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Pharmaceutical Sciences Faculty Patents Pharmaceutical Sciences
2-14-2017 Compounds Including Cox Inhibitor Moiety and Enhanced Delivery of Active Drugs Using Same Audra Stinchcomb
Kyung Bo Kim University of Kentucky, [email protected]
Ragotham Reddy Pinninti University of Kentucky, [email protected]
Priyanka Ghosh
Kalpana S. Paudel Right click to open a feedback form in a new tab to let us know how this document benefits oy u.
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Recommended Citation Stinchcomb, Audra; Kim, Kyung Bo; Pinninti, Ragotham Reddy; Ghosh, Priyanka; and Paudel, Kalpana S., "Compounds Including Cox Inhibitor Moiety and Enhanced Delivery of Active Drugs Using Same" (2017). Pharmaceutical Sciences Faculty Patents. 168. https://uknowledge.uky.edu/ps_patents/168
This Patent is brought to you for free and open access by the Pharmaceutical Sciences at UKnowledge. It has been accepted for inclusion in Pharmaceutical Sciences Faculty Patents by an authorized administrator of UKnowledge. For more information, please contact [email protected]. I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111 US009566341B 1 c12) United States Patent (10) Patent No.: US 9,566,341 Bl Stinchcomb et al. (45) Date of Patent: *Feb.14,2017
(54) COMPOUNDS INCLUDING COX INHIBITOR 2009/0291128 Al 11/2009 Stinchcomb et al. MOIETY AND ENHANCED DELIVERY OF 2010/0273895 Al 10/2010 Stinchcomb et al. 2011/0052694 Al 3/2011 Stinchcomb et al. ACTIVE DRUGS USING SAME 2011/0245288 Al 10/2011 Stinchcomb et al. 2011/0245783 Al 10/2011 Stinchcomb et al. (75) Inventors: Audra Stinchcomb, Baltimore, MD 2012/0034293 Al 2/2012 Stinchcomb et al. (US); Kyung Bo Kim, Lexington, KY (US); Ragotham Reddy Pinninti, OTHER PUBLICATIONS Lexington, KY (US); Priyanka Ghosh, Baltimore, MD (US); Kalpana S. Definition of "compound" and "composition" from the Grant & Paudel, Knoxville, TN (US) Hackh's Chemical Dictionary (1987) p. 148, McGraw-Hill, Inc.* Miranda, H.F., Prieto, J.C., Pinardi, G. (2005) Spinal synergy (73) Assignee: University of Kentucky Research between nonselective cyclooxygenase inhibitors and morphine Foundation, Lexington, KY (US) antinociception in mice. Brain Research, vol. 1049, p. 165-170.* Dhooper, Harpreet Kaur, "Opioid-Cannabinoid Codrugs With Enhanced Analgesic and Pharmacokinetic Profile" (2010). Univer ( *) Notice: Subject to any disclaimer, the term ofthis sity of Kentucky Doctoral Dissertations. Paper 98.
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0 20 30 4.0
Time (hr) Figure 10 U.S. Patent Feb.14,2017 Sheet 15 of 21 US 9,566,341 Bl
-+- naltrexone
1000 -11- diclofenac
100
10
1
0.1
0Jl1 0.001------0 2 4 10 pH
Figure 11 U.S. Patent Feb.14,2017 Sheet 16 of 21 US 9,566,341 Bl
0 ...... " ~~OH / 1 \ l ~\ /;----" '-----<. \ Cl NH \}-····./ // \\ t \;-·~c1 \::::::::::::../
b. Didofenac free acid
c. Codrug d. Codrug Hydrochloride fv1\J\l 618.17 MVv' 654.15
Figure 12 U.S. Patent Feb.14,2017 Sheet 17 of 21 US 9,566,341 Bl
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Figure 13 U.S. Patent Feb.14,2017 Sheet 18 of 21 US 9,566,341 Bl
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Figure 14B U.S. Patent Feb.14,2017 Sheet 19 of 21 US 9,566,341 Bl
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Figure 15
Figure 16 U.S. Patent Feb.14,2017 Sheet 20 of 21 US 9,566,341 Bl
~ .,.. ~I
Naltrexol flux from naltrexol base vs. cod rug
,-.... 4.00 ff. 3.50 E 0 3.00 -0 2.50 E e 2.00 -x :;:t 1,50 ; o VJO ....>< 050 z 0.00 ------) NTXolfrom NTXol from todrug naltrei Figure 18 U.S. Patent Feb.14,2017 Sheet 21 of 21 US 9,566,341 Bl ...... '''''''"""'' ,,,,'''''''"'''''' ...... I I ' I 11 l 1 \ C \ ~ \ ( J ...... '''''''""'''' ,,,,,,,,,,,,,,,...... •,•,•,•,',',',',','.',',',','•'''''''"'''''' "'"''""'''' ''''"''''''''''''''''"""'' ''''"''''''''''''''''""'''' 1 1 ...... t 'l "' l 1.' I< 1 ~<,'~'1 'Io I JI o I Figure 19 US 9,566,341 Bl 1 2 COMPOUNDS INCLUDING COX INHIBITOR 2003, the number of prescriptions for two buprenorphine MOIETY AND ENHANCED DELIVERY OF formulations for the treatment of opioid dependence ACTIVE DRUGS USING SAME increased from 48,000 ($5 million) to 1.9 million ($327 million) in 2007 (Mark, 2009). Unfortunately, the buprenor- RELATED APPLICATIONS 5 phine sublingual tablets have a very bitter taste and potential for "peak" plasma drug level related side effects (dizziness, This application claims priority from U.S. Provisional sedation, orthostatic hypotension), and the poor acceptance Application Ser. No. 61/517,874 filed Apr. 27, 2011, the of the drug has limited the use. entire disclosure of which is incorporated herein by this Alcohol use disorders (AUDs) are a major public health reference. 10 problem (Breese et al., 2005; Koob and Le Moal, 2005; Sinha, 2007), and alcohol abuse is one of the leading causes GOVERNMENT INTEREST of death in the United States, contributing to over 80,000 deaths annually (Mokdad et al., 2004). Alcohol consumption This invention was made with govermnent support under accounts for 9% of the disease burden in developed coun NIH R01DA13425 awarded by the National Institutes of 15 tries and is linked to more than 60 diseases including Health. The govermnent has certain rights in the invention. cancers, cardiovascular diseases, liver cirrhosis, neuropsy- chiatric disorders, injuries and fetal alcohol syndrome (Cass TECHNICAL FIELD well and Thamarangsi, 2009; Schuckit, 2009; WHO, 2004). Prescriptions for alcoholism increased from 393,000 ($30 The presently-disclosed subject matter relates to com 20 million) in 2003 to 720,000 ($78 million) in 2007. Disul pounds including a cyclooxygenase enzyme inhibitor moi firam, naltrexone, and acamprosate prescriptions dominate ety. In some embodiments, the compound includes a Cox the market (Mark, 2009). inhibitor moiety and an opioid. The compounds allow for The leading cause of preventable deaths in the United improved microneedle-enhanced delivery rates across skin, States is cigarette smoking, with over 440,000 premature as well as an increase in the pore lifetime or duration of 25 deaths occurring each year. Cigarette smoking is known to delivery. reduce appetite and increase the rate of cellular metabolism, leading to body mass stabilization or reduction. One of the INTRODUCTION main reasons for recidivism in persons trying to quit smok ing is the potential for weight gain, especially in women. According to the National Survey of Drug Use and Health 30 Regardless of the varied findings of the many studies on (NSDUH) of 2008, 7 .2 million Americans are in need of smoking cessation and weight gain, the primary recommen treatment for Substance-Related Disorders (SRDs), and a dation has been to incorporate weight loss treatment with large proportion of those need treatment for opiate pain smoking cessation. Naltrexone (NTX) may be a potential reliever and heroin addiction. Additionally, over 8.5% of the weight loss drug candidate, especially in combination with U.S. population and 75 million people worldwide meet the 35 bupropion (Greenway et al., 2009; Greenway et al., 2010; diagnostic criteria for alcohol use disorders (AUDs), with Ioannides-Demos et al., 2011); and the biochemical rela alcohol abuse being among the top three preventable public tionship between appetite and addictive craving is receiving health problems in the U.S. and the world. increasing attention (Reece, 2011). Interesting positive clini Opioid and alcohol abuse are major worldwide problems cal results have been seen when women smokers are treated connected with tremendous social and personal strife. The 40 with a combination ofNTX and nicotine patches (Epperson CDC reported in February 2011 that the abuse of and deaths et al., 2010; Roche et al., 2010; Shaw and al'Absi, 2010). from prescription opioid narcotics has reached epidemic Good clinical trial results and preclinical data have shown levels. In May 2009 the Columbia University National a reduction in amphetamine and methamphetamine use with Center on Addiction and Substance Abuse reported that NTX treatment (Grant et al., 2010; Haggkvist et al., 2009a; $468 billion dollars were spent by the Govermnent in 2005 45 Haggkvist et al., 2009b; Karila et al., 2010). Studies with on expenses related to smoking, alcohol abuse, and illegal many other drugs have shown a lack of success for meth drugs. Prevention, treatment and addiction research spend amphetamine dependence treatment, therefore NTX may be ing comprised only about 2% of that $468 billion. Taxpayers a very good option (Karila et al., 2010). would benefit if more of that budget were spent on treatment Naltrexone (NTX), an opioid antagonist, is currently options, rather than law enforcement expenses and the 50 prescribed as an oral tablet or 30-day depot injection form to healthcare cost burden that comes from patients who help maintain opioid addicts in a drug-free state. Unfortu develop cirrhosis, lung and cardiovascular disease, and a nately, postmarketing reports of serious injection site reac variety of cancers, not to mention the expense of emergency tions (cellulitis, abscess, and necrosis) have recently room visits for overdoses. required patient warning updates for the depot injection, and The latest NHSDA (National Household Survey on Drug 55 the oral dosage form is associated with many gastrointestinal Abuse) report conducted by the Substance Abuse and Men side effects. NTX is also FDA-approved for the treatment of tal Health Services Administration reported in July 2007 that alcohol dependence. Substantial clinical evidence exists for nearly one in 12 full time workers in the US have serious the benefits of NTX treatment in smoking cessation, espe enough drug/alcohol problems to require medical treatment. cially in women. Very intriguing clinical trial data has also The 2008 National Survey of Drug Use and Health (NS 60 been observed in amphetamine-dependent individuals, DUH) estimated that approximately 7.2 million Americans which is especially important as methamphetamine depen were in need of treatment for illicit drug use. Unfortunately, dence has no FDA-approved treatment options. Preclinical most of these individuals do not seek treatment. However, data also exists that suggests that NTX may benefit anabolic for those who do seek treatment, the third and fourth most androgenic steroid dependency. Treatment with NTX in the common substance abuse types are opiate pain reliever and 65 90's produced variable success rates in addicts; however, heroin, respectively. Providing recovery help for addicts current clinical NTX therapy is being optimized based on with pharmacological interventions has proven helpful. In human pharmacogenetic data. US 9,566,341 Bl 3 4 Transdermal delivery of NTX is desirable for addicts in Compounds of the presently-disclosed subject matter are order to help reduce side effects associated with oral (hepa useful for use in a therapeutically-successful delivery system totoxicity at high doses in hepatocompromised addicts) and that allows for less-frequent dosing. In some embodiments, injectable therapies, and improve compliance. Permeation the compounds can be used in an MN transdermal delivery through the skin allows the drug to directly enter the system to provide a therapeutic transdermal delivery rate of systemic circulation and avoid the first pass effect. NTX an opioid antagonist (e.g., naltrexone or naltrexol). Contem itself does not have the essential physicochemical properties plated delivery rates across the skin are improved because of that would allow a therapeutic dose of the drug to cross the optimized physicochemical properties for faster diffusion. human skin barrier. In some embodiments, the opioid from which the first Microneedle (MN)-enhanced transdermal delivery is an 10 moiety is derived is selected from: naltrexone, buprenor efficient and painless method for increasing the skin perme phine, butorphanol, codeine, cyclazocine, cyclorphan, ation of many drugs, including NTX. The major limitation oxilorphan, dihydrocodeine; dihydromorphine, ethymor of MN-enhanced delivery is that the micropore lifetime only phine, hydromorphone, levallorphan, levorphanol, nalbu allows delivery of drug for two to three days. Therapeutic phine, nalmefene, nalorphine, naloxone, naltrexol, phenazo- viability ofNTX treatment would be improved by a product 15 cine, and pholcodine. In some embodiments, the opioid that allows for less-frequent treatment. antagonist is naltrexsone or naltrexol. Accordingly, there remains a need in the art for novel In some embodiments, the COX enzyme inhibitor from compounds and methods that allow for a therapeutically which the second moiety is derived is selected from: successful drug delivery system that allows for less-frequent diclofenac, SC560, valeryl salicylate, mofezolac, administration. 