Methods for reducing addictive potential of opioid medications

Abstract

In certain embodiments, the present invention provides method of preventing synaptic and / or behavioral adaptations resulting from opioid use in a mammal in need thereof comprising administering a carbonic anhydrase (CA) inhibitor agent to the mammal. In certain embodiments, the present invention provides a method of reversing opioid-induced synaptic and / or network changes resulting from a first opioid use in a mammal in need thereof, comprising administering a combination of a carbonic anhydrase (CA) inhibitor acetazolamide (AZD) and a second opioid to the mammal. In certain embodiments, the present invention provides a method of reversing opioid-induced synaptic and / or network changes resulting from a cocaine use in a mammal in need thereof, comprising administering a combination of a carbonic anhydrase (CA) inhibitor acetazolamide (AZD) and an opioid to the mammal.

Description

[0001] VHPM 17023.304WO1 / UIRF 25040 METHODS FOR REDUCING ADDICTIVE POTENTIAL OF OPIOID MEDICATIONS

[0002] PRIORITY OF INVENTION

[0003] This application claims priority to United States Provisional Application Number 63 / 749,475 that was filed on January 24, 2025, and to United States Provisional Application Number 63 / 764,379 that was filed on February 27, 2025. The entire content of the applications referenced above are hereby incorporated by reference herein.

[0004] GOVERNMENT FU DING

[0005] This invention was made with government support under DA037216 and DA052953 awarded by the National Institutes of Health and BX004440 awarded by the U.S. Department of Veterans Affairs. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] The opioid crisis remains a leading public health challenge and overdose deaths still approach 100,000 per year. Opioids and their withdrawal produce a sustained and increasing desire for more drugs, often referred to as opioid-seeking or craving. Only a few medications are FDA-approved to treat opioid use disorder (OUD) and all of them target the mu opioid receptor (MOR). Importantly, these medications do not correct the biological mechanisms that cause drug seeking or craving. Thus, new treatments with non-opioidergic mechanisms of action could have substantial impact for clinical management of OUD.

[0008] SUMMARY

[0009] In one aspect, provided herein is a method of preventing synaptic and / or behavioral adaptations resulting from opioid use in a mammal in need thereof comprising administering a carbonic anhydrase (CA) inhibitor agent to the mammal.

[0010] In one aspect, provided herein is a method of reversing opioid-induced synaptic and / or network changes resulting from a first opioid use in a mammal in need thereof, comprising administering a combination of a carbonic anhydrase (CA) inhibitor and a second opioid to the mammal.

[0011] In one aspect, provided herein is a method of reversing opioid-induced synaptic and / or network changes resulting from a cocaine use in a mammal in need thereofe, comprising administering a combination of a carbonic anhydrase (CA) inhibitor and an opioid to the mammal.VHPM 17023.304WO1 / UIRF 25040

[0012] BRIEF DESCRIPTION OF DRAWINGS

[0013] Figs. 1A-1L. CA4 disruption protects against oxycodone withdrawal-induced synaptic changes at glutamatergic synapses in NAcC MSNs and reduces oxycodone conditioned place preference (CPP). (Fig. 1A) Experimental timeline: oxycodone (3mg / kg, i.p.) or saline (i.p.) was administered in the home cage each day for 5 days followed by 10 days of withdrawal, after which slice electrophysiology was performed. (Fig. IB) Diagram illustrating the location of MSN recordings in NAcC and electrical stimulation, ac: anterior commissure; NAcSh: NAc shell. (Fig. 1C) Representative traces of the AMPAR-mediated EPSC at -70 mV and the NMDAR-mediated EPSC at +50 mV from NAcC MSNs from Car4+ / +and Car4 / ' mice following withdrawal from oxycodone (Oxy) vs saline (Sal). (Fig. ID) AMPAR / NMDAR ratio increased after withdrawal from oxycodone (Oxy) in Car4+ / +but not in Car (n = 10-12 neurons from 5 mice / group). (Fig. IE) Example traces of AMPAR-mediated EPSCs at -70 mV and +50 mV in NAcC MSNs of Car4+ / +and CarV / _mice following withdrawal from oxycodone (Oxy) vs saline withdrawal (Sal). (Fig. IF) Current-voltage relationship (IV-curve): withdrawal from oxycodone produced inward rectification in Car4+ / +mice (n = 7 to 16 neurons from 3-5 mice / group). (Fig. 1G) Quantification of rectification index shows an increase after withdrawal from oxycodone in Car4+ / +but not in Cor mice (n = 7 to 16 neurons from 3 to 5 mice / group). (Fig. 1H) Representative traces of the AMPAR-mediated evoked EPSC at -70 mV before (black) and after (blue) NASPM treatment. (Fig. II) NASPM sensitivity increased after withdrawal from oxycodone in Car4+ / +but not in Car 4 mice (n = 6 to 7 neurons from 3 mice / group). (Fig. 1 J) Experimental timeline for oxycodone CPP in Car4+ / +and Car4~ / ~ mice. (Fig. IK) Oxycodone conditioned place preference is impaired in Car 4^ mice (n = 18, 20 mice / grp). (Fig. IL) Car4+ / +and Car 4^ mice were administered oxycodone (15mg / kg, i.p.) or saline (i.p.) in the home cage each day for 5 days, followed by 10 days of withdrawal, after which slice electrophysiology was performed. AMPAR / NMDAR ratio increased after withdrawal from oxycodone (Oxy) in Car4+ / +but not in Car 4^ (n = 5-9 neurons from 2-3 mice / group).

[0014] Figs. 2A-2J. Acetazolamide (AZD) reversed oxycodone withdrawal-induced changes at glutamatergic synapses in NAcC MSNs in Car4 mice but not in Car4~~ mice. (Fig. 2A) Experimental timeline: oxycodone (3mg / kg, i.p.) or saline (i.p.) was administered each day for 5 days, followed by 10 days of withdrawal. AZD (30 / mg / kg, i.p.) or vehicle (saline, i.p.) was administered, and 24-hrs later, slices were harvested for electrophysiological recording. (Fig.

[0015] 2B) Representative traces of AMPAR / NMDAR from Car4+ / +and Car 4^ mice after withdrawal from oxycodone (Oxy) or saline (Sal) and treatment with AZD vs. vehicle (Veh). (Fig. 2C) AZD significantly reduced oxycodone withdrawal -induced increase in AMPAR / NMDAR ratioVHPM 17023.304WO1 / UIRF 25040 in Car4+ / +mice (n = 9 to 12 neurons from 3 to 4 mice / group). (Fig. 2D) withdrawal from oxycodone and AZD did not affect AMPAR / NMDAR ratio in Car 4^ mice (n = 9 to 10 neurons from 3 to 4 mice / group). (Fig. 2E) Representative traces of AMPAR-mediated EPSCs at -70 and +50 mV from oxycodone withdrawn Car4+ / +and CarV / _mice after treatment with AZD vs. vehicle. (Fig. 2F) AZD treatment attenuated oxycodone withdrawal-induced increase AMP AR rectification in Car4+ / +mice (n = 7 to 16 neurons from 3 to 5 mice / group). (Fig. 2G) Oxycodone withdrawal and AZD had no effect on AMP AR rectification in Car7’ mice (n = 7 to 11 neurons from 3 to 4 mice / group). (Fig. 2H) Representative traces of the AMPAR-mediated evoked EPSC at -70 mV before (black) and after (blue) NASPM application from Car4+ / +and Car mice after withdrawal from oxycodone (Oxy) and treatment with AZD vs. vehicle. (Fig.

[0016] 21) AZD treatment attenuated oxycodone withdrawal-induced increase in NASPM sensitivity in Car4+ / +(n = 6 to 7 neurons from 3 mice / group). (Fig. 2J) withdrawal from oxycodone and AZD had no effect on NASPM sensitivity in CarV / _mice (n = 5 to 6 neurons from 3 mice / group).

[0017] Figs. 3A-3H. Effects of AZD on oxycodone-induced increases in AMPAR / NMDAR depend on ASIC1A and generalize to other opioids. (Fig. 3A) Experimental timeline for heroin, morphine, and AZD treatment. (Fig. 3B) Representative traces of AMPAR / NMDAR from Car4+ / +mice after heroin (H) or saline (Sal) withdrawal and treatment with AZD vs vehicle. (Fig. 3C) AZD treatment reversed the heroin-induced increase in AMPAR / NMDAR ratio relative to vehicle-treated controls (n = 8-11 neurons from 4 mice / group). (Fig. 3D) Representative traces of AMPAR / NMDAR from Car4+ / +mice after withdrawal from morphine (Mor) or Saline (Sal) and treatment with AZD vs. vehicle (Fig. 3E) AZD treatment reversed morphine-induced increase in AMPAR / NMDAR ratio compared to vehicle-treated controls (n = 7-9 neurons from 4 mice / group). (Fig. 3F) Experimental timeline: oxycodone (3mg / kg, i.p.) or saline (i.p.) was administered each day for 5 days, followed by 10 days of withdrawal. AZD or vehicle was administered, and 24-hrs later, slices were harvested for electrophysiological recording. (Fig. 3G) Representative traces of AMPAR / NMDAR from Asicla ^ mice withdrawn from oxycodone (Oxy) or saline (Sal) and treated with AZD vs. vehicle. (H) AZD had no effect in drug naive and oxycodone-withdrawn Asidcr mice (n = 8-10 neurons from 4 mice / group).

[0018] Figs. 4A-4F. Increase in AMPAR / NMDAR ratio in oxycodone withdrawn mice was specific to D1+ neurons. (Fig. 4A) Image of Dl+neurons in NAcC labeled with tdTomato. (Fig. 4B) Diagram illustrating the location of recordings of D1+ and non-Dl+MSNs recording in NAcC. (C) Representative traces of AMPAR / NMDAR ratio of D1+ neurons in oxycodone withdrawn (Oxy) and saline withdrawn (Sal) mice treated with AZD vs. vehicle. (Fig. 4D) Oxycodone withdrawn mice exhibit increases in AMPAR / NMDAR ratio Dl+neurons, and AZDVHPM 17023.304WO1 / UIRF 25040 administration reversed the measure to levels in vehicle-treated controls (n = 5-7 neurons from 3 mice / group). (Fig. 4E) Representative traces of AMPAR / NMDAR ratio of non-Dl+MSNs in oxycodone withdrawn (Oxy) and saline withdrawn (Sal) mice treated with AZD vs. vehicle. (Fig. 4F) Oxycodone withdrawn mice exhibited no change on AMPAR / NMDAR ratio in DP neurons, and AZD had no effect (n = 5-7 neurons from 3 mice / group).

[0019] Figs. 5A-5H. CA4 disruption and inhibition reduced cue-induced drug-seeking behavior following forced abstinence from oxycodone self-administration. (Fig. 5A) Experimental timeline for intravenous oxycodone self-administration and slice electrophysiology. (Fig. 5B) Lever presses (active (ALP) and inactive (ILP) across experimental sessions. There was no effect of genotype (n = 10 mice / per group). (Fig. 5C) Infusions across experimental sessions between the genotypes. There was no effect of genotype.

