stabilized under MMT program, then gradually reduce doses in tapping phase. However, before the addiction of methadone is formed, social/family supporting system can contribute to help patient gradually eliminate methadone (abstinence phase). Since we have delineated the relation of E2-CYP-EDDP/methadone-MMT efficacy, we applied a translational approach. We tested the hypothesis that co-administration of a SERM[43], in the chronic methadone injection procedure (tolerance phase) to observe SERM effect on methadone induced addiction by measuring CPP duration. Then gradually reduce methadone dose (tapping and abstinence phases) to see SERM effect on CPP duration. Our data showed methadone reward response can be observed after 7-day injection in both male and female mice; and co-administration of SERM could facilitate methadone-induced CPP duration at day-11 and day- 14. Furthermore, while we gradually reduce methadone dose in same mice and measure CPP duration reduction, we found the reduction velocity is faster in SERM co-administration mice compared to placebo coadministration. Together, these results indicate that targeting estrogen signal via SERM coadministration not only enhances rewarding response during tolerance phase, but also shorten tapping and abstinence phases of MMT program.

[039] Embodiment 5. Altering methadone metabolism to the management of

heroin/morphine addiction patients. Balancing withdrawal syndrome alleviation and liability prevention of MMT is one important principle in managing heroin/morphine addictive patients [24].

[040] In the drawing changes in methadone liability and craving during MMT are shown in three different treatment approaches - methadone alone, methadone+support system, or methadone+SERM+support system during the tolerance, tapering, and abstinence phases, which links to three outcome scenarios.. The solid line, representing methadone alone group indicated methadone liability quickly increases over the tolerance phase, declines over the taper phase, but then increases again during the abstinence phase. The dashed line, representing

methadone+support system group shows methadone liability increasing at a rate comparable to that of methadone alone in the tolerance phase and then declining in tapering and abstinence phases. The dashed line representing methadone+SERM+support system shows methadone liability increasing but to a lower degree than that it did in mice receiving methadone alone in the tolerance phase. It declined to even lower degree than it did in subjects receiving

methadone+support in both the tapering and abstinence phases

[041] Embodiment 6. From modulating estrogen signal to MMT outcome. Methadone prevents withdrawal and limits cravings [7] but additional methadone may increase cravings for heroin[44]. Methadone dosing is interfering by methadone metabolism, plasma level, heroin craving and withdrawal and many other factors. High dropout of MMT and heroin relapse was related with low dose of methadone[45] and methadone dose was highly variable among countries. We disclose that CYP2B6 and plasma E2 influenced methadone dosing, and that the measurement of E2 or CYP2B6 polymorphism can be useful for dosing adjustment for clinical methadone treatment. We also report that the ratio of S-EDDP/S -methadone was negatively correlated with heroin compulsive use behavior of HUC 7-14 rather than obsessive thought of questionnaire HUC 1-6. This implies that lower metabolic rate of S-methadone can drive the drug using behaviors to prevent the body suffering, even who was not want to get drugs in his/her mind.

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