20 FR122047, celecoxib, ibuprofen, and ketoprofen. In some embodiments, the COX enzyme inhibitor is diclofenac. SUMMARY In some embodiments, the opioid from which the first moiety is derived is selected from: naltrexone, buprenor The presently-disclosed subject matter meets some or all phine, butorphanol, codeine, cyclazocine, cyclorphan, of the above-identified needs, as will become evident to 25 oxilorphan, dihydrocodeine, dihydromorphine, ethymor those of ordinary skill in the art after a study of information phine, hydromorphone, levallorphan, levorphanol, nalbu provided in this document. phine, nalmefene, nalorphine, naloxone, naltrexol, phenazo This Summary describes several embodiments of the cine, and pholcodine; and the COX enzyme inhibitor from presently-disclosed subject matter, and in many cases lists which the second moiety is derived is selected from: variations and permutations of these embodiments. This 30 diclofenac, SC560, valeryl salicylate, mofezolac, Summary is merely exemplary of the numerous and varied FR122047, celecoxib, ibuprofen, and ketoprofen. embodiments. Mention of one or more representative fea In some embodiments, the opioid antagonist is naltrexone tures of a given embodiment is likewise exemplary. Such an or naltrexol and the COX enzyme inhibitor is diclofenac. embodiment can typically exist with or without the In some embodiments, the compound includes a linker feature(s) mentioned; likewise, those features can be applied 35 moiety. In some embodiments, the opioid antagonist and the to other embodiments of the presently-disclosed subject COX enzyme inhibitor are linked by a linker moiety. In matter, whether listed in this Summary or not. To avoid some embodiments, the linker moiety is cleavable. In some excessive repetition, this Summary does not list or suggest embodiments, the linker moiety is cleavable by hydrolysis all possible combinations of such features. and/or enzymatic digestion. In some embodiments, the Cyclooxygenase (COX) enzyme inhibitors, such as 40 linker moiety inparts improved aqueous solubility to the diclofenac, can prolong the lifetime of micropores of compound. microneedle (MN)-enhanced transdermal delivery systems, In some embodiments, the compound comprises a struc allowing delivery of a drug to extend beyond a few days, ture selected from a structure as set forth in FIG. 2. allowing for less-frequent patch dosing (e.g., once a week). The presently-disclosed subject matter further includes a Many of the COX inhibitors are weak acids, and opioid 45 method for treating a subject, including identifying a subject antagonists, such as naltrexone and naltrexol, and many in need of treatment for a condition selected from the group other desirable transdermal drugs are weak bases. It is consisting of pain, depression, narcotic dependence, alcohol difficult to formulate high concentrations of a weak base and dependence, amphetamine dependence, smoking depen a weak acid in the same system, due to chemical stability and dence, and anabolic-androgenic steroid dependence; and solubility issues. 50 delivering to the subject a therapeutically effective amount The presently-disclosed subject matter includes com of a compound of the presently-disclosed subject matter. pounds including a first moiety derived from an drug of In some embodiments, a method for treating a subject interest; and a second moiety derived from a cyclooxy includes identifying a subject in need of treatment for a genase (COX) enzyme inhibitor, wherein the first moiety is condition selected from the group consisting of pain, depres- linked to the second moiety to form a single chemical entity. 55 sion, narcotic dependence, alcohol dependence, amphet The compounds of the presently-disclosed subject matter amine dependence, smoking dependence, and anabolic-an improved microneedle-enhanced delivery rates across skin, drogenic steroid dependence; and delivering to the subject a as well as an increase in the pore lifetime or duration of therapeutically effective amount of a compound of the delivery. Without wishing to be bound by theory or mecha presently-disclosed subject matter, wherein following deliv- nism, the cox inhibitor component of the compound allows 60 ery to the subject, the compound is transformed into at least for pores to remain open for a longer time period, allowing two active drug molecules. In some embodiments, the for improved delivery of the first moiety/drug of interest. In transformation is by hydrolysis and/or enzymatic digestion. some embodiments, the first moiety is derived from a drug The compound can be delivered in a variety of manners. In with a phenolic hydroxyl or hydroxyl group. In some some embodiments, the compound is delivered transder- embodiment, the first moiety is derived from an opioid. In 65 mally. some embodiments, the first moiety is derived from an In some embodiments, the compound can be transder opioid antagonist. mally delivered by microneedle, wherein the compound US 9,566,341 Bl 5 6 achieves improved duration of microporation-assisted deliv microneedles that had pores that closed up did not show this ery of the drug of interest (from which the first moiety of the type of staining, so the effect means the pores are viable. compound is derived), as compared to the parent drug of This technique has been used by others to visualize micropo- interest (as administered independent from the compound res in other studies. Pores heal quickly after the patches are including first and second moieties as described herein). In 5 removed (Banks et al., 2010). some embodiments, the improvement of duration of FIG. 10 is a graph showing how plasma concentration microporation-assisted delivery of the drug of interest (e.g., changes with a change in flux rate. from a single patch) is an improvement of greater than 1, 2, FIG. 11 Solubility profile of naltrexone and diclofenac 3, 4, 5, 6, or 7 days. generated using predicted chemical property values from 10 Scifinder®. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 12 Structure of parent drugs, (a) naltrexone, (b) diclofenac (c) 3-0-ester codrug and (d) hydrochloride salt FIG. 1 is a graph showing solubility of naltrexone and form of 3-0-ester codrug. diclofenac as a function of pH; pH 5 is the ideal value for FIG. 13 Stability study profile for codrug in hairless topical formulation. 15 guinea pig plasma indicating pseudo first order kinetics. FIGS. 2A-2W are structures of exemplary embodiments (n=3). of compounds of the presently-disclosed subject matter, FIG. 14 In vitro diffusion study across full thickness where n is 1, 2, 3, or 4 in FIGS. 2C-2J, 2M, and 2N, and n Yucatan miniature pig skin of the base form of the com is 4, 5, or 6 in FIGS. 2K and 2L. pound of FIG. 2U (A) Steady state flux of naltrexone and FIG. 3 describes the specific decision making for com 20 diclofenac from the codrug at 48 hours post MN treatment. pounds of the presently-disclosed subject matter, as (n=3) (B) Skin concentration over time from the base form described in the present Examples. of the compound of FIG. 2U. Both naltrexone and FIG. 4 is a graph showing NTX flux comparison from a diclofenac concentration could be observed in the skin human study with MN and NTX patches (Wermeling et al., indicating the regeneration of the parent molecules. n=3 for 2008), the compound of FIG. 2A (codrug) salt, and an 25 12 h, 24 hand n=2 for 36 hand 48 h. optimized NTX HCl formulation with 10% propylene glycol FIG. 15 In vitro diffusion study across full thickness (PG). The human study used 60% PG, which causes a Yucatan miniature pig skin comparing the base form on the decrease in flux due to the viscosity increase (Milewski and codrug to the hydrochloride salt form. The steady state flux Stinchcomb, 2011). The optimized NTX HCl flux is a target values of naltrexone are reported from codrug base (n=3) for embodiments of the compound. 30 and codrug salt (n=4). FIG. 5 is a graph showing skin concentrations of FIG. 16 includes a graph showing flux ofnaltrexone from diclofenac after treatment with Solaraze gel in Yucatan different formulations of the compound of FIG. 2U. In vitro miniature pig skin in vitro with MN treatment and non-MN diffusion study across full thickness Yucatan miniature pig treated controls. Daily application of Solaraze was applied skin comparing the flux of naltrexone at steady state from to the skin to mimic the in vivo hairless guinea pig study 35 different donor formulations. Propylene glycol (PG) was where microneedle treated pores stayed open for 7 days used in different concentrations to optimize formulation for (FIG. 7). in vivo studies (n;;,;3) FIG. 6 includes graphs showing skin concentrations in FIG. 17 is a series of pictures of visualized hairless guinea Yucatan miniature pig skin in vitro after MN treatment and pig micropores (A) immediately following MN treatment, application of the compound of FIG. 2A (codrug). Experi- 40 (B) at day 4 following treatment with MN followed by ments were terminated and skin disposition measured at 12, application of NTX HCl gel under occlusion, and (C) 24, 36, and 48 h. Flux values correlated with the skin visualized by India ink staining wherein the HCl salt form of disposition data, as the flux of NTX from the codrug was the compound was used following MN treatment indicating twice as high as the diclofenac flux, further proving the that the pores can stay open up to a 7 day time point. higher retention of diclofenac in the skin. The flux of NTX 45 FIG. 18 is a graph showing flux of NTXol in a study was also measured over 48 h from the compound of FIG. 2A involving the compound of FIG. 2V. and its HCl salt. FIG. 19 In vitro diffusion study across full thickness FIG. 7 is a graph showing NTX plasma levels in hairless Yucatan miniature pig skin comparing the flux of naltrexone guinea pigs after application of NTX gel alone or NTX gel at steady state from naltrexone HCl, salt form of the codrug and Solaraze (diclofenac gel). A control containing the 50 and previously studied formulation for in vivo human phar hyaluronic acid in Solaraze was also tested, and an extension macokinetic studies (n>3). in micropore lifetime was not observed. No significant depletion from the NTX control gel was identified, as only DESCRIPTION OF EXEMPLARY a few percent of the total drug was delivered (Banks et al., EMBODIMENTS 2011). 