[0020] (Fig. 5D) Active lever presses at baseline (day 11) and after 30 days of forced abstinence (day 41). There was no significant time by AZD x genotype interaction, though there was a significant time x genotype interaction. In vehicle-treated Car4+ / +mice, active lever presses did not change from day 11 to day 41 (n = 9). In AZD-treated Car4+ / +mice (n = 10), vehicle-treated Car mice (n = 10), and AZD-treated Cor mice (n = 10), lever presses decreased from day 11 to day 41. (Fig. 5E) Day 41 lever presses normalized to mean day 11 lever pressing. Two-way ANOVA identified a significant genotype x AZD interaction. Planned contrast testing revealed that vehicle-treated Car4+ / +mice had greater cue-induced oxycodone seeking than AZD-treated Car4+ / +mice, vehicle-treated Cor mice, and AZD-treated CarV / _mice. (Fig- 5F) Inactive lever presses at baseline (day 11) and after 30 days of forced abstinence (day 41). There was no significant time by AZD x genotype interaction. Inactive lever pressing did not change significantly between day 11 and day 41 in vehicle-treated Car4+ / +mice, AZD-treated Car4+ / +mice, and vehicle-treated CarV / _mice. Inactive lever pressing did decrease in AZD23 treated Car mice. (Fig. 5G) Representative traces of AMP AR (-70 mV) and NMD AR (+50 mV) from NAcC of Car4+ / + and Car4 / _mice treated with AZD vs. vehicle and recorded after forced abstinence from oxycodone self-administration and behavioral testing of drug seeking.

[0021] (Fig. 5H) AZD treatment reversed AMPAR / NMDAR ratio in Car4+ / +mice without affecting Car4 / _mice (n = 8-10 neurons from 3 to 6 mice / group).

[0022] Fig. 6. Effects of oxycodone withdrawal and CA4 are retained when quantifying AMPAR / NMDAR ratio using peak NMD AR amplitude instead of sustained NMD AR current. Car4+ / +and Car4 / _mice were administered 15 mg / kg ip oxycodone vs. saline for 5 days, followed by 10 days of withdrawal, then AMPAR / NMDAR was assessed. Here peak NMD AR amplitude was used to quantify AMPAR / NMDAR ratio instead of NMD AR amplitudeVHPM 17023.304WO1 / UIRF 25040 60 ms after EPSC onset as in other figures. With this alternative approach a significant oxycodone by genotype interaction was still observed (n = 6-8 neurons from 3 mice / group).

[0023] Fig. 7. AMPAR / NMDAR ratio results separated by sex, genotype, and AZD treatment. Although there were strong effects of oxycodone withdrawal, Car4 genotype, and AZD, there were no significant interactions between these factors and sex (n = 5 to 6 neurons from 2 to 3 mice / group).

[0024] Figs. 8A-8B. Oxycodone withdrawal-induced increase in AMPAR / NMDAR ratio in NAcC MSNs required an extended abstinence period (>24 hrs). (A) Oxycodone (oxy) dosing paradigms administered to Car4+ / +mice to assess effects on AMPAR / NMDAR ratio: (i) 5 injections and 10 days of withdrawal (ii) 5 days of injections and 24 hrs withdrawal (iii) a single injection and 24 hrs withdrawal. (B) AMPAR / NMDAR ratio increased in group (i), but not in groups (ii) or (iii) (n = 7-13 neurons from 3 to 4 mice / group).

[0025] Figs. 9A-9F. In vitro and in vivo administration of the CA inhibitor AZD reversed effects of opioid withdrawal on AMPAR / NMDAR ratio. (A) Experimental timeline: oxycodone (3mg / kg, i.p.) or saline (i.p.) was administered each day for 5 days, followed by 5 days of withdrawal. Brain slices were prepared, and AZD lOOpM or vehicle (ACSF) was added to the recording chamber for 60 minutes before testing. (B) The AMPAR / NMDAR ratio increased after oxycodone withdrawal (Oxy) in Car4+ / +mice. Applying AZD to the recording chamber reversed values to control levels (n = 7-12 neurons from 3-4 mice / group). (C) Experimental timeline: oxycodone (3 mg / kg) vs. saline (i.p, 5 days), followed by 5 days of withdrawal. 3 hrs later, AZD (30 mg / kg) vs. vehicle was administered in vivo, and slices were harvested for electrophysiological recording. (D) AZD treatment reversed the oxycodone withdrawal-induced increase in AMPAR / NMDAR ratio (n = 7-8 neurons from 3 mice / group). (E) Experimental timeline: morphine (lOmg / kg), or saline (i.p.) was administered for 5 days, followed by 5 days of withdrawal. AZD (30 / mg / kg) or vehicle was administered by i.p. injection in vivo, and 3 hrs later, slices were harvested for electrophysiological recording. (F) In vivo administration of AZD reversed AMPAR / NMDAR ratios following morphine abstinence to control levels (n = 6-9 neurons from 3 mice / group).

[0026] Figs. 10A-10B. Low-dose heroin (2mg / kg) followed by withdrawal did not affect AMPAR / NMDAR ratio in NAcC MSNs of Car4* / +mice. (A) Experimental timeline of heroin treatment and electrophysiology. (B) Abstinence from low-dose heroin did not change the AMPAR / NMDAR ratio and AZD had no effect (n = 9-12 neurons from 4 mice / group).

[0027] Figs. 11A-11B. AMPAR / NMDAR ratio was increased in NAcC MSNs of Car4+ / +mice after 5 days of morphine withdrawal, and AZD normalized it to control levels. (A) Experimental paradigm of morphine treatment and electrophysiology. (B) AMPAR / NMDARVHPM 17023.304WO1 / UIRF 25040 ratio was increased after 5 days of morphine withdrawal and was normalized by AZD (n = 7-13 neurons from 4 mice / group).

[0028] Figs. 12A-12F. Oxycodone withdrawal and AZD had no effect on dendritic spine densities in NAcC MSNs. (A) Experimental timeline: oxycodone (3mg / kg, i.p.) or saline (i.p.) was administered in the home cage each day for 5 days, followed by 10 days of withdrawal, after which Dil labeling was performed. (B) Representative images (projected z-stack) of dendritic spines in NAcC MSNs from Car4+ / +mice withdrawn from oxycodone (Oxy) vs saline (Sal) and treated with AZD vs. vehicle. (C) Total spine density (average # spines / um per neuron) was unchanged by oxycodone withdrawal and AZD (n = 9 - 16 neurons per group from 3 - 4 mice and 2 - 4 dendritic segments (50 - 60 pm) averaged per neuron. (D) Stubby spine density was unchanged by oxycodone withdrawal and AZD (Oxy by AZD interaction. (E) Thin spine density was unchanged by oxycodone withdrawal and AZD. (F) Mushroom spine density was unchanged by oxycodone withdrawal and AZD.

[0029] Fig. 13. Study of acetazolamide (AZD) as a prophylactic therapy to mitigate withdrawal-induced synaptic adaptations in opioid use disorder (OUD).

[0030] Bar Graph Description:

[0031] • Groups Analyzed:

[0032] o Sal-Veh: Saline-treated, vehicle control.

[0033] o Oxy-Veh: Oxycodone-treated, vehicle control.

[0034] o Oxy-AZD: Oxycodone-treated, acetazol ami de-treated.

[0035] o Sal-AZD: Saline-treated, acetazol ami de-treated.

[0036] • Y-Axis: AMPAR / NMDAR ratio, indicating synaptic plasticity. Higher values suggest increased AMPA receptor activity relative to NMDA receptors, which can be linked to changes in excitatory synaptic strength, drug seeking, and relapse.

[0037] DETAILED DESCRIPTION

[0038] The nucleus accumbens core (NAcC) is a central hub in the brain circuits underlying responses to drugs of abuse and is thought to play a key role in the development of OUD. The majority of neurons (-95%) in NAcC are medium spiny neurons (MSNs), which are thought to be critical for drug-seeking behaviors. Drugs of abuse including cocaine and opioids have been found to produce changes in synapses onto NAcC MSNs that persist well beyond acute drug exposures and are thus thought to bias neural circuits towards the seeking out of drugs in the future, thus likely promoting vulnerability to relapse. For example, cocaine withdrawal increases AMPAR / NMDAR ratio and Ca2+-permeable AMPARs (CP-AMPAR) at synapses onto NAcC-MSNs, which is thought to promote cocaine-seeking behavior. Synaptic responses to opioids areVHPM 17023.304WO1 / UIRF 25040 less well-characterized, although some rearrangements similar to those evoked by cocaine have been reported. For example, withdrawal from non-contingent morphine administration increased AMPAR / NMDAR ratio in Dl+MSNs in NAc shell and withdrawal from non-contingent administration of morphine, heroin, and oxycodone increased AMPAR / NMDAR ratio in NAcC-MSNs. Supporting an important role for synaptic AMPARs in NAcC in promoting opioidseeking behaviors, AMP AR antagonists delivered to the NAcC reduced reinstatement in rats following heroin self-administration.

[0039] Acid-sensing ion channels (ASICs) were recently identified as a novel molecular mechanism in cocaine and opioid-seeking behaviors. ASICs are cation channels activated by extracellular acidosis that consist of trimeric assemblies of differing combinations of ASIC1A, ASIC2A, and ASIC2B subunits. The ASIC1 A subunit is required for activation by pH changes within a physiologically relevant range (below pH 7.4 to pH 5). In NAcC MSNs, it was found that these channels are activated by protons released from glutamate-containing presynaptic vesicles during synaptic transmission. Loss of ASIC 1 A increased sensitivity of NAcC synapses to rearrangements induced by cocaine and opioids, and also increased conditioned place preference (CPP) to cocaine and opioids. Together, these findings of heightened drug vulnerability in mice lacking ASIC 1 A, suggest the intriguing possibility that increasing ASIC function might oppose synaptic and behavioral responses to both cocaine and opioids.

[0040] Consistent with this possibility, overexpressing ASIC1 A in NAcC in rats reduced cocaine selfadministration. Subsequent work, however, suggested that overexpressing ASIC1 A in NAcC in rats following cocaine self-administration and withdrawal increased reinstatement of cocaine-seeking behavior. Thus, behavioral effects of potentiating ASIC function may be complex and depend on timing and neuron specificity.