55 FIG. 8 is a graph showing NTX plasma concentrations in The presently-disclosed subject matter includes com Yucatan miniature pig (n=3 trials) after topical dosing for 72 pounds including a first moiety derived from a drug of h with an NTX hydrogel and a single MN application. Time interest; and a second moiety derived from a cyclooxy points from 72 to 96 hours are after patch removal. No NTX genase (COX) enzyme inhibitor, wherein the first moiety is was detected when the same formulation (& patch area) 60 linked to the second moiety to form a single chemical entity. applied to the pig without MN treatment. Similar to human Compounds of the presently-disclosed subject matter are results, showing the need for micropore closure inhibition. sometimes referred to herein as codrugs. A codrug com Intravenous NTX was evaluated in the pigs in order to prises two different drugs within a single chemical entity. determine the clearance of NTX and NTXOL. The two drugs may be connected either directly or by means FIG. 9 is a picture of hairless guinea pig micropores 65 of a cleavable, biolabile covalent linker. Many diseases are visualized with India ink at the end of the 7 day experiment treated by a combination of therapeutic agents that are with the compound of FIG. 2A (codrug). Skin treated with co-administered in separate dosage forms. However; there US 9,566,341 Bl 7 8 are potential advantages in delivering the co-administered In some embodiments, the opioid antagonist and the COX agents as a single chemical entity. One advantage is that enzyme inhibitor are joined by a linker moiety or more than often, when the two drugs are chemically linked together in one linker moiety. The linker moiety(ies) can be a cleavable the codrug structure, the resulting physicochemical and linker moiety(ies) such as ester, thioester, carbonate, car pharmacokinetic properties of the codrug are superior to bamate, thiocarbamate, amide, thioamide, ureide, or any those of the individual parent drugs. other suitable chemical moieties, providing that the chem The term "derived from" indicates that the first and istry is feasible. It will be recognized by the skilled artisan second moieties of the compounds have a structure of a that desirable linker moieties can be bioconvertible or bio parent drug of interest and COX inhibitor that have been labile. In some embodiments the linker moiety(ies) is cleav- 10 able via hydrolysis or enzymatic digestion. modified as is necessary to be connected to the remainder of Exemplary compounds of the presently-disclosed subject the compound, while maintaining some or all of the activity matter are set forth in FIGS. 2A-2W. associated with the parent compound, or while obtaining The presently disclosed subject matter also includes meth enhanced activity relative to the parent compound. For ods of treating a subject for conditions including pain, example, a moiety derived from a drug of interest and COX 15 depression, and substance dependence, including narcotic inhibitor can have the structure of the drug of interest and dependence, alcohol dependence, amphetamine depen- COX inhibitor, less a leaving group (e.g., less a hydrogen, dence, smoking dependence, and anabolic-androgenic ste less a hydroxyl, less a covalent bond, or less another leaving roid dependence. As used herein, dependence is inclusive of group) or including a connecting group, as will be apparent addiction and is inclusive of any disorder associated with to one of ordinary skill in the art. 20 dependence and/or addiction; as such, alcohol dependence, The compounds of the presently-disclosed subject matter for example, is inclusive of alcohol abuse and alcoholism. provide for improved microneedle-enhanced delivery rates In some embodiments, a method of the presently-dis- across skin, as well as an increase in the pore lifetime or closed subject matter includes identifying a subject in need duration of delivery. Without wishing to be bound by theory of treatment for a condition selected from the group con or mechanism, the cox inhibitor component of the com- 25 sisting of pain, depression, narcotic dependence, alcohol pound allows for pores to remain open for a longer time dependence, amphetamine dependence, smoking depen- period, allowing for improved delivery of the first moiety/ dence, and anabolic-androgenic steroid dependence; and drug of interest. In some embodiments, the first moiety is delivering to the subject a therapeutically effective amount derived from a drug with a phenolic hydroxyl or hydroxyl of a compound of the presently-disclosed subject matter. group. In some embodiment, the first moiety is derived from 30 Without wishing to be bound by mechanism, following an opioid. In some embodiments, the first moiety is derived delivery to the subject, the compound is transformed into at from an opioid antagonist. least two active drug molecules. Exemplary first moieties of the compounds of the pres Subjects who can benefit from the methods of the present ently-disclosed subject matter can be derived from opioids invention include, for example, mammals, such as humans, including, but not limited to naltrexone, buprenorphine, 35 particularly humans who are suffering from pain, depres butorphanol, codeine, cyclazocine, cyclorphan, oxilorphan, sion, and/or substance dependence. dihydrocodeine, dihydromorphine, ethymorphine, hydro "Treatment" or "treating," as used herein, refers to com- morphone, levallorphan, levorphanol, nalbuphine, nalme plete elimination as well as to any clinically or quantitatively fene, nalorphine, naloxone, naltrexol (including 6-beta nal measurable reduction in condition for which the subject is trexol and 6-alpha naltrexol), phenazocine, and pholcodine, 40 being treated. The methods of the presently-disclosed sub and other opioids known to those skilled in the art. ject matter include delivering a therapeutically effective Exemplary second moieties of the compounds of the amount of the compound. A "therapeutically effective presently-disclosed subject matter can be derived from COX amount," as used herein, refers to an amount, determined by enzyme inhibitors including, but not limited to diclofenac, one skilled in the art, sufficient for treating the condition for SC560, valeryl salicylate, mofezolac, FR122047, celecoxib, 45 which the subject is being treated. ibuprofen, ketoprofen, and other COX inhibitors known to The compound can be delivered to a subject transder those skilled in the art, including non-steroidal anti-inflam mally, intravenously, orally, buccally, sublingually, by topi matory drugs (NSAIDs), COX-1 inhibitors and COX-1- cal creams, subdermally, as a sustained release depot, oph specific inhibitors, and COX-2 inhibitors and COX-2-spe thalmically, intranasally, aurally, by inhalation, rectally or cific inhibitors. 50 vaginally. In some embodiments of the compound, the opioid In some embodiments, transdermal delivery is used. In an antagonist is naltrexone or naltrexol, and the COX enzyme exemplary method for transdermal delivery of the com inhibitor is selected from diclofenac, SC560, valeryl salicy pound, the steps comprise contacting a section of skin with late, mofezolac, FR122047, celecoxib, ibuprofen, and keto the compound and biotransforming the compound into two profen. In some embodiments of the compound, the opioid 55 drugs by skin enzymes or by hydrolysis of the compound in antagonist is naltrexone or naltrexol, and the COX enzyme the skin. A transdermal patch comprising a suitable substrate inhibitor is diclofenac. In some embodiments of the com and a layer of the compound can be employed to deliver the pound, the opioid antagonist is naltrexol and the COX compound to the skin. enzyme inhibitor is diclofenac. The compound can be administered as a pharmaceutical In some embodiments, the compound has more than one 60 composition comprising pharmaceutically acceptable carri first moiety derived from an opioid antagonist and/or more ers, diluents, and/or excipients, which are vehicles com than one second moiety derived from a COX inhibitor, monly used to formulate pharmaceutical compositions for wherein each of the first moieties and each of the second animal or human administration. The carriers, diluents and/ moieties are independently selected (e.g., each of the more or excipients are not intended to have biological activity than one first moieties need not be derived from the same 65 themselves, and are selected so as not to affect the biological opioid antagonist and/or each of the more than one second activity of the compound and any other active agent(s). A moieties need not be derived from the same COX inhibitor. pharmaceutically acceptable carrier, diluent, and/or excipi- US 9,566,341 Bl 9 10 ent as used herein includes both one and more than one such 7 days. Such a bandage, pad, or other type of patch which carrier, diluent, and/or excipient. Examples include but are can be used in the practice of the method of the present not limited to distilled water, saline, physiological phos invention can take the form of an occlusive body. In practice, phate-buffered saline, Ringer's solutions, dextrose solution, the occlusive body which includes the compound is posi and Hank's solution. Depending upon the manner of intro tioned on the subject's skin under conditions effective to duction, the compound can be formulated as, for example, a transdermally deliver the compound to the subject's skin. sterile injectable formulation comprising aqueous solutions In some embodiments, the methods of the presently and/or suspensions containing the active materials in admix disclosed subject matter can make use oftransdermal deliv ture with suitable carriers, diluents, and/or excipients. ery using microneedles. The microneedle strategy suitable The concentration of compound, the formulation (i.e., a 10 for use in the presently-disclosed subject matter is not formulation that is therapeutically effective to the subject to particularly limited, but includes any microneedle strategy which it is administered) and the dose administered can be that can be employed to facilitate the transdermal delivery of readily determined by a person of ordinary skill in the art. Typically, dosages used in vitro and in animal models, such compounds described herein. As used herein, the term "microneedles" refers to a as in the experiments provided in the present application, 15 can provide useful guidance in the amounts useful for in plurality of elongated structures that are sufficiently long to vivo administration. penetrate through the stratum corneum skin layer and into The compound can be applied to the skin as a topical the epidermal/dermal layer, yet are also sufficiently short to cream, salve, ointment, gel, or other topical formulation; not activate nerve endings and cause pain. and/or by using delivery devices such as bandages, occlu- 20 Various microneedles that can be employed to facilitate sive bodies, patches, and/or the like. The area of skin to transdermal delivery of compounds of the presently-dis which the compound is applied can optionally be pre-treated closed subject matter are described in printed publications with microneedles or other microporation devices, as are and are known to those of ordinary skill in the art. Suitable known to those of ordinary skill in the art. microneedles have been fabricated from many materials, Illustratively, a composition of the compound that is 25 including silicon, metals, and polymers. applied to the skin can be formulated as a topical cream, The microneedles can be solid or hollow. If solid salve, gel, or ointment. The topical formulations can include microneedles are used, channels can be made by poking the inert diluents and carriers as well as other conventional skin with a microneedle array, followed by removal of the excipients, such as wetting agents, preservatives, and sus needles and then application of the drug. Alternatively, the pending and dispersing agents. In addition to the above, 30 solid microneedles can be inserted and left in the skin, generally non-active components, topical formulations con allowing diffusion through the gaps between the taining compound can further include other active materials, microneedles and the surrounding tissue. Solid microneedles particularly, active materials which have been identified as can also be coated with the compound prior to insertion into useful in the treatment of the condition for which the subject the skin. Alternatively, microneedles containing a hollow is being treated, for example substance addiction, and which 35 bore can be used to transport drugs through the interior of can usefully be delivered transdermally to the subject. For the needles by diffusion or pressure-driven flow. Hollow instance, such other active materials can include acampro microneedles can act as mini hypodermic injection needles. sate, disulfiram, topiramate, sertraline, rivastigmine, citalo In some embodiments, the microneedles can be used to pram, and doxepin. The topical formulation can be applied create a microneedle-treated site prior to applying the com- directly to the skin and then optionally covered (e.g., with a 40 pound to the microneedle-treated site. As noted above, the bandage of gauze) to minimize the likelihood of its being microneedles can be inserted and left in the skin. In some disturbed. Alternatively, the topical formulation can be embodiments, a microneedle array comprised of solid coated on the surface of a bandage, gauze, etc., and the microneedles can be used to increase skin permeability by bandage, gauze, etc. can then be applied to the skin of the inserting the microneedles into the skin and then removing subject such that the topical formulation is in direct contact 45 the microneedles to create a microneedle-treated site prior to with the subject's skin. applying the compound to the microneedle-treated site. Alternatively, the compound can be delivered transder Optionally, the microneedles can be repeatedly inserted and mally to the subject by formulating compound into a ban removed, at the same site. Typical microneedle arrays com dage, pad, or other type of patch which can be applied to the prise 15-200 microneedles, but can include any number of subject's skin. 50 