[0041] Another potential strategy for increasing ASIC activation is to reduce pH buffering. Extracellular pH is buffered by the reaction (H++ HCO3 -^CCh + H2O) catalyzed by carbonic anhydrases (CA). Among more than 14 in mammals, CA4 was focused ontas a candidate for regulating synaptic ASICs based on several of its established characteristics. CA4 is abundantly expressed in brain neurons, including NAc MSNs, it is anchored in the cell membrane facing the extracellular compartment, and it had been previously suggested to buffer synaptic pH. Indeed, disrupting CA4 in post-synaptic MSNs increased ASIC-mediated EPSCs. Furthermore, loss of CA4 prevented synaptic rearrangements following cocaine withdrawal including the associated increase in AMPAR / NMDAR ratio, CP -AMPARs, mEPSC frequency, and dendritic spine density. Consistent with these synaptic effects of CA4 disruption, there were also behavioral effects. Car^- mice self-administered a similar amount of cocaine as Car4+ / +mice, though after 4 weeks of forced abstinence, Car4^^ mice had reduced active lever presses (unreinforcedVHPM 17023.304WO1 / UIRF 25040 by drug). CA4 disruption also reduced locomotor responses to acute cocaine challenge following cocaine withdrawal.

[0042] In this study, effects of CA4 disruption on opioid withdrawal-induced synaptic adaptations and dendritic spine morphology in NAcC-MSNs were investigated, as well as oxycodone-seeking behavior. Pharmacological inhibition of CA4 with acetazolamide (AZD), a carbonic anhydrase inhibitor used clinically for a variety of illnesses, was also tested. It was hypothesized that CA4 disruption and pharmacological inhibition would protect against effects of oxycodone withdrawal on glutamatergic synapses in NAcC and drug-seeking behavior. The results suggest that CA4 may offer a new target for mitigating drug seeking and relapse in OUD that is unlike traditional opioid replacement therapies that target the mu opioid receptor.

[0043] In one aspect, provided herein is a method of preventing synaptic and / or behavioral adaptations resulting from opioid use in a mammal in need thereof comprising administering a carbonic anhydrase (CA) inhibitor agent to the mammal.

[0044] In certain aspects, the CA inhibitor agent is acetazolamide (AZD), topiramate, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, and / or zonisamide.

[0045] In certain aspects, the CA inhibitor is AZD.

[0046] In certain aspects, the opioid is capable of mu receptor agonist activity.

[0047] In certain aspects, the opioid is oxycodone, heroin, morphine, methadone, hydrocodone, hydromorphone, buprenorphine, fentanyl, talpentadol and / or pethidine.

[0048] In certain aspects, the opioid is oxycodone.

[0049] In certain aspects, the AZD is administered prior to, simultaneously with, or subsequent to, the administration of the opioid.

[0050] In certain aspects, the AZD is administered prior to the administration of the opioid. In certain aspects, the AZD is administered simultaneously with the administration of the opioid.

[0051] In certain aspects, the AZD is administered subsequent to the administration of the opioid.

[0052] In certain aspects, the AZD is administered with the final dose of the opioid.

[0053] In certain aspects, the CA inhibitor is administered via a topical, oral, or IV route.

[0054] In certain aspects, the mammal is a human.

[0055] In one aspect, provided herein is a method of reversing opioid-induced synaptic and / or network changes resulting from a first opioid use in a mammal in need thereof, comprisingVHPM 17023.304WO1 / UIRF 25040 administering a combination of a carbonic anhydrase (CA) inhibitor and a second opioid to the mammal.

[0056] In certain aspects, the CA inhibitor agent is acetazolamide (AZD), topiramate, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, and / or zonisamide.

[0057] In certain aspects, the CA inhibitor is AZD.

[0058] In certain aspects, the opioid is capable of mu receptor agonist activity.

[0059] In certain aspects, the second opioid is oxycodone, heroin, morphine, methadone, hydrocodone, hydromorphone, buprenorphine, fentanyl, talpentadol and / or pethidine.

[0060] In certain aspects, the second opioid is oxycodone.

[0061] In certain aspects, the AZD is administered prior to or simultaneously with administration of the second opioid.

[0062] In certain aspects, the AZD is administered prior to the administration of the second opioid.

[0063] In certain aspects, the AZD is administered simultaneously with the administration of the second opioid.

[0064] In certain aspects, the combination of AZD and an opioid is administered at least one day after cessation of the first opioid use.

[0065] In certain aspects, the first opioid and the second opioid are different.

[0066] In certain aspects, the first opioid is oxycodone and the second opioid is methadone. In certain aspects, the mammal is a human.

[0067] In one aspect, provided herein is a method of reversing opioid-induced synaptic and / or network changes resulting from a cocaine use in a mammal in need thereofe, comprising administering a combination of a carbonic anhydrase (CA) inhibitor and an opioid to the mammal.

[0068] In certain aspects, the CA inhibitor agent is acetazolamide (AZD), topiramate, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, and / or zonisamide.

[0069] In certain aspects, the CA inhibitor is AZD.

[0070] In certain aspects, the opioid is capable of mu receptor agonist activity.

[0071] In certain aspects, the opioid is oxycodone, heroin, morphine, methadone, hydrocodone, hydromorphone, buprenorphine, fentanyl, talpentadol and / or pethidine.

[0072] In certain aspects, the opioid is oxycodone.

[0073] In certain aspects, the CA inhibitor is administered simultaneously with the administration of the opioid.VHPM 17023.304WO1 / UIRF 25040 In certain aspects, the combination of CA inhibitor and the opioid is administered at least one day after cessation of the cocaine.

[0074] CARBONIC ANHYDRASE (CA) INHIBITORS

[0075] In certain embodiments, the CA inhibitor CA inhibitor agent is acetazolamide (AZD), topiramate, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, and / or zonisamide. In certain embodiments, the CA inhibitor agent is AZD.

[0076] OPIOIDS

[0077] The mu (p) receptors are a class involved in neuromodulating different physiological functions. Mu receptors are responsible for supraspinal analgesia, respiratory depression, euphoria, sedation, decreased gastrointestinal motility, and physical dependence. Mu receptors are found primarily in the brainstem and medial thalamus.

[0078] In certain aspects, the opioid is capable of mu receptor agonist activity. In certain embodiments, the opioid capable of mu receptor agonist activity is oxycodone, heroin, morphine, methadone, hydrocodone, hydromorphone, buprenorphine, fentanyl, talpentadol and / or pethidine. In certain embodiments, the opioid is oxycodone.

[0079] METHODS OF TREATMENT

[0080] The compounds of the present invention can be used as prophylactic or therapeutic agents for treating synaptic and / or behavioral adaptations resulting from opioid use (e.g., addiction to opioids). “Synaptic and / or behavioral adaptations resulting from opioid use” is used herein to mean to mitigate the symptoms of addiction to, or withdrawal from, opioids. An “effective amount” of an agent is intended to mean that amount of compound that, when administered to a mammal in need of such treatment, is sufficient to effect treatment for synaptic and / or behavioral adaptations resulting from opioid use. Thus, for example, a therapeutically effective amount of an agent is a quantity sufficient to modulate, regulate, or inhibit the Synaptic and / or behavioral adaptations resulting from opioid use such that an addiction condition which is mediated by the opioid is reduced or alleviated.

[0081] The terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder (e.g., addiction or withdrawal symptoms). For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of symptoms, stabilized (i.e., not worsening) symptoms, delay or slowing of symptoms, and amelioration or palliation of the symptoms. Those in need ofVHPM 17023.304WO1 / UIRF 25040 treatment include those already with the condition or symptoms, prone to have the condition or symptoms, or those in which the condition or symptoms are to be prevented. The terms “treating,” “treat,” and “treatment” embrace both preventative, i.e., prophylactic, and palliative treatment.

[0082] Patients that can be treated with compositions of the present invention include, for example, patients that have been diagnosed as having addiction to an opioid. As used herein, “patient” can mean a human or veterinary patient.

[0083] COMBINATION THERAPY

[0084] In certain embodiments, the present invention utilizes a combination of a carbonic anhydrase (CA) inhibitor and an opioid. The doses of the CA inhibitor agent and the opioid are appropriately selected based on clinically employed doses. The proportion of the CA inhibitor agent and the opioid are appropriately determined according to the administration subject, the administration route, the clinical condition, the combination, and other factors. In cases where the administration subject is a human, for instance, the opioid may be used in an amount of 0.01 to 100 parts by weight per part by weight of the CA inhibitor agent.

[0085] The CA inhibitor agent and the opioid may be administered together in a unitary pharmaceutical composition or separately and, when administered separately this may occur simultaneously or sequentially in any order. Such sequential administration may be close in time or remote in time. The amounts of the CA inhibitor agent and the opioid, and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.

[0086] Suitable dosages for any of the above co-administered agents are those presently used or may be lowered due to the combined action (synergy) of the CA inhibitor agent and the opioid.

[0087] The combination therapy may provide “synergy” and prove “synergistic,” i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. For example, a lower dose of the opioid may be used and still achieve the desired effect of mitigating addiction and / or withdrawal symptoms. A synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administeredVHPM 17023.304WO1 / UIRF 25040 sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[0088] ADMINISTRATION OF CA INHIBITORS AND OPIOIDS

[0089] The compounds of the invention (i.e., CA inhibitors and, in certain embodiments, opioids) may be administered by any route appropriate to the condition to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intrathecal and epidural), transdermal, rectal, nasal, topical (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal. It will be appreciated that the preferred route may vary with for example the condition of the recipient. Where the compound is administered orally, it may be formulated as a pill, capsule, tablet, etc. with a pharmaceutically acceptable carrier or excipient. Where the compound is administered parenterally, it may be formulated with a pharmaceutically acceptable parenteral vehicle and in a unit dosage injectable form, as detailed below.

[0090] The pharmaceutical compositions of the invention are formulated, dosed and administered in a fashion, i.e., amounts, concentrations, schedules, course, vehicles and route of administration, consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The therapeutically effective amount of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the disorder. The compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to enable patient compliance with the prescribed regimen.

[0091] The composition for use herein is preferably sterile. In particular, formulations to be used for in vivo administration must be sterile. Such sterilization is readily accomplished, for example, by filtration through sterile filtration membranes. The compound ordinarily can be stored as a solid composition, a lyophilized formulation or as an aqueous solution.

[0092] Pharmaceutical formulations of the compounds of the present invention may be prepared for various routes and types of administration. For example, the bulk drug substance (i.e., compound of the present invention or stabilized form of the compound (e.g., complex with a cyclodextrin derivative or other known complexation agent) is dissolved in a suitable solvent in the presence of one or more excipients. The particular carrier, diluent or excipient used willVHPM 17023.304WO1 / UIRF 25040 depend upon the means and purpose for which the compound of the present invention is being applied.

[0093] Sustained-release preparations of the CA inhibitor agent may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3 -hydroxybutyric acid.

[0094] The pharmaceutical compositions of CA inhibitor agent may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3 -butanediol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

[0095] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0096] The compositions of the invention may also be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder)

[0097] Suitable pharmaceutically-acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; bindingVHPM 17023.304WO1 / UIRF 25040 agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and / or appearance, in either case using conventional coating agents and procedures well known in the art.

[0098] Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

[0099] Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), coloring agents, flavoring agents, and / or sweetening agents (such as sucrose, saccharine or aspartame).

[0100] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0101] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavoring and coloring agents, may also be present.VHPM 17023.304WO1 / UIRF 25040 The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring and preservative agents.