microneedles that is desired. Illustratively, matrix-type transdermal patches, in which The microneedles can be inserted and removed to create the compound is disposed in an adhesive matrix, can be a microneedle-treated site on the subject's skin under con employed. The matrix-type transdermal patch can further ditions effective to transdermally deliver the compound include other active materials for transdermal delivery to the upon application of the compound. Such conditions can subject with the compound. Suitable adhesives for use in 55 include, for example, positioning the microneedle-treated such matrix-type transdermal patches include polyisobuty site on a portion of the subject's skin which is not covered lenes, acrylates, silicone, hydrogels, and combinations with hair, and/or shaving the hair from the selected portion thereof. Still other patches suitable for use in the practice of of the subject's skin. the present invention are known to those of ordinary skill in The details of one or more embodiments of the presently- the art. 60 disclosed subject matter are set forth in this document. In another illustrative embodiment, the bandage, pad, or Modifications to embodiments described in this document, other type of patch can be one which is capable of control and other embodiments, will be evident to those of ordinary ling the release of the compound such that transdermal skill in the art after a study of the information provided in delivery of the compound to the subject is substantially this document. The information provided in this document, uniform and sustained over a period of at least 12 hours, 65 and particularly the specific details of the described exem such as at least 24 hours, at least 48 hours, at least 3 days, plary embodiments, is provided primarily for clearness of at least 4 days, at least 5 days, at least 6 days, and/or at least understanding and no unnecessary limitations are to be US 9,566,341 Bl 11 12 understood therefrom. In case of conflict, the specification of and development for improved pharmacologic treatments this document, including definitions, will control. for drug and alcohol abuse are very important. Naltrexone While the terms used herein are believed to be well (NTX), an opioid antagonist, is currently prescribed in oral understood by one of ordinary skill in the art, definitions are tablet and injectable form to help maintain opioid addicts in set forth to facilitate explanation of the presently-disclosed a drug free state. Most recently, naltrexone has been indi subject matter. cated as an adjunct in the treatment of alcohol dependence, Unless defined otherwise, all technical and scientific as well as reported to reduce alcohol craving in certain terms used herein have the same meaning as commonly alcoholic populations. Unfortunately, postmarketing reports understood by one of ordinary skill in the art to which the of serious injection site reactions (cellulitis, abscess, and presently-disclosed subject matter belongs. Although any 10 necrosis) have recently required patient warning updates for methods, devices, and materials similar or equivalent to the 30-day NTX depot injection. Additionally, high oral those described herein can be used in the practice or testing doses that may be needed for therapeutic effect in addicts of the presently-disclosed subject matter, representative can cause hepatotoxicity. Substantial clinical evidence exists methods, devices, and materials are now described. for the benefits of NTX treatment in smoking cessation, Following long-standing patent law convention, the terms 15 "a", "an", and "the" refer to "one or more" when used in this especially in women (Epperson et al., 2010; Roche et al., application, including the claims. Thus, for example, refer 2010; Shaw and al'Absi, 2010; Toll et al., 2010). Very ence to "a cell" includes a plurality of such cells, and so intriguing clinical trial data has also been observed in forth. amphetamine dependent individuals, which is especially Unless otherwise indicated, all numbers expressing quan 20 important as methamphetamine dependence has no FDA tities of ingredients, properties such as reaction conditions, approved treatment options (Grant et al., 2010; Haggkvist et and so forth used in the specification and claims are to be al., 2009a; Haggkvist et al., 2009b; Karila et al., 2010). understood as being modified in all instances by the term Preclinical data also exists that suggests that NTX may "about". Accordingly, unless indicated to the contrary, the benefit anabolic-androgenic steroid dependent people (Ka- numerical parameters set forth in this specification and 25 nayama et al., 2010a; Kanayama et al., 2010b). Treatment claims are approximations that can vary depending upon the with NTX in the 90's and early 2000's produced variable desired properties sought to be obtained by the presently success rates in addicts; however, current clinical NTX disclosed subject matter. therapy is being optimized based on human pharmacoge As used herein, the term "about," when referring to a netic data (Barr et al., 2010; Fortin et al., 2010; Ray et al., value or to an amount of mass, weight, time, volume, 30 2010a; Ray et al., 2010b). Transdermal delivery ofnaltrex concentration or percentage is meant to encompass varia one is desirable for addicts and alcoholics in order to help tions of in some embodiments ±20%, in some embodiments reduce side effects associated with oral (bypass first-pass ±10%, in some embodiments ±5%, in some embodiments effect) and injectable therapy and improve compliance. ±1%, in some embodiments ±0.5%, and in some embodi Naltrexone itself does not have the essential physicochemi- ments ±0.1 % from the specified amount, as such variations 35 cal properties that would allow a therapeutic dose of the drug are appropriate to perform the disclosed method. to cross the human skin barrier. Microneedle-enhanced As used herein, ranges can be expressed as from "about" transdermal delivery is an efficient and painless method for one particular value, and/or to "about" another particular increasing the skin permeation of many drugs, including value. It is also understood that there are a number of values naltrexone (Prausnitz, 2004; Wermeling et al., 2008). The disclosed herein, and that each value is also herein disclosed 40 major limitation of microneedle-enhanced delivery is that as "about" that particular value in addition to the value itself. the micropore lifetime only allows delivery of drug for two For example, ifthe value "10" is disclosed, then "about 10" to three days (Banks et al., 2010; Milewski et al., 2010; is also disclosed. It is also understood that each unit between Wermeling et al., 2008). two particular units are also disclosed. For example, if 10 Diclofenac and other relevant cyclooxygenase (COX) and 15 are disclosed, then 11, 12, 13, and 14 are also 45 enzyme inhibitors can prolong the lifetime of the micropores disclosed. to seven days, allowing for once a week patch dosing, the The presently-disclosed subject matter is further illus transdermal delivery ideal goal (Banks et al., 2011 ). Many of trated by the following specific but non-limiting examples. the COX inhibitors are weak acids, and naltrexone and many The following examples may include prophetic descriptions, other desirable transdermal drugs are weak bases. It is and/or compilations of data and information that are repre 50 difficult to formulate high concentrations of a weak base and sentative of data and information gathered at various times a weak acid in the same system, due to chemical stability and during the course of development and experimentation solubility issues. The present inventors disclose herein com related to the present invention. positions (sometimes referred to herein as codrugs), which are covalently linked yet biolabile combinations of an opioid EXAMPLES 55 antagonist (e.g., naltrexone) and a COX inhibitor (e.g., diclofenac ), in order to make a therapeutically successful Example 1 once a week drug delivery system. It is contemplated that codrugs of naltrexone in combination with microneedle An objective of the exemplary studies described herein treatment will provide a 7-day therapeutic transdermal deliv- include improvement of drug therapy for opioid, smoking, 60 ery rate of naltrexone, and that these codrugs will make methamphetamine, alcohol and other relevant addiction excellent research tools for investigating quantitative struc recovery by creating transdermal dosage form with longer ture permeability relationships (QSPR) for transdermal flux intervals between dosing, e.g., at least 7-day (once-a-week and optimization of flux and micropore lifetime in combi dosing). nation with microneedle enhancement. These codrugs/mi- Alcoholism and drug addiction lead to major health 65 croneedles should improve naltrexone delivery rates across related problems and societal costs for people in the United the skin because of optimized physicochemical properties States and the rest of the world as well. Therefore, research for faster diffusion. US 9,566,341 Bl 13 14 A series compounds (FIG. 2) were designed to elucidate injection or patch treatment. In contrast to depot dosage fundamental QSPRs for microneedle-enhanced transdermal forms, transdermal systems offer a patient freedom from flux optimization and subsequent bioconversion rates. Char injections (a 380 mg NTX as 1.1 g microsphere intramus acterization of the physicochemical properties of the cod cular injection), surgical implantations, and the healthcare rugs, including molecular volume, lipophilicity, hydrogen costs associated with implant dosing. Injection site reactions bonding potentials, melting points, heats of fusion, are one of the most common Vivitrol adverse events. hydrolysis kinetics, and solubilities in select solvents is Another advantage of transdermal dosing is the ability to contemplated. The present inventors also contemplate mea terminate the drug dose input at any time by simply remov suring the drugs' penetration, concurrent bioconversion, and ing the patch. The process of terminating drug input from the COX inhibitor deposition in skin in vitro with microneedle 10 depot injection requires surgical removal of microspheres use. Also contemplated is characterization of the pharma from the muscle, which might be necessary if a patient cokinetics of the compounds in animals (e.g., guinea pigs requires opiate treatment for pain in an emergency situation and Yucatan miniature pigs in vivo, and humans) with that occurs within the 30-day window where plasma levels microneedle use. Correlation of the in vitro data with the in of naltrexone are released. A transdermal patch provides vivo model will aid in the creation of a reliable QSPR 15 sustained-release of a drug for a maximum of about one database that is useful to determine relative efficacy of week. Once-a-week dosing is certainly more desirable than compounds for clinical use. The studies will incorporate a the three to seven times a week dosing of the currently parallel pharmacodynamic (micropore closure) and drug available ReVia®. The transdermal patch delivery system delivery/pharmacokinetic approach to identify novel codrug suits the addict population well, being similar to the patch- treatments for significant clinical problems, such as opiate, 20 wearing population of nicotine addicts. A transdermal sys smoking, methamphetamine and alcohol addiction. tem that delivered a therapeutic dose of naltrexone into the As noted herein naltrexone (NTX) is an opioid antagonist body for one week would certainly be consistent with a that is currently used to help maintain opioid addicts in a weekly visit to an addict recovery clinic or probation officer. drug-free state. Most recently, naltrexone has been indicated As compliance is the major concern in nonmotivated crimi- for use in treatment of other substance dependencies, e.g., 25 nal addicts, use of an adhesive that sticks to the skin only alcohol dependence, including use to reduce alcohol craving once could be a helpful clue for a probation officer looking in certain alcoholic populations (Garbutt, 201 O; Gue for the noncompliant addict. This could deter an addict from orguieva et al., 2010; Martinotti et al., 2010; Morley et al., removing his/her patch during the week, knowing that 2010; O'Malley, 1996; O'Malley et al., 1992; Soyka and punishment for showing up with a non-stick patch would be Rosner, 2010; Volpicelli et al., 1992; Zarkin et al., 2010). 