[0102] Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavoring and / or coloring agent.

[0103] Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0104] Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedures well known in the art.

[0105] Compositions for transdermal administration may be in the form of those transdermal skin patches that are well known to those of ordinary skill in the art.

[0106] Formulations suitable for intrapulmonary or nasal administration have a particle size for example in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in increments microns such as 0.5, 1, 30 microns, 35 microns, etc.), which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis disorders as described below.

[0107] The pharmaceutical composition (or formulation) for application may be packaged in a variety of ways depending upon the method used for administering the drug. For example, anVHPM 17023.304WO1 / UIRF 25040 article for distribution can include a container having deposited therein the pharmaceutical formulation in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings. The formulations may also be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.

[0108] The invention further provides veterinary compositions comprising a CA inhibitory agent together with a veterinary carrier. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

[0109] The amount of a compound of this invention that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the subject treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. In one embodiment, a suitable amount of a CA inhibitory agent is administered to a mammal in need thereof. Administration in one embodiment occurs in an amount between about 0.001 mg / kg of body weight to about 60 mg / kg of body weight per day. In another embodiment, administration occurs in an amount between 0.5 mg / kg of body weight to about 40 mg / kg of body weight per day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day.

[0110] The invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and areVHPM 17023.304WO1 / UIRF 25040 compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

[0111] The amount of a compound of this invention that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the subject treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. In one embodiment, a suitable amount of a CA inhibitory agent is administered to a mammal in need thereof. Administration in one embodiment occurs in an amount between about 0.001 mg / kg of body weight to about 60 mg / kg of body weight per day. In another embodiment, administration occurs in an amount between 0.5 mg / kg of body weight to about 40 mg / kg of body weight per day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day.

[0112] PHARMACEUTICAL FORMULATIONS

[0113] In order to use a CA inhibitory agent for the therapeutic treatment (including prophylactic treatment) of mammals including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition. According to this aspect of the invention there is provided a pharmaceutical composition that comprises a CA inhibitory agent, in association with a pharmaceutically acceptable diluent or carrier.

[0114] ARTICLES OF MANUFACTURE

[0115] In another embodiment of the invention, an article of manufacture, or “kit,” containing materials useful for the treatment of the disorders described above is provided. In one embodiment, the kit comprises a container comprising a CA inhibitory agent. The kit may further comprise a label or package insert on or associated with the container. The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products. In one embodiment, the label or package inserts indicates that the composition comprising a CA inhibitory agent can be used to treat one or more of the disorders or conditions disclosed herein.

[0116] In one embodiment, the kit further comprises a container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The container may be formed from aVHPM 17023.304WO1 / UIRF 25040 variety of materials such as glass or plastic. The container may hold a CA inhibitory agent that is effective for treating one or more conditions or disorders disclosed herein and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0117] The kit may further comprise directions for the administration of the first pharmaceutical composition (i.e., CA inhibitory agent) and, if present, the second pharmaceutical formulation (i.e., opioid). For example, when the kit comprises a first composition comprising a CA inhibitory agent and an opioid, the kit may further comprise directions for the simultaneous, sequential or separate administration of the first and second pharmaceutical compositions to a patient in need thereof.

[0118] In another embodiment, the kits are suitable for the delivery of solid oral forms of a CA inhibitory agent, such as tablets or capsules. Such a kit can include a number of unit dosages. Such kits can include a card having the dosages oriented in the order of their intended use. An example of such a kit is a “blister pack.” Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If desired, a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered.

[0119] According to one embodiment, an article of manufacture may comprise (a) a first container with a first pharmaceutical formulation (e.g., a CA inhibitory agent) contained therein; and optionally (b) a second container with a second pharmaceutical formulation (e.g., opioid) contained therein. Alternatively, or additionally, the article of manufacture may further comprise a third container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0120] In certain other embodiments, wherein the kit comprises first pharmaceutical formulation and a second pharmaceutical formulation, the kit may comprise a container for containing the separate compositions such as a divided bottle or a divided foil packet, however, the separate compositions may also be contained within a single, undivided container. Typically, the kit comprises directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

[0121] The invention will now be illustrated by the following non-limiting Examples.VHPM 17023.304WO1 / UIRF 25040

[0122] EXAMPLE 1

[0123] Acetazolamide inhibition 1 of carbonic anhydrase 4 reverses opioid-induced synaptic rearrangements in nucleus accumbens and reduces drug-seeking behavior ABSTRACT

[0124] Persistent vulnerability to drug-seeking is driven by enduring synaptic adaptations, yet current p-opioid receptor-targeting pharmacotherapies provide limited efficacy against these neuroadaptations. Thus, there is critical need for mechanistically distinct, non-opioid interventions. We recently found that carbonic anhydrase 4 (CA4) disruption reduces cocaine induced synaptic adaptations and drug seeking. Building on this foundation, we sought to determine whether deleting CA4 or pharmacological inhibition with acetazolamide (AZD) a clinically employed carbonic anhydrase inhibitor — could mitigate opioid withdrawal-associated plasticity and thus might reduce relapse vulnerability. We studied synaptic and behavioral adaptations to withdrawal from oxycodone in mice and found prolonged withdrawal from oxycodone increased AMPAR / NMDAR ratio and promoted synaptic incorporation of Ca2+permeable AMPARs in nucleus accumbens core (NAcC) medium spiny neurons (MSNs). We found synaptic changes after protracted withdrawal from multiple opioids which were most pronounced in DI -expressing MSNs, and were prevented by CA4 disruption. Moreover, AZD reversed withdrawal-induced synaptic alterations both in vitro and in vivo, in a CA4- and acid sensing ion channel 1 A (ASIC1 A)-dependent manner. Unlike withdrawal from cocaine, withdrawal from oxycodone did not alter dendritic spine density in NAcC MSNs, suggesting a distinct mode of plasticity. Finally, following oxycodone self-administration, both CA4 deletion and a single systemic AZD dose reduced drug seeking after prolonged abstinence. Together, these findings identify CA4 as a regulator of opioid-induced synaptic adaptations and suggest AZD as a promising, readily translatable pharmacological intervention. By targeting a pathway independent of classical opioid receptor signaling, CA4 inhibition represents a mechanistically distinct strategy that may reduce relapse vulnerability in OUD.

[0125] INTRODUCTION

[0126] The opioid crisis is ongoing, and overdose deaths remain unacceptably high. Opioids and their withdrawal produce a sustained, increasing desire for drug, referred to as opioid-seeking or craving. Few medications are FDA-approved to treat opioid use disorder (OUD), and all target the mu opioid receptor. While these medications mitigate withdrawal and reduce relapse risk, they fail to correct the persistent synaptic rearrangements that underpin relapse. Thus, newVHPM 17023.304WO1 / UIRF 25040 treatments with mechanisms distinct from the mu opioid receptor could have a substantial impact on the clinical management of OUD.

[0127] The nucleus accumbens (NAc) is a central hub in the brain circuits underlying responses to drugs of abuse and is thought to play a key role in OUD. -95% of neurons in NAc are medium spiny neurons (MSNs), which are critical for drug-seeking behaviors. Drugs of abuse, including cocaine and opioids, produce changes in synapses onto NAc MSNs that persist beyond acute drug exposures and may thus bias neural circuits towards future drug seeking, promoting vulnerability to relapse. For example, withdrawal from cocaine has been reported to increase AMPAR / NMDAR ratio at synapses in NAc core (NAcC) and shell (NAcS), and to increase Ca2+-permeable AMPARs (CP-AMPARs) in NAcC and NAcS; and such changes have been suggested to promote cocaine-seeking. Synaptic responses to opioids are less well characterized, although similar rearrangements have been reported. For example, withdrawal from noncontingent morphine increased AMPAR / NMDAR ratio in Dl+MSNs in NAc shell, and withdrawal from non-contingent morphine, heroin, and oxycodone increased AMPAR / NMDAR ratio in NAcC-MSNs. Increases in CP-AMPARs have been reported following withdrawal from morphine 1, fentanyl, and oxycodone. Supporting an important role for synaptic AMPARs in NAcC in promoting opioid-seeking behaviors, AMP AR antagonists delivered to the NAcC reduced reinstatement in rats following heroin self-administration.

[0128] We recently identified acid-sensing ion channels (ASICs) as a novel molecular mechanism in cocaine and opioid-seeking behaviors. ASICs are cation channels activated by extracellular acidosis and comprised of trimeric assemblies of combinations of ASIC1A, ASIC2A, and ASIC2B subunits. The ASIC1 A subunit is required for activation by acidic extracellular pH within a physiologically relevant range (pH 7.2 to pH 5). In NAcC MSNs, ASICs are activated by protons released from glutamate-containing presynaptic vesicles during synaptic transmission. ASIC 1 A disruption increased sensitivity to synaptic rearrangements induced by cocaine and opioids, and also increased conditioned place preference (CPP) to cocaine and opioids. Together, these findings suggest the possibility that increasing ASIC function might oppose synaptic and behavioral responses to cocaine and opioids. Consistent with this possibility, overexpressing ASIC1 A in NAcC in rats reduced cocaine selfadministration. Subsequent work, however, suggested that overexpressing ASIC1 A in NAcC in rats following cocaine self-administration and withdrawal increased reinstatement of cocaine-seeking. Thus, behavioral effects of potentiating ASIC function may be complex and depend on timing and neuron specificity.

[0129] Another strategy for increasing ASIC activation is to reduce pH buffering. ExtracellularVHPM 17023.304WO1 / UIRF 25040 pH is buffered by the reaction (H++ HCO3 -^CCh + H2O) catalyzed by carbonic anhydrases (CA). Among more than 14 isoforms in mammals, we focused on CA4 as a candidate for regulating synaptic ASICs based on its established characteristics. CA4 is abundantly expressed in brain neurons, including NAc MSNs, is anchored in the cell membrane facing the extracellular compartment, and has been previously suggested to buffer synaptic pH. Indeed, disrupting CA4 in postsynaptic MSNs increased acidosis and ASIC-mediated EPSCs. The CA4 inhibitor acetazolamide (AZD) also increased acidosis and potentiated ASIC5 mediated EPSCs, and had no added effect on ASIC-mediated EPSCs in Car^' mice, suggesting actions of AZD on ASICs depend on CA4. Furthermore, loss of CA4 prevented synaptic rearrangements following withdrawal from cocaine, including increased AMPAR / NMDAR ratio, CP-AMPARs, mEPSC frequency, and dendritic spine density. Consistent with these synaptic effects of CA4 disruption, there were also behavioral effects. Car4^^ mice self administered a similar amount of cocaine as Car4+ / +mice, though after 4 weeks of abstinence, Car4~ / ~ mice had reduced active lever presses (unreinforced by drug). CA4 disruption also reduced locomotor responses to acute cocaine challenge following withdrawal from cocaine. Here, we investigated effects of CA4 disruption in mice on opioid withdrawal-induced synaptic adaptations and dendritic spine morphology in NAcC-MSNs. We also assessed oxycodone-seeking behavior using an operant model resembling a procedure in rats that produced incubation of oxycodone-seeking behavior. Additionally, we tested pharmacological inhibition of CA4 with acetazolamide (AZD), a carbonic anhydrase inhibitor used clinically for a variety of illnesses. We hypothesized that CA4 disruption and pharmacological inhibition would protect against effects of withdrawal from oxycodone on glutamatergic synapses in NAcC and drugseeking behavior. Our results suggest AZD may offer a new pharmacological agent for mitigating drug seeking and relapse in OUD that is unlike traditional opioid replacement therapies that target the mu opioid receptor.