30 likely. Pharmacogenetic analysis has been shown to improve NTX A very promising use of a transdermal NTX system is as treatment outcomes in clinical trials for alcoholism (Barr et an adjunct in the treatment of alcohol dependence. Trans al., 2010; Fortin et al., 2010; Kranzler and Edenberg, 2010; dermal delivery of the potential hepatotoxin naltrexone will Kranzler et al., 2000; Ray et al., 2010a; Ray et al., 2010b). bypass first-pass liver metabolism and also decrease gastro- NTX Hydrochloride is currently commercially available in 35 intestinal side effects. A relatively motivated alcoholic the United States as a 50-mg oral tablet (ReVia®), as well would certainly want the ease of use and reduction of side as in generic form. Naltrexone undergoes extensive first effects possible with transdermal delivery. In fact, compli pass metabolism and has oral bioavailability estimates rang ance has been a critical issue in the response of alcoholics to ing from five to forty percent (PDR). Naltrexone is also a naltrexone therapy in recent clinical trials (Volpicelli et al., hepatotoxin that has the capacity to cause dose-related 40 1997). Alcohol use has been shown to decrease by about hepatocellular injury. This hepatotoxicity limits dosage one-half in adult alcoholics taking naltrexone (Pettinati et increases in those addicts who may benefit from more than al., 2010; Volpicelli et al., 1997). Additionally, there is one a 50-100 mg per day oral dose. Transdermal delivery would case report of an adolescent male that describes 30-day circumvent these liver-related problems by allowing lower abstinence during naltrexone alcohol-dependence treatment. doses to be used, because first-pass metabolism is bypassed. 45 The prevalence rate of adolescent alcohol dependence Certainly, the ability to decrease the drug dosage would appears to be about four to ten percent, according to some benefit the already hepato-compromised alcoholic or nar self-reported epidemiological studies. Thus, transdermal cotic addict. Many of the adverse effects seen with Re Via® delivery of naltrexone may also prove useful in the adoles- oral therapy (abdominal pain, constipation, nausea, and cent alcohol-dependent population, as well as in adults. vomiting) could also be reduced by transdermal therapy. 50 Naltrexone itself does not have the essential physico- Compliance, the major problem with addict recovery phar chemical properties that would allow a therapeutic dose of macological intervention, is likely improved when side the drug to cross the human skin barrier. Prodrugs contain effects are reduced and drug doses are adequate to reduce covalently bound chemical moieties added to a drug to make craving (O'Malley, 1996; O'Malley et al., 1992). One goal it more permeable, but these prodrugs are rapidly biocon- of this research is to enhance the therapeutic value of 55 verted back to the active parent compound after dosing or naltrexone by reducing side effects and improving addict absorption. A codrug contains two active drug molecules compliance with transdermal delivery that allows for dosing covalently linked together to improve the delivery or other of at least about 7 days. Several once-a-week transdermal properties of one or both drugs (Kiptoo et al., 2006; Kiptoo patches are on the market and are very popular. et al., 2008; Strasinger et al., 2008). In this case, the acid The major problems with addict naltrexone use are moti 60 functionality is being removed (temporarily masked) from vation and compliance, which have prompted the develop one of the drugs by forming a codrug so that the weak acid ment of 30-day depot dosage forms (Garbutt et al., 2005; COX inhibitor and the weak base NTX can be combined at Kranzler et al., 2000). The depot dosage form (Vivitrol, high concentrations in one dosage form without stability or approved by the FDA in the summer of 2006) has been solubility incompatibility issues (FIG. 1). The naltrexone shown to be effective in clinical trials with opiate addicts and 65 delivery rate will be enhanced with codrugs designed to alcoholics, which bodes well for the patch, as the plasma optimize microneedle-assisted delivery for NTX flux as well drug concentration profiles would be similar after depot as micropore lifetime. US 9,566,341 Bl 15 16 Verification of codrug NTX delivery enhancement with an simple calculated and experimentally measured parameters applicable in vitro/in vivo transdermal delivery correlation could save the pharmaceutical chemist valuable time and will help identify the most promising codrug for eventual effort. human use. This validation of in vitro/in vivo correlation With skillful formulation and addition of skin penetration will be especially important in microneedle-assisted deliv- 5 enhancers, some borderline delivery candidates can be made ery, where optimal and efficient testing methods are just into transdermally useful therapeutic agents. Penetration being developed for this new and rapidly growing area of enhancers are chemicals that alter the structure of the drug delivery. Increased understanding of the quantitative stratum corneum temporarily in order to open up a pathway structure-permeability relationships (QSPRs) for transder for faster diffusion of drugs through skin. A major disad mal codrug flux and concurrent bioconversion in vivo will 10 vantage to penetration enhancer use in transdermal delivery be attained by statistically comparing calculated codrug systems is the potential for skin irritation and allergic physicochemical parameters, experimentally-derived physi reaction. Skin irritation is a very common reason why many cochemical parameters, and diffusion study data. This way topical and transdermal dosage forms do not make it to the the data gathered here can be applied to the design of other market. A major advantage of using transdermal prodrugs/ transdermal codrugs for use with microneedle-assisted 15 codrugs with prompt bio-activation is that their skin irrita delivery. tion and allergenic potential should only mirror the profile of Skin Permeation: The skin consists of two major layers, the parent drug(s), without the possible added toxicities of the outer epidermis and the inner dermis. The stratum penetration enhancers. corneum, the outermost 10-15 µm of the epidermis, is Successful delivery of NTX has been shown in humans responsible for the skin's excellent diffusional resistance to 20 using microneedle enhancement for two to three days (Wer the transdermal delivery of drugs. Most of the skin's enzy meling et al., 2008). Use of microneedles with optimized matic activity lies in the basal cell layer of the viable codrugs will enable naltrexone delivery rates to increase epidermis. Many researchers (Potts et al., 1997; Potts and multiple-fold over passive delivery patches, without irritat Guy, 1992; Roberts and Sloan, 1999) have developed skin ing enhancers, and with the added advantage of improving permeability relationships based on the physicochemical 25 the delivery duration from one patch to 7 days, the optimal parameters (molecular weight, molecular volume, lipophi transdermal dosing goal. licity, hydrogen-bonding potentials, polarity, etc.) of skin Therapeutic Goal: From the standpoint of making NTX penetrants. In the proposed project, these useful permeabil- transdermally useful, the present inventors contemplated a ity models will be correlated with transdermal codrug dif need for a temporary form of NTX with optimized proper 2 fusion, while taking into account complications of concur- 30 ties to substantially increase its flux (from 3 nmol/cm /hr to rent bioconversion of the codrug. about 12-90 nmol/cm2/hr). The most recent depot injection Transdermal Prodrugs and Codrugs (Mutual Prodrugs): clinical trial for alcohol dependence indicates that the Studies performed by R. J. Scheuplein et al. in the 1960's required flux would be 34-68 nmol/cm2 /hr from a 20 cm2 spurred pharmaceutical scientists to make use of new infor patch (Garbutt et al., 2005). If the patch size is doubled, the mation concerning passive permeability and metabolism 35 required flux rate would be reduced by one half. With properties of the skin. Since transport is a highly restricting microneedle treatment alone, the present inventors found factor to successful topical use of drugs and since it is the that NTX could be delivered at the lower end of the physical properties of a drug that largely determine the upper therapeutic range for opiate receptor blockade in humans, 2 limit to transport, attention was soon focused on manipu ng/mL (Verebey et al., 1976). lating the physical properties of drugs to improve delivery. 40 The main therapeutic goal is to achieve a substantial Models for evaluating transdermal prodrug delivery began increase in the flux of the codrug across the skin to reach to appear in the pharmaceutical literature in the late 1970's effective levels in the body. This codrug would also supply (Yu et al., 1979). Over the last thirty years some other an equimolar dose exposure to diclofenac (or other future scientists have described possible transdermal prodrug COX inhibitors of choice). The minimum daily effective delivery for drugs such as fluorouracil, naproxen, acyclovir, 45 systemic therapeutic dose of diclofenac is 75 mgx0.55 oral indomethacin, morphine, estradiol, and theophylline. A few bioavailability (Micromedex 1.0), or 41 mg per day. This people have written comprehensive reviews about transder translates to a required systemic delivery rate of 270 nmol/ mal prodrug delivery, but this usually appears as a minor cm2/hr from a 20 cm2 patch, which is several-fold higher subject in broadly based articles on prodrugs. than the required systemic NTX flux. One transdermal delivery review incorporated codrugs 50 Therefore, since the required NTX flux is the target for and prodrugs (Strasinger et al., 2008). Prodrugs specifically systemic therapy, the diclofenac absorbed would be sub to be used with microneedle-enhanced transdermal delivery therapeutic because its use in the codrug is only to deposit are also not readily available in the literature yet (Milewski into the skin and maintain the pore lifetime through a et al., 201 Oa; Milewski et al., 201 Ob). After three decades of localized effect. As diclofenac is a potent COX inhibitor, transdermal prodrug/codrug delivery study, the field still 55 many other COX inhibitors could be used that would have seems relatively young with relatively small amounts of a local effect and very little systemic effect. literature published in the area, and no products on the 6-~-Naltrexol (NTXOL) is the major metabolite formed market. Synthesizing specific codrugs with varying physi by rapid reduction ofNTX in humans (Cone et al., 197 4) and cochemical properties and systematically studying how the in several other animal species, including guinea pigs transport and bioconversion of these codrugs are affected by 60 (Valiveti et al. 2004). NTXOL may contribute to the thera these physicochemical properties will give us a greater peutic action ofNTX in drug and alcohol dependence due to database of transdermal codrug knowledge. Publication of its significant plasma concentrations (Ferrari et al., 1998; the results of these carefully controlled in vitro and in vivo Meyer et al., 1984; Wall et al., 1981) and longer terminal diffusion experiments with corresponding statistical rela half life (McCaul et al., 2000a; McCaul et al., 2000b; tionships to physicochemical codrug properties (QSPR) will 65 Rukstalis et al., 2000; Wall et al., 1981) in humans compared greatly add to the prodrug/codrug literature. The ability to to NTX. The NTXOL half-life ranged from 7.5 to 13 hours choose the design of a successful prodrug/codrug based on compared to 2-10 hours for NTX in these human subjects. US 9,566,341 Bl 17 18 Additionally, NTXOL exerted a 4-9 times longer duration of codrugs have been proposed for use in connection with action than NTX in different animal model experiments microneedle systems heretofore. In order to obtain the involving rats and mice. NTXOL is also an opioid antagonist fastest drug delivery rates possible, optimization of codrug with a similar affinity for the opioid receptors but with a solubility, hydrogen bonding, and bioconversion rates will lower potency than NTX in in vivo studies. However, the need to be completed. lower potency can be expected to be compensated by the In order to increase understanding of the QSPRs for presence of a much higher plasma concentration, suggesting transdermal codrug flux and concurrent bioconversion, one that NTXOL might contribute a major role in the efficacy of should start with the basics. The original 3-alkyl-ester, NTX. carbonate, and carbamate prodrug series for passive trans- Recent studies have provided evidence that NTXOL may 10 dermal permeation served as the first building block in the be of future relevance with respect to treatment of alcoholics design of better prodrugs (Pillai et al., 2004; Stinchcomb et as well. A preclinical study by Stromberg et al. showed that al., 2002; Vaddi et al., 2009; Vaddi et al., 2005). This newly when given alone, NTXOL reduced alcohol consumption in proposed microneedle-enhancement project provides a vali rats in a dose-dependent manner. This was also confirmed in dated methodology of studying the effects of a simple and a second study (McCaul et al., 2000a; McCaul et al., 2000b ). 