[0130] MATERIALS AND METHODS

[0131] Mice

[0132] All mice were on C57BL / 6 genetic background. Car 4 mice (stock no. 008217) and 4 Drdla-tdTomato mice (stock #016204) were obtained from Jackson Laboratory.

[0133] Asicla~'~ mice were generated. Mice were housed in groups of 2-5 littermates with free access to standard chow and water. Mice were maintained on 12-hour light-dark cycle, experiments performed during light phase. Experimental groups were matched by sex and age (10-15 weeks). Experimentally naive mice were randomly assigned to conditions. All procedures wereVHPM 17023.304WO1 / UIRF 25040 approved by the University of Iowa Animal Care and Use Committee and complied with NIH guidelines.

[0134] Oxycodone, heroin, morphine and AZD

[0135] Oxycodone, heroin, and morphine were supplied by NIDA. Acetazolamide (XGen, USA) was purchased from University of Iowa Hospital pharmacy. Timing, dosing, and delivery of drugs are described in figures.

[0136] Slice preparation and electrophysiology

[0137] 300 pm coronal NAc slices were prepared from mice (8-12 weeks old) in cold buffer (in mM: 225 Sucrose, 26 NaHCOs, 1.2 KH2PO4, 1.9 KC1, 10 D-glucose, 1.1 CaCh, 2 MgSO4) with 95% O2 and 5% CO2. Slices were transferred to oxygenated ACSF (in mM: NaCl 127, 26 NaHCOs, 1.2 KH2PO4, 1.9 KC1, 10 D-glucose, 2.2 CaCh, 2 MgSO4) held at 32°C for 30-mins followed by 30-min room temperature. Whole-cell recordings were performed in NAcC MSNs using 2.5-4 MQ pipettes. Data were obtained with Axopatch 200B amplifier ( 1 Axon Instruments) analyzed off-line by Clampfit (Axon software). Internal solution mM: 125 cesium methanesulfonate, 20 CsCl, 10 NaCl, 2 Mg-ATP, 0.3 Na-GTP, 10 HEPES, 0.2 EGTA, 2.5 QX314, pH 7.3 adjusted with CsOH. EPSCs were evoked using a bipolar tungsten electrode positioned -200 pm from neuron. Stimulation intensity was adjusted to -half-maximum EPSC response. For AMPAR / NMDAR ratios, peak AMPAR-EPSCs were measured at -70 mV and NMDAR-EPSCs were measured 60-ms after onset at +50 mV with CNQX (20 pM) added to the bath. Picrotoxin (100 pM) was added for all recordings. Alternative assessment of AMPAR / NMDAR ratios using peak NMD AR currents preserved effects of CA4 and AZD (Fig.

[0138] 6). AMP AR rectification was determined by recording at -70, -30, +30, and +50 mV in the presence of picrotoxin (100 pM), APV (100 pM). AMP AR rectification index was calculated as ratio of current at -70 mV to +50 mV. For NASPM sensitivity, cells were held at -70 mV, with 20-25 baseline sweeps collected in presence of picrotoxin (100 pM) and APV (100 pM).

[0139] NASPM (200 pM, Alomone Lab) was applied, and 15-min later, 20-25 sweeps collected. In vitro effects of CA4 inhibition were tested by applying AZD (100 pM) for 1-hr.

[0140] Dil labeling, dendritic spine imaging, and analysis

[0141] Mice were perfused, coronal slices cut, and NAcC MSNs stained with Dil. Dendritic segments were imaged, and spine density and morphology analyzed using Neuron Studio. Each experimental group consisted of 3-4 animals, with a total of 9-16 neurons / group, 3-4 neurons analyzed / animal. Each neuron was averaged over 2-4 dendritic segments with each segment -50-60 pm in length. Results were calculated as average density (number of spines / pm dendritic length) per neuron. Number of spines assessed by group: 1 Car4+'+saline-veh (3850), Car4+'+saline-AZD (4646), Car4 oxycodone-veh (3007), and Car4 oxycodone- AZD (3960).VHPM 17023.304WO1 / UIRF 25040 Oxycodone Conditioned Place Preference (CPP)

[0142] For Oxycodone CPP, mice underwent pre- and post-tests when they were allowed to explore a two-chamber CPP apparatus (Med Associates) for 20-min. For 3 days between pre-and post-tests, mice underwent two training sessions daily during which oxycodone (15 mg / kg, i.p.) and saline were paired with opposite compartments. Preference on oxycodone-paired side was calculated by subtracting pre-test from post-test.

[0143] Oxycodone Self-Administration (SA)

[0144] First, mice underwent jugular vein catheterization. Catheter patency was verified postsurgery and during the course of the SA protocol. Mice were fasted overnight prior to SA training and restricted to 85-90% pre-fasting bodyweight during days 1-10. Training occurred in operant chambers (Med Associates) equipped with cues (light and tone) and two levers (active vs. inactive). Mice received oxycodone (0.25 mg / kg / infusion) on a fixed-ratio 1 (FR1) schedule during daily 6-hour sessions for 10 days minimum. Active lever presses (ALPs) triggered oxycodone infusions and 20 seconds of cues during which no additional infusions could be obtained. Mice meeting criteria of at least 10 infusions / day for final 3 days, were included in further testing. 24-hrs after final SA session, baseline drug seeking was assessed by returning mice to operant chambers for 30-mins during which ALPs triggered cues but no drug. After 30 days of forced abstinence, mice received AZD (30 mg / kg, i.p.) or vehicle (saline). 24-hrs later, drug-seeking behavior was again assessed as at baseline.

[0145] Statistical analyses

[0146] Student’s t-test was used to assess differences between two groups, while two-way ANOVA was used to assess significance for experiments involving 2 independent factors.

[0147] Within the context of the ANOVA, planned contrast testing (Fisher’s LSD) was used to test a priori hypothesized relationships between groups. To assess significance of lever press data following oxycodone SA, three-way ANOVA with repeated measures was used to compare between-subject factors (genotype, AZD) and within-subject factor (time). Paired-sample t-tests were used to compare same groups of mice tested at two different time points (baseline vs postwithdrawal). ROUT (Q= 1%) was used to screen for outliers. F-test was used to compare variances between groups, and Welch’s correction was applied for unequal variances. P < 0.05 was considered significant. Bar graphs express values as mean ± S.E.M. Statistical analyses were performed using GraphPad Prism.

[0148] RESULTS CA4 disruption prevented oxycodone-withdrawal-induced increases in AMPAR / NMDAR ratio and CP-AMPARs, and attenuated CPPVHPM 17023.304WO1 / UIRF 25040 To test whether oxycodone withdrawal induces synaptic rearrangements that are sensitive to CA4 disruption, we delivered oxycodone (3 mg / kg, i.p.) vs. saline control once daily for 5 days to Car 4+1+ and Car 4-1- mice and then withheld oxycodone for 10 days (Fig. 1A). We then harvested brain tissue and tested glutamatergic transmission onto NAcC-MSNs in acute slices (Fig. IB). We found that withdrawal from oxycodone increased AMPAR / NMDAR ratio in Car4+ / + mice but not in Car4-I- mice (Figs. 1C, ID; drug x genotype interaction, F(l,38)=10.55, p=0.0024), suggesting 1 loss of CA4 prevented the oxycodone-induced change. Effects of oxycodone and CA4 disruption were independent of sex (Fig. 7). To test if extended withdrawal was required to increase AMPAR / NMDAR ratio, we tested 24 hrs of withdrawal after 5 doses (once daily x 5 days) and after one dose (Fig. 8A). These 24-hr withdrawal was insufficient to change AMPAR / NMDAR ratios (Fig. 8B), suggesting an extended withdrawal period was required.

[0149] An increase in GluA2-lacking CP-AMPARs in postsynaptic membrane is one mechanism that may contribute to increased AMPAR / NMDAR ratios. CP-AMPARs are more inwardly rectifying; therefore, we tested rectification index in NAcC MSNs by measuring current-voltage relationships of synaptic AMP AR responses (Fig. IE). Indeed, withdrawal from oxycodone increased rectification index in Car4+I+mice (vs. saline) but not in Car4 mice (Figs. IE, F, G; drug x genotype interaction, F(l,40)=6.625, p=0.0139). We also tested sensitivity to the CP-AMPAR-specific antagonist NASPM and found increased NASPM sensitivity in Car4+l+mice following withdrawal from oxycodone (Figs. 1H, II; drug x genotype interaction, F(l,22)=10.40, p=0.0039). Consistent with a protective effect of CA4 disruption, NASPM sensitivity was unchanged in Car 4 mice. Together, these findings suggest that withdrawal from oxycodone increases CP-AMPARs in the postsynaptic membrane of NAcC MSNs and that loss of CA4 prevents this increase. These findings highlight CA4 disruption as a potential strategy for preventing synaptic rearrangements associated with withdrawal from oxycodone.

[0150] To assess whether CA4 disruption affects drug-associated memory, we tested oxycodone CPP (15 mg / kg, i.p.) (Fig. 1J), a dose that produced exaggerated CPP n A icla mice. We found that both Car4+'+and Car4~ / _mice preferred the oxycodone-paired chamber, although this preference was reduced in Car4~ / _mice (1 Fig. IK, t(31.31)=2.197, p=0.0355). We further tested effects of this oxycodone dose on AMPAR / NMDAR ratio; we administered oxycodone (15 mg / kg) vs saline daily for 5 days followed by 10 days of withdrawal. We found an increase in AMPAR / NMDAR ratio in oxycodone-withdrawn Car4+ / +mice vs. saline-treated controls, which was absent in Car4 ^ mice (Fig. IL; drug x genotype interaction, F(l,24)=5.987,VHPM 17023.304WO1 / UIRF 25040 p=0.0221). Together, these results indicate CA4 disruption attenuates oxycodone CPP and protects against synaptic rearrangements following withdrawal from oxycodone.

[0151] AZD reversed CA4-dependent oxycodone-induced synaptic changes

[0152] To explore whether pharmacologically inhibiting CA4 produces effects similar to CA4 disruption, we used AZD. AZD is approved to treat a variety of illnesses in humans and its properties make it ideal. AZD potently blocks CA4 (~10 nM affinity), as well as other CAs. AZD readily crosses the blood-brain barrier. It is rapidly metabolized and excreted by the kidneys, with a ~1 hr half-life in mice. Moreover, AZD potentiates ASIClA-mediated synaptic currents in NAcC MSNs in a CA4-dependent manner.

[0153] To determine whether AZD produces effects similar to CA4 disruption, we first tested its effects in brain slices in Car4+ / + mice. We delivered oxycodone (3 mg / kg, i.p. vs. saline) daily for 5 days, followed by 5 days of withdrawal (Fig. 9A). We then assessed AMPAR / NMDAR ratio in NAcC MSNs. As before, withdrawal from oxycodone increased AMPAR / NMDAR ratio. Compared to vehicle, applying AZD (100 pM) to the bath for 1 hr reduced AMPAR / NMDAR ratio in oxycodone withdrawn mice (Fig. 9B, oxy x AZD interaction, F(l,37)=8.729, p=0.0054). Importantly, AZD had no effect on AMPAR / NMDAR ratio in saline-treated mice. These data suggest that acute AZD application normalizes opioid-induced increases in AMPAR / NMDAR ratio within 1 hr.

[0154] We next tested whether administering AZD in vivo has similar effects. We estimated an AZD dose of 30 mg / kg, i.p. should achieve a systemic concentration similar to that used in our brain slice experiments and analogous to that used in humans. We administered oxycodone (3 mg / kg, i.p. vs. saline) daily for 5 days. After 5 days of withdrawal (Fig. 9C) we gave AZD vs. vehicle. Three hours later, we harvested brain slices and assessed AMPAR / NMDAR ratio in NAcC MSNs. Similar to our observations in vitro, administering AZD in vivo after withdrawal from oxycodone reduced AMPAR / NMDAR to levels comparable to that of vehicle-treated saline-withdrawn counterparts (Fig. 9D, oxycodone x AZD interaction, F(l,26)=21.69, p<0.0001), while AZD had no effect in mice not exposed to oxycodone. To test generalizability to other opioids, we also tested effects of in vivo AZD administration after 5 days of withdrawal from morphine and observed similar results (Fig. 9F, Mor x AZD interaction, F(l,24)=10.34, p=0.0196).

[0155] Next, we tested whether effects of in vzvo-administered AZD lasted beyond 3 hrs. We administered AZD vs. vehicle following withdrawal from oxycodone and tested AMPAR / NMDAR 24 hrs later (Fig. 2A). Withdrawal from Oxycodone again increased AMPAR / NMDAR ratio in Car4+I+ mice, and importantly AZD reduced it back to baseline levels (Figs. 2B, 2C; oxycodone x AZD interaction, F(l,37)=4.966, p=0.032). As with CA4VHPM 17023.304WO1 / UIRF 25040 disruption, AZD effects were sex-independent (Fig. 7). Together, these findings suggest that AZD reverses opioid-induced synaptic rearrangements. Moreover, because of its short half-life, effects of AZD likely persisted beyond its clearance.

[0156] We hypothesized that AZD effects on AMPAR / NMDAR ratio were mediated through CA4 inhibition rather than another target. Therefore, we also tested AZD in Car4- / - mice and found no effect of oxycodone or AZD on AMPAR / NMDAR ratio (Fig. 2D). We similarly tested effects of AZD on oxycodone-withdrawal-induced changes in AMP AR rectification and NASPM sensitivity. In Car4+ / +mice, AZD reversed oxycodone-associated increases in both rectification (Figs. 2E, 2F; oxy x AZD interaction, F(l,41)=6.862, p=0.0123) and NASPM sensitivity (Figs. 2H, 21, oxy x AZD interaction, F(l,23)=18.38, p=0.0003), suggesting that AZD also reverses oxycodone-induced increases in CP-AMPARs. In contrast, in Car4 mice, neither rectification index or NASPM sensitivity were affected by withdrawal from oxycodone or AZD (Figs. 2E, 2G, 2H, 2J). These results are consistent with our hypothesis; however, because there were no oxycodone-induced changes to reverse in Cad mice, the absence of an AZD effect in this background cannot alone establish causality, but it supports the interpretation that AZD acts through CA4 rather than an off-target mechanism.

[0157] AZD also reversed changes in AMPAR / NMDAR induced by heroin and morphine To test whether the protective effects of AZD generalized to other opioids, we also tested withdrawal from heroin (8mg / kg for 5 days) and morphine (lOmg / kg for 5 days) (Fig. 3A). After 10 days of withdrawal, Car4+ / +mice received AZD or vehicle, and 24 hours later AMPAR / NMDAR ratio was assessed. Withdrawal from heroin (Figs. 3B, 3C) and morphine (Figs. 3D, 3E) both increased AMPAR / NMDAR ratio in NAcC-MSNs (heroin, F(l,32)=6.156, p=0.0006; morphine, F(l,26)=7.179, p=0.0025) and AZD reversed these effects.

[0158] Withdrawal from a lower heroin dose (2 mg / kg, i.p.) did not increase AMPAR / NMDAR ratio (Figs. 10A, 10B), suggesting that dose matters for at least some of the opioid-induced synaptic rearrangements. In another experiment we tested just 5 days of withdrawal from morphine (10 mg / kg) and found it was sufficient to increase AMPAR / NMDAR (Figs. 11A, 11B) (F(l,36)=2.697, p=0.0102), suggesting that withdrawal periods as short as 5 days, but longer than 24 hrs, are sufficient to alter AMPAR / NMDAR ratio. Together, these observations suggest that synaptic adaptations evoked by withdrawal from oxycodone and the normalizing effects of AZD are not specific to oxycodone but also generalize to other opioids.

[0159] AZD had no effect on AMPAR / NMDAR ratios in Asida mice

[0160] We next tested withdrawal from oxycodone and AZD in Asida mice. We previously reported that Asida mice have an elevated AMPAR / NMDAR at baseline and exposure to cocaine or oxycodone normalizes it towards baseline levels seen in drug-naive Asicla+ / +VHPM 17023.304WO1 / UIRF 25040 controls. Thus, we wondered whether AZD could impact either the elevated baseline AMPAR / NMDAR ratio in Asiclcr' mice, or the oxycodone-induced reduction. We delivered oxycodone (3 mg / kg, i.p. vs. saline) daily for 5 days, followed by 10 days of withdrawal, then treated with AZD vs. vehicle and 24 hours later tested AMPAR / NMDAR ratio in NAcC MSNs. AZD had no effect on either the elevated baseline or the oxycodone-withdrawn AMPAR / NMDAR ratios (Figs. 3G, 3H). Together, these results suggest AZD’s ability to reduce AMPAR / NMDAR ratio depends on ASIC 1 A and are consistent with the actions of AZD and CA4 on ASIClA-mediated currents.

[0161] Effects of oxycodone withdrawal and AZD on AMPAR / NMDAR were observed in Dl+MSNs but not DI- MSNs

[0162] NAcC MSNs are subclassified by expression of dopamine receptor subtypes (DI vs. D2). Conventionally, these MSN subtypes have been thought to play differing roles, with Dl+NAcC-MSNs promoting drug-seeking behaviors, and D2+MSNs opposing drug seeking, although more recent observations suggest more nuanced roles of these neuron types in reward and aversion. To test whether the effects of oxycodone and AZD described above were specific to MSN subtype, we leveraged mice expressing tdTomato selectively in Dl+MSNs (Fig. 4A).

[0163] We tested responses in AMPAR / NMDAR ratio in Dl+-NAcC-MSNs versus non-Dl+-NAcC-MSNs (not expressing tdTomato) using the same oxycodone and AZD exposures as before (Fig.

[0164] 2A). We recorded from Dl+- and non-Dl+-NAcC-MSNs as illustrated (Fig. 4B). We found withdrawal from oxycodone evoked an increase in AMPAR / NMDAR ratio in Dl+MSNs which was reversed by AZD (Figs. 4C, 4D, oxy x AZD interaction, F(l,19)=8.578, p=0.0086).

[0165] However, in non-Dl+NAcC-MSNs, withdrawal from oxycodone did not change AMPAR / NMDAR ratio and AZD had no additional effect (Figs. 4E, 4F). Potential effects on CP-AMPARs were not tested. These results suggest effects of withdrawal from oxycodone on AMPAR / NMDAR ratios are more selective for Dl+-MSNs.

[0166] Dendritic spine density in NAcC MSNs was unaffected by withdrawal from oxycodone and AZD

[0167] Along with functional changes in glutamatergic synapses in the NAcC (e.g.

[0168] AMPAR / NMDAR ratio), changes in dendritic spine density following withdrawal from cocaine have also been reported. However, the direction and magnitude of spine changes have varied across studies and exposure procedures, and causal relationships between synaptic physiology, spine densities, and drug-seeking behaviors have not been clearly established. Fewer studies have investigated effects of withdrawal from opioid on dendritic spine density, leaving open the question of whether withdrawal from opioid produces structural changes similar to those reported with cocaine. Supporting differing effects of opioids versus cocaine, we recentlyVHPM 17023.304WO1 / UIRF 25040 observed no effects of withdrawal from oxycodone on spine density in either wild-type or mice lacking ASIC 1 A. Thus, we were interested in whether inhibiting CA4 with AZD would affect dendritic spine density following withdrawal from opioid. To test this possibility, we administered oxycodone for 5 days, followed by 10 days of withdrawal. We then administered AZD or vehicle and 24 hrs later harvested brain tissue for dendritic spine analyses (Fig. 12A).

[0169] We found that total spine density was similar between oxycodone- and saline-withdrawn Car4+ / +mice (Figs. 12B, 12C), with no differences in the density of stubby (Fig. 12D), thin (Fig. 12E), or mushroom spines (Fig. 12F). These results were consistent with our recently reported absence of effect of withdrawal from oxycodone on dendritic spine density in NAcC-MSNs. Importantly, in both oxycodone- and saline-withdrawn mice, AZD had no effect on these measures versus vehicle (Figs. 12B-12F). Together, these results suggest that withdrawal from oxycodone exerted little or no effect on dendritic spine density in NAcC-MSNs, and thus differ from effects we have recently seen following a similar protocol of withdrawal from cocaine. In addition, these data suggest that the above alterations in glutamatergic neurotransmission evoked in NAcC-MSNs by withdrawal from oxycodone were not accompanied by changes in dendritic spine density.

[0170] Drug-seeking and elevated AMPAR / NMDAR ratios following withdrawal from oxycodone SA were reduced by CA4 disruption and AZD

[0171] To examine potential behavioral consequences of the above-described effects of CA4 disruption and AZD, we next turned to oxycodone SA. SA procedures allow rodents to control the amount of drug they consume, and thus model human drug use and seeking. Car4+ / +mice and Car mice were implanted with jugular catheters for oxycodone SA (0.25 mg / kg / infusion) in response to active lever presses (ALP) on a FR1 schedule. Mice were allowed to SA oxycodone for 6 hours daily for 10 days (Fig. 5A). A 30-minute baseline test of cue induced drug-seeking behavior was assessed on day 11, in which ALPs produced the light and tone cues but no drug infusion. After 30 days of abstinence (experimental day 40), mice received a single administration of AZD (30 mg / kg, i.p.) or vehicle and 24-hours later (day 41) again underwent a cue-reinforced drug-seeking session for comparison to baseline testing. Overall, we found mice of both genotypes similarly acquired the SA task (days 1 through 10) and both genotypes developed a similar preference for ALPs versus inactive lever presses (ILPs) (Fig. 5B).