15 relatively stable skin esterase enzyme system on di-conju In addition, serum concentrations of NTXOL have been gation of the NTX molecule, to determine if significant shown to be associated with reductions in number of drinks enhancement is achieved over and above the enhancement per month (O'Malley et al., 1992) and with potentially seen with the mono-conjugated codrugs of NTX. The pre important negative subjective effects of alcohol in humans cise balance among lipophilicity, hydrophilicity, and meta- (McCaul et al., 2000a; McCaul et al., 2000b ). In a random 20 bolic bioconversion needs to be found for these codrugs, in ized, double-blind clinical trial of NTX, subjects with a order to maximize NTX flux rates across the skin and NTXOL level of more than 40 ng/ml did not experience an optimize COX inhibitor deposition into the skin simultane alcohol relapse (O'Malley et al. 1992), indicating that the ously. Different polar and/or hydrophilic codrugs with vary relapse rate might be lower in patients treated with NTXOL. ing properties will be especially suitable for the microneedle In addition, since NTXOL is mainly excreted through the 25 flux optimization (Banks et al., 2010; Banks et al., 2008), as renal route, it would be a safer alternative than NTX in diverse prodrug forms are known to significantly change hepato-compromised patients. All this indicates that absorption rates of injectable drugs, if one considers NTXOL would be a useful therapy in drug addiction and microneedle delivery to be a micro-injection. alcoholism. Thus, the present inventors contemplate ana There are currently no transdermal prodrug, codrug (mu logues of NTXOL for transdermal delivery, and consider- 30 tual prodrug), or microneedle products on the market. The ation of the present inventors' codrug designs with NTXOL, present inventors contemplate the following innovative fea to see if significant flux enhancement for this promising drug tures in connection with embodiments of the presently can be achieved (Paudel et al., 2005). disclosed subject matter. Microneedle Enhancement: Microneedle delivery is in its Microneedles--carefully controlled models for transder infancy stages of development for transdermal drug deliv- 35 mal delivery candidate selection, formulation, and ery, but the microelectromechanical systems (MEMS) class micropore lifetime optimization will be evaluated using the that microneedles stemmed from is a multibillion dollar "poke and patch" technique for systemic drug delivery. Most market (McAllister et al., 2003). While microneedle inser prior research in microneedle-assisted transdermal delivery tion for transdermal drug delivery is new, it has shown has been for vaccines, biodegradable microneedles or coat promising results that show increased drug permeation; it is 40 ings for very potent drugs, or in optimization of needle also a relatively painless drug delivery procedure (Kaushik engineering. Currently, the present inventors' human et al., 2001). There are currently many other devices on the microneedle NTX pharmacokinetic study is the only pub market, such as the ImplaJect™, Luer-Jet™, Iject® and lished report in this area using the "poke and patch" tech Mini-Ject™, to deliver therapeutics systemically by needle nique (Prausnitz and Langer, 2008; Wermeling et al., 2008). free injection. The overriding problems associated with 45 There is also an ongoing clinical trial for microneedle these models are that they are costly to produce, require assisted delivery of parathyroid hormone and a human proof medical supervision for application, and importantly are not of concept study for insulin delivery using hollow pain-free (Furness). microneedles (Prausnitz and Langer, 2008). The microneedle would, on the other hand, cost much less Transdermal codrugs--carefully controlled design, syn to produce and purchase, require no office visit for applica- 50 thesis and optimization of codrug structures based on chemi tion, and would be pain-free. Initial studies with cal stability, enzymatic stability, and solubility. Few microneedles have shown significant increases in the per examples exist in the literature concerning optimization and meation oflarge molecules such as insulin (McAllister et al., utility of transdermal codrugs (Cynkowski et al., 2008; 2003). The design of the microneedle is based on the Kiptoo et al., 2006; Kiptoo et al., 2008; Strasinger et al., composition of skin, because it must pass through the 55 2008). stratum corneum (10-15 µm) and into viable epidermis or Micropore lifetime-carefully optimized pharmacoki dermal layers. In theory, the microneedle must only be long netic evaluation of micropore lifetime in order to further enough to pass through the first 15 µm of skin, but most evaluate the effect of diclofenac and potentially other COX microneedles are significantly longer. It is pertinent that a inhibitors. Previous studies on micropore lifetime have used microneedle works to produce a significant aqueous channel 60 many other visualization evaluation techniques (Kalluri and through the epidermis so that continual drug delivery Banga, 2009), but only a few have examined pharmacoki through this channel can occur before reversal (channel netic analysis (Banks et al., 201 O; Banks et al., 2011 ). closing) is observed. Pharmacokinetic evaluation of micropore lifetime is the Examples of microneedle systems include BioValve's most useful and realistic applied method of evaluation. Micro-Trans™ microneedle array patch and 3M's Micro- 65 Drugs that are targets for microneedle-assisted delivery structured Transdermal Systems (MTS). Several could impede or enhance the micropore closure rate, microneedle systems are in late-stage clinical trials. No depending on their mechanisms of action. Additionally, very US 9,566,341 Bl 19 20 few reports occur in the literature describing ways to delay microneedle enhancement, as compounds as large as insulin the micropore closure, so this in itself is an extremely unique have been delivered through micropores (McAllister et al., aspect of this project (Banks et al., 2011; Milewski et al., 2003). However, the present inventors have learned that an 2010a). increase in viscosity, roughly correlated with the molecular Transdermal codrugs specifically for microneedle-as weight of a compound, can decrease the diffusion rate sisted delivery-the overall goal of the project is something through the microneedle pores (Milewski et al., 2010a; that has never been done before. The present inventors' Milewski and Stinchcomb, 201 O; Milewski and Stinchcomb, research has shown some of the important aspects of what 2011; Milewski et al., 201 Ob), therefore polyethylene glycol type of prodrug/drug is best for microneedle-assisted trans (PEG, 11, 12) units will not be increased to over six. Codrug dermal delivery (Banks et al., 2010; Banks et al., 2008; 10 design can be limited to smaller molecular weights, if the Milewski et al., 201 Oa; Milewski and Stinchcomb, 201 O; solubility increase does not overshadow the molecular Milewski and Stinchcomb, 2011; Milewski et al., 201 Ob), weight dependency with microneedle delivery. but the addition of the codrug that can have optimized delivery rates as well as duration is very new in drug The addition of the synthesis of the di-conjugated codrugs and their comparison with the successful mono-conjugated delivery research. 15 Development of an improved 7-day transdermal system codrug moieties is novel. Ester, carbamate, and carbonate for NTX-this would improve patient compliance and the ester linked prodrugs are successful prodrugs, so these side-effect profile, as the depot injection and tablet have linkages have been maintained. All of the codrug structures many drawbacks. The translational goal of the research is to will be optimized to solubilize and permeate through the develop an improved dosage form for naltrexone delivery 20 aqueous channels created by microneedle delivery. These for addicts and alcoholics. After the proof-of-concept microneedle-assisted codrugs will therefore be very hydro- research is complete, preclinical toxicology tests could be philic, but it is certain that a relatively fast bioconversion completed, an IND filed, and a Phase I clinical trial could rate will still be an important property of these codrugs so begin at the end of three years. By this time, other that the diclofenac can be deposited into the skin. A more microneedle systems should be on the market, so the pri- 25 hydrophilic NTX codrug should more readily enter the mary regulatory hurdle would be the safety and efficacy of microneedle-created pore, and then diffuse to the capillaries the codrug itself. Seven day transdermal patches on the for systemic circulation uptake. More hydrophilic com market are very popular products. pounds should be taken up by the circulation more readily Exemplary Compounds. than lipophilic compounds, as can be seen in the sustained Synthesis and testing of the compounds of FIGS. 2A and 30 release injectables body of literature, where changes in 2B is now described, and contemplated with regard to other absorption rate correlate with lipophilicity and bioconver- compounds in FIG. 2. The detailed synthetic methods are in sion. For example, furanose sugar (17), L-malic acid (18, the Synthetic Schemes provided hereinbelow. These two 20), and phosphate acetic acid ester (15, 16, 19) promoieties compounds have an ester linking naltrexone or naltrexol to will be studied to determine the flux enhancement with diclofenac. The chemical stability half-life of Compound 2A 35 in buffer (skin pH 5.0 and skin surface temperature 32° C.) microneedles. was around 4.3 days and the enzymatic stability half-life in If the phosphate acetic acid ester is too chemically the hairless guinea pig plasma was around 6 minutes, unstable, a regular phosphate ester group can be substituted, indicating that any intact codrug that reached systemic and sodium salts of these could also be made. Salts of all of circulation would regenerate the parent drugs as soon as it 40 the compounds are contemplated, as the present inventors enters the body. have found that this is the best form to afford an increase in In both stability studies, regeneration of the parent com polarity and aqueous solubility (Banks et al., 2008). The pounds was observed with the degradation of the codrug. preferred initial salt form is hydrochloride (Scheme 1), but Compound 2A bioconverted completely in the skin to form lactate and glycolate salts would also be good candidates, for only the parent compounds in the receiver and plasma in the 45 example. Lactate and glyco late salts are common in topical in vitro diffusion and in vivo studies, respectively. Com preparations at high concentrations. Acetate salts would not pound 2A made a very good proof-of-concept drug to test in be practical because of potential odor. The acetate, lactate, vitro and in vivo in order to see if sufficient diclofenac could and glyco late salts have higher solubilities in the pH range be deposited into the skin for therapeutic micropore closure of interest for topical application, pH 4-6. Higher flux has delay, and also to see if it cleaved in the skin to the parent 50 been seen from NTX glycolate than NTX HCl through compounds. Compound 2B was synthesized next, as the microneedle-treated skin in vitro, because one can achieve a ester linkage to NTXOL at the 6-0 position has been found higher solubility with glyco late. There is a maximum effect to be more chemically stable than linkage at the 3-0 position of solubility on flux when the viscosity outweighs the of the phenolic ring. This is another reason to use NTXOL, solubility effect (Milewski and Stinchcomb, 2011). because the 6-0 position in NTX is a ketone and not a 55 The goal is to categorize how the physicochemical prop hydroxyl that can be easily made into a prodrug. However, erties of these codrug moieties when conjugated at the C-3 other structures have been proposed to help improve the phenolic hydroxyl group or at the C-6 (naltrexol) influence chemical stability of the 3-0 phenolic hydroxyl prodrug stability and flux measurements, and then to evaluate any linkage, e.g. Compound 2C with the addition of carbons in trends that may become evident from physicochemical prop between to help slow the hydrolysis, and Compound 2G and 60 erties and flux enhancement. Otherwise, physicochemical Compound 21 with carbamate and carbonate linkages. properties are altered only by the codrug link in between the Experimental Design The detailed synthetic are set forth two drugs via pegylation (11, 12), or by changing the hereinbelow. All the reactions utilize established reagents chemical and enzymatic stability with ester, carbonate, car and protecting chemistries. Hydrogen bonding capacity will bamate (1-10), and anhydride (13, 14) groups. The addition be especially important in the design of the codrugs to be 65 of one to four carbon atoms (3-10, 13,14) in between the used with the aqueous pores formed by the microneedles. ester, carbonate, carbamate, and anhydride linkages is pro- Molecular size can be somewhat increased in the case of the posed to help improve chemical stability if necessary. Other US 9,566,341 Bl 21 22 design permutations, besides the 20+ proposed basic struc can also be determined in skin homogenates, to ascertain if tures, are possible once data is gathered and optimization of the values are useful in the QSPR multiple regression flux and stability begins. analysis. The kinetics of codrug enzymatic degradation in A goal is to optimize solubility characteristics, chemical skin homogenates will be done using abdominal plastic stability, and bioconversion rates, the most critical prodrug surgery samples. Enzymatic reaction rates will be deter parameters identified. With these physicochemical attributes mined at several different codrug concentrations. The skin is maximized, a therapeutic transdermal drug delivery rate homogenized (Polytron tissue homogenizer and ground may be achieved (FIG. 4). The maximum metabolic trans glass homogenizer fitted with a glass pestle) in HEPES formation rate may be limited at a Michaelis-Menten maxi buffered Hanks' balanced salt solution and the supernatant is mum rate, or Vmax, providing the enzymatically labile 10 obtained. The enzymatic hydrolysis rates are determined in codrug is chemically stable enough to cross the micropores the homogenate at 37° C. The codrug and its metabolites intact. Altering the linkages in the codrugs for polarity and will be obtained by solid-phase extraction, or another appro bioconversion should significantly enhance the transdermal priate extraction method. Naltrexone and any other metabo drug delivery rate. The general codrug design and synthesis lite formed will be assayed by HPLC and expressed as moles methodology will allow for this flexibility. Many codrugs 15 of metabolite per minute per milligram of protein. Protein are proposed, but data collection will drive the decisions for concentrations in the supernatant will be measured by the which groups of compounds that will/will not be synthesized Bradford method. Controls containing only codrug and and tested, similar to what we have done with the currently HEPES-buffered Hanks' balanced salt solution at 37° C. will funded proposal. In four years a maximum of 100 com also be monitored to determine non-enzymatic hydrolysis. pounds could be synthesized. This number of compounds is 20 The kinetics of codrug enzymatic degradation in skin well within the capabilities of our synthetic chemistry exper homogenates will also be carried out using freshly der tise, as we have already demonstrated in our currently matomed guinea pig skin samples. Immediately after the funded studies. animals are euthanized, at the completion of their pharma Calculated codrug physicochemical parameters will cokinetic studies, untreated dorsal skin will be harvested for include: molecular weight and volume (Bondi), octanol/ 25 homogenate metabolism and in vitro diffusion studies. water partition coefficients (Daylight® 4.51 Software), These metabolism studies will help to quantify any signifi hydrogen-bonding potentials, relative thermodynamic activ- cant differences in the codrug enzymatic reaction rates ity and solubility parameters (Stinchcomb et al., 1995). A between the guinea pig skin and human skin. Additionally, wide range of partition coefficients will provide a sufficient codrugs may metabolize to other compounds, besides just group of data for QSPR analysis. With every carbon addition 30 the parent drugs. The same procedure can also be done for to the alkyl chain, the clogP value usually increases by about pig skin. 0.5. Generally, the greater a drug's innate tendencies to Cox inhibitors other than diclofenac are contemplated. In dissolve, the more likely it is that the drug can be delivered some cases, a COXl-specific inhibitor may be better, in at an adequate rate across any membrane, including the skin. which case SC560, valeryl salicylate, mofezolac, FR122047 Therefore, experimentally determined prodrug physico 35 or another COXl-specific inhibitor can be used. In some chemical parameters will include: octanol/water partition cases, a COX2-specific inhibitor may be better, in which coefficients, melting points and heats of fusion (important case, celecoxib, for example, can be used. In some cases, a physicochemical determinants of solubility), and solubilities less potent nonspecific COX inhibitor could be used, in in select solvents at 32° C. (buffer and other vehicles used which case ibuprofen, ketoprofen, or others are available. in the permeation experiments). Skin surface temperature is 40 Such COX inhibitors have carboxylic acid moieties, and approximately 32° C. Difficulties sometimes arise in deter chemistry for synthesis does not change substantially. Cele mining the octanol/water partition coefficients of readily coxib is an exception with an amine functional group, so hydrolyzed esters or extremely hydrophobic drugs. Experi different chemistry would be needed (Schemes), as would be mentally determined octanol/water partition coefficients for recognized by one of ordinary skill in the art. some of the codrugs may not be practical, therefore relative 45 Drug penetration, concurrent bioconversion, and COX HPLC retention times could substitute for partitioning esti- inhibitor deposition in skin in vitro with microneedle use. mation. Compound 2A was tested in the in vitro diffusion study Solubility studies are carried out by equilibrating large using Yucatan miniature pig skin with microneedle treat excesses of each drug with the solvent studied. The experi ment. A study was conducted with Compound 2A, and a ments will be done at skin surface temperature (32° C.). To 50 study was conducted with its HCl salt. FIG. 6 shows the skin hasten the attainment of equilibrium, each slurry will be concentrations obtained at different time points. NTX and continuously shaken or stirred. Samples will be taken, diclofenac build to steady-state concentrations as the codrug filtered with an appropriate filter for the solvent, measured hydrolyzes. More diclofenac is retained in the skin because with respect to volume, and either assayed directly by HPLC it is more hydrophobic than NTX, and therefore diclofenac or evaporated to dryness and reconstituted for assay. Binding 55 skin levels are higher than NTX as NTX diffuses into the of the compounds to the filter will be checked to eliminate receiver compartment. the possibility of drug adsorption on the filter and/or filter FIG. 5 shows a diclofenac effective skin concentration casing. All toxic solvents and other chemicals will be goal, and Compound 2A did not provide that level, however, handled with caution, and fume hoods will be used when the codrug was successful in the hairless guinea pig phar- appropriate. 60 macokinetic study at keeping open the pores for 7 days, so The chemical stability of the prodrugs to hydrolysis and it was most likely that more Solaraze (diclofenac) was being solvolysis at the appropriate temperatures will be deter applied than necessary in the initial guinea pig study (FIG. mined by measuring concentrations over time in the buffers 7). Therefore, a diclofenac skin concentration goal of 0.1 and solvents used in the solubility and diffusion studies. µmol/g of tissue may be all that is needed for effect. At any Chemical degradation rate constants will be important in 65 rate, the codrug is working as designed, to allow flux of distinguishing enzymatic degradation from non-enzymatic NTX and retain diclofenac to have an effect on the degradation. Codrug enzymatic metabolism rate constants micropore closure rate. The present inventors are testing the US 9,566,341 Bl 23 24 effect of Solaraze ( diclofenac) on the micropore closure rate voir ofNTX gel on top. NTX plasma profiles after one-time in human volunteers, and have seen similar results. application of MN's in hairless guinea pigs with one-time FIG. 6 also shows the substantial NTX flux improvement application of0.5 g of 16% NTX.HCl (control) compared to obtained from the compound salt as compared to the com daily application of 0.5 g of 16% NTX.HCl and approxi pound free base, consistent with previous data (Banks et al., mately 100 µl of Solaraze® topical gel (3% Diclofenac 2008). Therefore, the focus will be on codrug salt forms, but sodium), 6.7 cm2 area (n=4) are shown in FIG. 7. Micropore non-salt forms are contemplated as well. This codrug com lifetime was extended up to 7 days in the presence of pletely hydrolyzed in the skin and only NTX and diclofenac diclofenac, but NTX without diclofenac had complete were detected in the receiver compartment of the in vitro micropore closure in a similar timeframe to the human study, diffusion study. 10 2-3 days ((Banks et al., 2011) Appendix). Full thickness Yucatan miniature pig skin in vitro studies Compound 2A in its HCl salt form was used to treat 4 to measure diffusion and bioconversion will provide the hairless guinea pigs. Since flux from this codrug was low, most accurate absorption data with microneedle (MN) treat ment. It is not necessary to use human skin when using four patches were placed on the guinea pigs with 7% saturated codrug gel after a single pretreatment with microneedles, since the pig skin is the same thickness and 15 the primary route of diffusion is through the MN pores and microneedles on Day 1. Microneedle treatment at each patch 2 not the stratum corneum lipid bilayers. Human skin can be site contained 100 micropores created by a 1 cm 50 needle used in final experiments in order to make sure bioconver stainless steel array rotated 90° and inserted twice. A new gel sion is similar, however, the present inventors are primarily patch was applied at 3.5 days, as the chemical stability of taking advantage of esterase activity and have not seen a 20 Compound 2A is not ideal. Plasma samples were obtained difference in skin metabolism for this enzyme system in from a jugular catheter over seven days and analyzed for humans vs. pigs. Pig skin is purchased from Sinclair, or drug concentration by LC/MS (Methods). In three out of the dermatome from sacrificed pigs used in the pharmacokinetic four guinea pigs significant NTX plasma levels were seen studies once the skin has returned to normal. The human skin for 7 days, and the pores in one guinea pig closed at 6 days. in these experiments will be healthy full-thickness (subcu 25 Micropores were visualized on the skin of the animals at 7 taneous fat removed) abdominal plastic surgery samples days by staining the skin with India ink, and the 100 pores obtained from the Cooperative Human Tissue Network can be seen clearly, including the pattern from the 90° (CHTN). Universal Precautions (OSHA standards) will be rotation and re-insertion FIG. 9. Intact codrug was not used when handling the human skin samples, in order to detected in the plasma samples. Significant micropore stain- protect the researcher from any