[0172] Additionally, both genotypes received a similar number of oxycodone infusions (Fig. 5C). These results suggest that CA4 disruption did not affect oxycodone consumption. After 30 days of abstinence, vehicle-injected Car4+ / +mice maintained a similar amount of cue-reinforced ALPs relative to the baseline (Fig. 5D), suggesting that the desire to obtain oxycodone was sustained. In contrast, we found effects of CA4 disruption and AZD on ALPs (Fig. 5D;VHPM 17023.304WO1 / UIRF 25040 genotype x time interaction, F(l,35)=4.733, p=0.0364) (Fig. 5E; genotype vs. AZD interaction, F(l,35)=4.252, p=0.0467). AZD-injected Car4+ / + mice reduced ALPs by half (Fig. 5D, E, t(9)=3.333, p=0.0088). ALPs in Q / / '- / - -mice were also reduced by half on day 41 relative to baseline testing (day 11) (Figs. 5D, 5E, t(9)=2.529, p=0.0323), and AZD had no additional effects (Figs. 5D, 5E). ILPs were largely unaffected by abstinence, CA4 disruption, or AZD (Fig. 5F; no interaction effects), although there was reduction in ILPs following abstinence in Car4~ / ~ mice treated with AZD (Fig. 5F, t(l 9)=3.591, p=0.0058). Together, these data suggest that after 30 days of abstinence, AZD and CA4 disruption both suppressed oxycodone-seeking behavior and that effects of AZD depended on CA4.

[0173] We next harvested brain tissue following SA on days 42 to 46 to test whether behavioral effects of AZD and CA4 might be related to oxycodone withdrawal-induced differences in glutamatergic transmission. Indeed, we found that AZD treatment in Car4+ / +mice significantly attenuated the AMPAR / NMDAR ratio in NAcC-MSNs compared to non-AZD treated Car4+ / +mice (Figs. 5G, 5H, genotype x AZD interaction, F(l,51)=5.126, p=0.0278). Importantly, AZD did not impact the AMPAR / NMDAR ratio in Car 4- / - mice, and AMPAR / NMDAR ratio was lower in vehicle-treated Car4~ / ~ mice compared to vehicle-treated Car4+ / +mice (F(l ,51)=5.126, p=0.0006). AMP AR rectification and NASPM sensitivity were not tested in this procedure. Together, these results parallel behavioral changes observed during drug-seeking (Figs. 5D, 5E) and support the possibility that behavioral effects of AZD and CA4 disruption are related to their effects on glutamatergic synapses in NAcC-MSNs.

[0174] DISCUSSION

[0175] These results provide new insights into synaptic adaptations in NAcC MSNs induced by withdrawal and abstinence from opioids, which may contribute to persistent drug seeking. These results further reveal a novel role for CA4 in opioid-induced synaptic adaptations and suggest that medications that inhibit CA4, such as AZD, may hold promise for reducing the risk of relapse in people with OLD.

[0176] Increases in CP-AMPARs likely contribute to oxycodone-induced increases in AMPAR / NMDAR ratio but other mechanisms are possible

[0177] We found that oxycodone withdrawal increased AMPAR / NMDAR ratio and increased CP-AMPARs at glutamatergic synapses in NAcC MSNs in Car4+ / + mice, assessed via rectification index and NASPM sensitivity. Additionally, CP-AMPARs have a higher conductance. Thus, it is reasonable to conclude that increases in CP-AMPARs likely underlie the increase in AMPAR / NMDAR ratio, at least in part. However, it is also possible that other factors contribute. For example, non-CP-AMPARs or NMDAR-mediated currents could changeVHPM 17023.304WO1 / UIRF 25040 following drug withdrawal. Additionally, formation of silent synapses or their unsilencing could contribute. Our method for assessing AMPAR / NMDAR provides reliable comparison between these two EPSC components but does not provide a rigorous quantification of their absolute magnitudes. Miniature EPSCs, quantification of evoked AMP AR- and NMDAR-mediated EPSC components in response to varying stimulus intensities, and assessment of silent synapses would provide important insights into relative contributions of AMPARs vs. NMDARs to the changes observed here. We previously found increases in AMP AR miniature EPSCs in NAcC MSNs following withdrawal from cocaine that were prevented by CA4 disruption, thus bolstering the interpretation that changes in AMPARs contribute to opioid effects observed here.

[0178] Temporal limits, drug-administration contingency, and neural specificity of oxycodone induced changes

[0179] We do not yet know the temporal limits of the withdrawal from oxycodone induced neuroadaptations, but changes in AMPAR / NMDAR required more than 24-hrs, emerged within 5 days of withdrawal, and persisted for more than 30 days following oxycodone SA and were thus relatively long-lasting. Previous studies in mice found similar changes in AMPAR / NMDAR ratio and / or CP-AMPARs at 7-10 days post-withdrawal from experimenter-administered cocaine. The above-described effects of oxycodone on AMPAR / NMDAR ratio occurred with both experimenter- and SA drug, suggesting that the effect depended on the drug itself rather than how it was administered. Although we did not assess CP-AMPARs after SA, we might expect a similar increase; recent studies in rats suggest that oxycodone SA increases CP AMPARs in NAcC MSNs after 15 days of abstinence. Interestingly, analogous studies with psychostimulants in rats suggest increases in CP-AMPARs following SA may follow a slower time course. However, it is also important to consider that differences between our findings of drug-induced effects in mice versus previous studies in rats might also reflect species-dependent differences, drug doses, or withdrawal times. Here, the increase in AMPAR / NMDAR ratio following withdrawal from oxycodone mapped to Dl+MSNs and not to non-Dl+MSNs, consistent with earlier views that Dl+MSNs promote reward- and drug-seeking behaviors. However, growing evidence suggests that the roles of Dl+versus D2+MSNs are not strictly dichotomous. Rather, both populations can contribute to reward- and aversion-related behaviors in a circuit- and context dependent manner and may function cooperatively within distributed motivational networks. Interestingly, oxycodone incubation in rats was recently reported to increase CP-AMPARs in both Dl+and D2+MSNs, whereas psychostimulant-induced increases in CP-AMPARs appear more selective to Dl+MSNs. Additional work will be required to determine the mechanisms that generate the Dl-selective effects observed here and to clarify their implications for drug-seeking behavior.VHPM 17023.304WO1 / UIRF 25040 Our findings that oxycodone effects generalized to other mu receptor agonists, morphine and heroin, are not surprising. More surprising is that our results with these opioids also paralleled, at least in part, those observed previously in NAcC-MSNs following abstinence from cocaine. Despite binding to different targets, opioids and cocaine share some converging molecular effects. For example, exposure to both opioids and cocaine alters dopamine signaling, which has been suggested to contribute to neuroadaptations in glutamate receptor trafficking in NAc MSNs. In contrast, we did not observe an effect of withdrawal from oxycodone on dendritic spine density, consistent with our previous observation that withdrawal from oxycodone did not alter spine density in NAcC MSNs, but instead reduced spine volume and neck diameter. Differential effects of opioids and psychostimulants on dendritic spines have been reported previously. Thus, although some synaptic consequences of opioids may be shared across drug classes, our findings suggest that adaptations in spine structure are not universal and may differ between opioids and cocaine. Here, we did not distinguish effects in Dl+versus D2+MSNs, leaving open the possibility of cell-type specific effects. Nonetheless, the functional synaptic adaptations observed here (e.g., changes in AMPAR / NMDAR ratio and CP -AMP AR accumulation) likely occurred in the absence of detectable changes in spine density.

[0180] Global CA4 disruption and systemic AZD administration reduced synaptic and behavioral responses to opioid withdrawal and abstinence

[0181] Importantly, following opioid withdrawal, behavioral changes and adaptations at NAcC MSN glutamatergic synapses were prevented by CA4 disruption. However, it is not yet clear how withdrawal from opioid produces these changes or how CA4 disruption prevents them. Prior studies suggest that prolonged withdrawal from cocaine in rats decreases mGluRl signaling and promotes CP-AMPAR trafficking to the postsynaptic membrane, presumably via in PLC-mediated IPs-dependent Ca2+release and PKC. Supporting this mechanism, pharmacological activation of mGluRl reduced synaptic CP-AMPARs in a PKC5 dependent manner. More recently, disinhibition of retinoic acid synthesis, which can result from low intracellular Ca2+, was implicated in homeostatically increasing synaptic CP-AMPARs following withdrawal from cocaine. We observed similar withdrawal from opioid induced synaptic adaptations in our study. We therefore speculate that withdrawal from opioid may likewise reduce mGluRl signaling, impairing Ca2+release, and homeostatically increasing CP-AMPARs into NAcC synapses. Within this framework, we previously showed that extracellular acidification drives robust Ca2+increases in NAc MSNs via ASIC1 A in mice via voltage-gated Ca2+channels. Thus, enhancing ASIC1 A-mediated Ca2+entry via CA4 disruption or its pharmacological inhibition with AZD may thereby oppose withdrawal-induced deficits in Ca2+signaling and CP-AMPAR trafficking.VHPM 17023.304WO1 / UIRF 25040 Another important consideration is that the global CA4 disruption employed in these experiments leaves the precise site of CA4 action in these phenomena undefined. Our previous work demonstrated that NAcC-specific CA4 disruption prevented analogous withdrawal from cocaine induced synaptic changes in NAcC MSNs and attenuated cocaine-evoked behavior, suggesting that NAcC MSNs are likely sites of CA4 action. However, the effects observed here with global deletion are also a strength because they indicate that systemically disruption or inhibition of CA4 is sufficient to blunt opioid-induced maladaptive plasticity and thus more precise interventions may be unnecessary to target CA4 therapeutically.

[0182] We previously found that disrupting or pharmacologically inhibiting CA4 increased ASIClA-mediated synaptic currents in NAcC MSNs by lowering synaptic pH buffering capacity. Additionally, because loss of ASIC1 A in NAcC MSNs increased sensitivity to cocaine and opioid-induced synaptic rearrangements, it seems reasonable to expect that the capability of CA4 disruption to enhance ASIClA-mediated synaptic currents would produce the opposite effect and reduce sensitivity to drug-evoked synaptic changes. Consistent with the expectation that effects of CA4 disruption depend on ASIC 1 A, here AZD failed to affect AMPAR / NMDAR ratio in Asiclcr'~ mice, which exhibit increased baseline AMPAR / NMDAR ratios that are reduced in response to cocaine and opioids.

[0183] It has been suggested that some opioid medications may modulate specific ASICs. For example, morphine has been reported to inhibit ASIC3 currents in rat DRG neurons.

[0184] Alternatively, oxycodone has been suggested to increase sustained ASIC3 currents in rat sensory neurons. It is unlikely that these observations could explain the present results because in mouse brain ASIC3 expression is low or absent. It is unknown if opioids affect ASIC 1 A function in brain. The synaptic effects of opioids (e.g. on AMPAR / NMDAR ratio) still occur \n Asicla and may be heightened, thus ASIC1 A does not seem to be required for those rearrangements. AZD blocks other carbonic anhydrases in addition to CA4. Thus, AZD effects might arise from effects on other CAs. The absence of AZD effects in Car4_ / ~ mice and in saline-treated Car4+ / +mice argues against substantial off-target effects. Still, because Car4^ mice did not exhibit oxycodone-evoked synaptic changes, their lack of AZD response does not by itself confirm CA4 as the target. Nevertheless, in Car4+ / +mice AZD produced the same effects as genetically disrupting CA4. Taken together with the previous finding that AZD effects on synaptic pH buffering depend on CA4, the results here support a model in which AZD reverses oxycodone-induced plasticity by inhibiting CA4. SA remains the gold standard for modeling opioid consumption and seeking in rodents. Interestingly, CA4 disruption did not affect the acquisition of oxycodone SA, nor the total number of drug infusions. Although Car4+ / +control mice did not exhibit incubation of seeking behavior after extended abstinence as recently described inVHPM 17023.304WO1 / UIRF 25040 rats, they did maintain the same amount of ALPs after 30 days of abstinence compared to baseline. However, in sharp contrast cue-induced drug-seeking in Car4^ mice fell to half of baseline levels. Thus, CA4 disruption weakened cue-induced oxycodone seeking, which was sustained in Car4+ / +mice during an extended abstinence. These results echo previous effects of CA4 disruption on drug-seeking and AMPAR / NMDAR ratio following withdrawal and abstinence from cocaine SA, suggesting that targeting CA4 may reduce seeking behavior for both drugs. Importantly, administering AZD 24 hrs prior to testing, at a dose used safely in humans, significantly reduced active lever presses in Car4+ / +mice and had no effect in Car 4^ mice. These results with AZD suggest that inhibiting CA4 acutely can elicit effects similar to chronic CA4 disruption. Because the half-life of AZD in mice is ~1 hour, the effects of AZD treatment likely lasted beyond its bioavailability.

[0185] Together, these observations strengthen the possibility that CA4 might be an effective non-opioid therapeutic target for reducing relapse in substance use disorders. Moreover, by directly modulating synaptic plasticity rather than engaging opioid receptors like current treatments, AZD may reduce the risk of misuse or dependence, providing a safer option for management of OUD. Additionally, supporting its safety profile AZD’s effects appeared to be confined to drug-induced synaptic plasticity, as AZD did not alter glutamatergic transmission in drug-naive conditions. In conclusion, these results raise the exciting possibility that AZD or similar carbonic anhydrase inhibitors, some already approved for use in humans, might be readily repurposed to reduce drug-seeking and relapse in OUD and other substance use disorders.

[0186] EXAMPLE 2

[0187] Experimental Setup:

[0188] • Treatment Timeline (Fig. 13):

[0189] o Days 1-5: Oxycodone (3 mg / kg) or saline was administered.

[0190] o 10 min after the last oxycodone injection: Groups received acetazolamide (AZD, 30 mg / kg) and saline (Sal).

[0191] o Withdrawal Periods: Data were collected at 10 and 20 days of withdrawal (lOd WD, 20d WD).

[0192] o Electrophysiology (Ephys): Measurements were taken on these withdrawal days.

[0193] Observations:

[0194] 1. 10 Days Withdrawal:VHPM 17023.304WO1 / UIRF 25040 o Oxy-Veh showed a significant increase in the AMPAR / NMDAR ratio compared to Sal-Veh (p<0.001).

[0195] o Oxy-AZD had a lower AMPAR / NMDAR ratio than Oxy-Veh (p<0.01), suggesting that AZD mitigated the synaptic changes caused by oxycodone withdrawal.

[0196] o Sal-AZD had ratios similar to Sal-Veh, indicating no significant AZD effect in saline-treated controls.

[0197] 2. 20 Days Withdrawal:

[0198] o Oxy-Veh maintained an elevated AMPAR / NMDAR ratio compared to Sal-Veh (p<0.01)

[0199] o Oxy-AZD showed a similar trend of reduced ratios compared to Oxy-Veh but less pronounced than at 10 days withdrawal.

[0200] o Sal-AZD remained comparable to Sal-Veh.

[0201] Key Findings:

[0202] • Oxycodone withdrawal induces a sustained increase in AMPAR / NMDAR ratio, indicating altered synaptic transmission.

[0203] • Acetazolamide treatment appears to counteract these changes, at 10 and 20 days of withdrawal.

[0204] This study highlights the potential of acetazolamide (AZD) as a prophylactic therapy to mitigate withdrawal-induced synaptic adaptations in opioid use disorder (OUD) (Figure 8). Withdrawal from oxycodone (3 mg / kg) was associated with a significant and sustained elevation in the AMPAR / NMDAR ratio, reflecting heightened excitatory synaptic activity, which contributes to withdrawal symptoms, and relapse vulnerability. Mice were treated with oxycodone (3 mg / kg) for 5 consecutive days, and AZD (30 mg / kg) was administered 10 minutes after the last oxycodone injection. Electrophysiological recordings were performed at 10 and 20 days of withdrawal to assess synaptic plasticity via the AMPAR / NMDAR ratio, a well-established indicator of excitatory synaptic strength. Vehicle-treated oxycodone-withdrawn animals exhibited a significant increase in the AMPAR / NMDAR ratio at both 10 and 20 days of withdrawal, indicative of persistent excitatory synaptic potentiation and altered glutamatergic signaling, which are hallmarks of withdrawal-associated neuroplasticity.

[0205] However, when AZD (30 mg / kg) was administered prophylactically — immediately after the final oxycodone dose — it effectively prevented the development of these maladaptive synaptic changes, as evidenced by a normalized AMPAR / NMDAR ratio up to 20 days of withdrawal. This early intervention disrupted the initiation of synaptic remodeling processesVHPM 17023.304WO1 / UIRF 25040 critical to withdrawal neurobiology, highlighting its potential to stabilize synaptic balance in key brain regions such as nucleus accumbens.

[0206] In certain embodiments, ass a prophylactic therapy, AZD is integrated into clinical management strategies for OUD to prevent the neurobiological changes that drive withdrawal symptoms and relapse. This approach has significant clinical implications, as it targets the root cause of withdrawal-induced neuroplasticity rather than merely alleviating symptoms, providing a novel pharmacological strategy to enhance recovery and reduce relapse risk in individuals with OUD.

[0207] Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0208] All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

[0209] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms ( / .< ., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0210] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. TheVHPM 17023.304WO1 / UIRF 25040 inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.

[0211] Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. VHPM 17023.304WO1 / UIRF 25040 WHAT IS CLAIMED IS:

1. A method of preventing synaptic and / or behavioral adaptations resulting from opioid use in a mammal in need thereof comprising administering a carbonic anhydrase (CA) inhibitor agent to the mammal.

2. The method of claim 1, wherein the CA inhibitor agent is acetazolamide (AZD), topiramate, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, and / or zonisamide.

3. The method of claim 2, wherein the CA inhibitor is AZD.

4. The method of any one of claims 1-3, wherein the opioid is capable of mu receptor agonist activity.

5. The method of claim 4, wherein the opioid is oxycodone, heroin, morphine, methadone, hydrocodone, hydromorphone, buprenorphine, fentanyl, talpentadol and / or pethidine.

6. The method of claim 5, wherein the opioid is oxycodone.

7. The method of any one of claims 1-6, wherein the AZD is administered prior to, simultaneously with, or subsequent to, the administration of the opioid.

8. The method of claim 7, wherein the AZD is administered prior to the administration of the opioid.

9. The method of claim 7, wherein the AZD is administered simultaneously with the administration of the opioid.

10. The method of claim 7, wherein the AZD is administered subsequent to the administration of the opioid.

11. The method of any one of claims 1-7, when the AZD is administered with the final dose of the opioid.VHPM 17023.304WO1 / UIRF 25040 12. The method of any one of claims 1-11, wherein the CA inhibitor is administered via a topical, oral, or IV route.

13. The method of any one of claims 1-12, wherein the mammal is a human.

14. A method of reversing opioid-induced synaptic and / or network changes resulting from a first opioid use in a mammal in need thereof, comprising administering a combination of a carbonic anhydrase (CA) inhibitor and a second opioid to the mammal.

15. The method of claim 14, wherein the CA inhibitor agent is acetazolamide (AZD), topiramate, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, and / or zonisamide.

16. The method of claim 14, wherein the CA inhibitor is AZD.

17. The method of any one of claims 14-16, wherein the second opioid is capable of mu receptor agonist activity.

18. The method of claim 17, wherein the second opioid is oxycodone, heroin, morphine, methadone, hydrocodone, hydromorphone, buprenorphine, fentanyl, talpentadol and / or pethidine.

19. The method of claim 18, wherein the second opioid is oxycodone.

20. The method of any one of claims 14-19, wherein the AZD is administered prior to or simultaneously with administration of the second opioid.

21. The method of any one of claims 14-19, wherein the AZD is administered prior to the administration of the second opioid.

22. The method of any one of claims 14-19, wherein the AZD is administered simultaneously with the administration of the second opioid.

23. The method of claim 14-19, wherein the combination of AZD and an opioid is administered at least one day after cessation of the first opioid use.VHPM 17023.304WO1 / UIRF 2504024. The method of any one of claims 14-23, wherein the first opioid and the second opioid are different.

25. The method of claim 24, wherein the first opioid is oxycodone and the second opioid is methadone.

26. The method of any one of claims 14-25, wherein the mammal is a human.

27. A method of reversing opioid-induced synaptic and / or network changes resulting from a cocaine use in a mammal in need thereofe, comprising administering a combination of a carbonic anhydrase (CA) inhibitor and an opioid to the mammal.

28. The method of claim 27, wherein the CA inhibitor agent is acetazolamide (AZD), topiramate, methazolamide, dorzolamide, brinzolamide, diclofenamide, ethoxzolamide, and / or zonisamide.

29. The method of claim 27, wherein the CA inhibitor is AZD.

30. The method of any one of claims 27-29, wherein the opioid is capable of mu receptor agonist activity.

31. The method of claim 30, wherein the opioid is oxycodone, heroin, morphine, methadone, hydrocodone, hydromorphone, buprenorphine, fentanyl, talpentadol and / or pethidine.

32. The method of claim 31, wherein the opioid is oxycodone.

33. The method of any one of claims 27-32, wherein the CA inhibitor is administered simultaneously with the administration of the opioid.

34. The method of any one of claims 27-32, wherein the combination of CA inhibitor and the opioid is administered at least one day after cessation of the cocaine.

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