potential harm. IRB 30 ing was also seen at days 1, 4, 5, 6, and 7. approval is not required by NIH to use non-identified A goal is to quantitatively characterize the transdermal surgical waste tissue. Microneedle arrays purchased from delivery of selected naltrexone codrugs in vivo using phar 1Ox Technology or another manufacturer will be used for all macokinetic determinants. This will be accomplished by studies, see letter of support attached. comparing the kinetic profiles and parameters of intravenous Guinea pig skin samples will also be used in some in vitro 35 diffusion and metabolism studies. We want to compare the bolus and topical applications of codrugs using the chroni transdermal flux rates seen in the animal in vivo data with cally annulated hairless guinea pig model. Hairless guinea animal in vitro data. It will also be important to see the pigs are a typical animal model used for transdermal deliv differences between in vitro guinea pig skin and in vitro ery experiments. More importantly, guinea pigs have been human/pig skin diffusion data. Since we are attempting to 40 shown to have a metabolic profile more similar to humans validate our in vitro human skin diffusion data with an in than rats or rabbits. Fifteen of the most promising codrug vivo animal model, we need to know the basic magnitude of candidates screened in the in vitro experiments will be fully difference in the skin permeabilities among the 3 species, if characterized in the guinea pig model. Plasma samples will any. Micropore closing will not take place in vitro, so these be analyzed by LC/MS. Detailed procedures for hairless experiments are used for flux optimization only. Detailed 45 guinea pig pharmacokinetic studies and data analysis are in procedures for diffusion studies are in the publications in the the Appendix publications and many previous publications Appendix and in our other publications. Simple analysis of from our group (Valiveti et al., 2005a; Valiveti et al., 2005b ). variance (ANOVA) will determine significant differences Five to six animals (guinea pigs and pigs) per treatment among the codrugs' flux values. group will be used, and dosing can be done in a crossover In some studies it is contemplated to increase the 50 fashion, 2 routes of administration, intravenous vs. percu microneedle array area (and needle number), which can taneous. A codrug will be considered successful if it can increase flux, as there is a direct increase in flux with number deliver a similar or greater flux than that achieved in the of micropores. In some cases a 1 cm2 (50 needles) array is human study with NTX HCl (Wermeling et al., 2008). Four used, but in other cases a 10 cm2 array, and potentially an of the most promising codrug candidates screened in the even larger array could be designed. Since the therapeutic 55 hairless guinea pigs will be examined in the Yucatan min delivery rate in humans with NTX HCl and 400 micropores iature pig, as the thickness of the skin is more similar to has been achieved, it is believed that achievement of a 7-day humans, important for microneedle application. This dose codrug therapeutic flux is highly likely as long as a solubility finding proof-of-concept study in the pigs will provide data similar to NTX HCl is achieved (FIG. 4). Some chemical that can be included in an IND (Investigational New Drug) stability issues can be resolved by limiting the water in the 60 application, and optimizing the dosing of the best codrugs final patch formulation that would be used in a microneedle will help to prepare for pig toxicology studies required for product. the IND. ANOVA statistical analysis will be used in order to Characterization of the Pharmacokinetics of the Drugs in determine statistical significance among treatment groups. Guinea Pigs and Yucatan Miniature Pigs In Vivo with Skin infection is not a concern with microneedle treat- Microneedle Use. 65 ment and has not been a problem in the animal or human A study was done in hairless guinea pigs using diclofenac studies. Skin is swabbed with alcohol prior to microneedle (Solaraze) gel applied daily to the micropores with a reser- treatment and formulations can contain microbicidal addi- US 9,566,341 Bl 25 26 tives, like benzyl alcohol used in our human study (Wer shipping to the University of Kentucky. Pigs will be trans meling et al., 2008). Microneedles are also very sturdy and ported and acclimated for 7 days once at the university do not break in the skin. before initiation of any pharmacokinetic studies. Miniature LC-MS/NIS conditions: Chromatography colunm used pigs (n=5-6 per test compound) are used typically as a large was Waters Atlantis Hilic Silica 5 µm, 2.lx150withamobile 5 animal model for topical/transdermal studies. This breed of phase consisting of acetonitrile:buffer 80:20 (v/v). The aque pig is ideal for topical/transdermal studies due to minimal ous buffer consisted of 1 mM ammonium acetate with 0.1 % body hair compared to other breeds. The pig will be placed acetic acid and 5% MeOH. The injection volume was 20 µ!. in a sling for dosing and sampling. Pigs will be dosed with Liquid chromatography was carried out in the isocratic topical drug doses after MN application and blood sampling (24 pre-determined time points) will be done for up to 168 mode at flow rate 0.3 ml/min with a total run time of 14 min. 10 hours. A jacket (Lomir Biomedical Inc) is placed on the pig The retention times for NDC, NTX, NTXol, DIC and NAL to prevent the pig from removing the patches. Lidocaine were as follows: 6.7 min, 9.6 min, 11.4 min, 7.4 min, and 1.6 (4%) cream is applied to the port area for 10 min prior to min, respectively. blood collection to minimize discomfort to the pig. Special The LC-MS-MS system consisted of a HPLC Waters ized Huber needles are used for flushing and sampling to Alliance 2695 Separations Module, Waters Micromass® 15 maintain the integrity of the ports. Blood removal will not Quattro Micro™ API Tandem Mass Spectrometer. Positive exceed 10% of the animal's total blood volume. Plasma electrospray ionization was used for detection of all com samples are harvested and analyzed by LC/MS/MS. Weights pounds (ESI+), except DIC was analyzed through negative are obtained prior to initiation of studies and monitored ionization (ESI-). Multiple reaction monitoring (MRM) was weekly. Animals are euthanized after completion of the carried out with the following parent to daughter ion tran 20 experiments. sitions for Compound 2A (NDC), NTX, NTXOL, diclofenac Skin irritation in hairless guinea pigs (n=5-6 per test (DIC) and naloxone internal standard (NAL): m/z compound): Erythema from topical drug treatments can be 619~342, m/z 342-324, m/z 344~626, m/z 293~250, and quantified with a Minolta ChromaMeter (Kobayashi, m/z 328~310, respectively. Capillary voltage was 3.5 kV, Hosaka et al. 1997; Sutinen 2000). A colorimetric measure- RF lens 0.3 V, source temp 130° C., desolvation temperature 25 ment of the skin reflection is taken before and after the 270° C. and cone voltage 29 V for NTX, NTXOL and NAL, topical drug treatment, in order to assess skin irritation 18 V for DIC and 38 V for NDC. The collision gas was 19 (Kobayashi, Hosaka et al. 1997). Trans-epidermal water loss eV for NTX, 10 eV for DIC and 20 eV for NDC, NTXOL (TEWL) measurements will also be taken before and after patch application, as a relative measure of skin damage. and NAL. Nitrogen gas was used as a nebulization and These two skin measurements are complimentary, and drying gas at flow rates of 50 and 450 L/h, respectively. 30 should provide a good prediction of potential treatment Calibration plots were prepared by a linear regression analy- irritation (Kobayashi, Hosaka et al. 1997; Sutinen 2000). sis of the ratio of the drug-to-internal standard areas versus The irritation potential of the drugs will be evaluated by nominal drug concentration. Quadrupoles were set on unit taking erythema and trans-epidermal water loss (TEWL) mass resolution. Based on the peak-to-noise ratios of3:1 for readings at the induction sites after patch removal. Erythema LOD the following limits of detection were estimated: 35 from topical drug treatments will be quantified with a NDC IN HCI. Compound 22 and naltrexone (23) will be coupled HRMS: Calculated for C34H320 5 N2 Cl2 618.1688 and using conventional coupling approach to yield a Boe-pro- found 618.1697. Scheme-2 HO Formarnidine sulfonic acid Benzylbromide N~ AqNaOH N~ Acetone/K2C03 0 23 24 22 DCC/DMAP N~ N~ Cl Boe l N 25 ee Cl 26 1. H2, Pd-C 12. H+ US 9,566,341 Bl 33 34 -continued HO 2MHC1 1,4-Dioxane Reflux NH 0 C1)0vXe 0 N~ ~~~o ~~~o ucX) ucX) 2 27 Naltrexol (24) was synthesized with a reported process -continued (Decosta, Iadarola et al. 1992) and phenolic hydroxyl group 20 Cl was protected with benzyl bromide to get compound (25) 1. 23 (Pelotte, Smith et al. 2009). Benzyl protected naltrexol (25) _,..Boe DCC will be coupled with Boe-protected diclofenac acid (22) to y: N 0 DMAP provide naltrexol-diclofenac codrug-(2). This codrug on II 2. H+ treatment with hydrochloric acid produced the salt form of 25 Cl(JYO~I ~OH codrug-(27). Codrug-2: Diclofenac acid was dissolved in dry THF and ~ 0 activated with DCC/DMAP for about two hours on an ice 30 bath. Later on 3-0Bn-6-~-naltrexol was added drop wise in 30 NC! dry THF, and reaction mixture allowed to stir over night under nitrogen atmosphere. Completion of the reaction was ~NH 0 monitored with TLC and crude compound washed with ethyl acetate to get rid of dicyclohexy urea and purification ci~o00 on silica column gave titled compound-2 (Scheme-2) in 60% 35 yield. u~ Chemical Formula: C34H34Cl2 N2 0 5 , Exact Mass: 620.18, Molecular Weight: 621.55 1 H-NMR (CDC1 , 500 MHz) ll 7.34-7.30 (m, 2H), 7.26- N'7 3 40 7.7.24 (m, lH), 7.16-7.07 (m, lH), 6.97 (s, 2H), 6.88-6.85 0 (m, lH), 6.70-6.68 (m, lH), 6.56 (m, lH), 4.74-4.62 (m, 2H), 3.85 (s, 2H), 3.08 (s, lH), 3.02-2.98 (m, lH), 2.64-2.55 (m, 2H), 2.36-2.23 (m, 3H), 2.12-2.07 (m, lH) 2.01-1.63 (m, lH), 1.79-1.76 (m, lH), 1.59-1.57 (m, lH), 1.46-1.40 (m, 45 2H), 1.25 (m, 3H), 0.87-0.83 (m, 2H), 0.83-0.51 (m, 2H), Commercially available benzyl protected hydroxy acids 0.11 (s, 2H) (28) will be used in this synthesis. If not required, starting MS-spec: calcd. 621.2 (M+Ht. found 621.2. material will be synthesized according to reported proce dures (Sunazuka, Tsuzuki et al. 1992). Precursor (29) will be obtained on DCC coupling ofbenzyl protected hydroxy acid 50 Seheme-3 (28) and Boc-diclofenac acid (22). Hydrogenation of com pound 29 followed by coupling with naltrexone (23) will 0 22 give codrug-(3). DCC HO II DMAP ~OBn 55 - Seheme-4 28 Cl (YC! _,..Boe y _,..Boe 60 1. 25 N 0 DCCI DMAP 0 Cf0 ' 0 2. H+ ~o~OBo 0°0H UM 65 UM 29 30 US 9,566,341 Bl 35 36 -continued -continued ,,?' Cl BnO~: (I( 0 OH YNH 0 N~ H1/Pd-C c100~ 0 u~ 1. 22 31 10 EDC HO 2. H+ NC! N~ - YNH 0 N~15 34 ClITTOI ~o ~ ~ 0 20 4 Parallel protocol will be used to make codrug-( 4). Deben rl zylation via hydrogenolysis of intermediate (31) will give Cl~Cl iarget codrug-( 4). 25 NH 0 ~~00 Scheme-5 ug 30 ~OH 23 Boc-HN DCC/DMAP 11 0 N'7 32 0 0 35 Boe-RN II ~o 40 Boe protected amino acids (32) will be used to make TFA codrugs-(5) and -(6) as outlined in schemes-5 and -6. Once N~ - Boe protected amino acid is attached to naltrexone (23) with 45 DCC mediated coupling to get (33), Boe group will be 0 removed with well-known deprotection methods. Amino 33 compound (34) and naltrexone (23) will be coupled to yield codrug-(5). Scheme-6 BnO ~OH 25 Boc-HN DCC/DMAP TFA 11 - 0 0 32 N~ Boc-HN II ~o 35 US 9,566,341 Bl 37 38 -continued 1. 22 EDC -2. H+ N~ N~ 36 37 N~ To make codrug-(6) with ester and amide link, benzyl protected naltrexol (25) will be used. Debenzylation of (37) will yield codrug-(6). Scheme-7 c'~c' NH H H N'f...... l 0 N~ 1. 25/Triph7sgene 2. H2/Pd-C 7 3. 22/DCC/DMAP 4. H+ 0 O_)lN~NH H 2 ()# 1. 25/Triphosgen\ 2. 22/DCC/DMAP 3. H+ US 9,566,341 Bl 39 40 -continued c'~c' NH N~ H1! Pd-C cnHN~NyO H - 0 39 HO c'~c' NH N~ cnHN~NyO H 0 Mono benzyl amine protected starting materials will be 2007). Triphosgene treated naltrexone (Hamad, Kiptoo et al. used to produce amide-amide linked spacer codrug-(7). 2006) (23) on action with (38) will give compound 39 Unavailable starting amino compounds will be synthesized (Scheme 7). As outlined in scheme-7, codrug-(8) will be according to published methods (Krivickas, Tamanini et al. synthesized by hydrogenation of compound 39. Scheme-8 22 DCC/DMAP 40 Bno 41 25 Triphosgene o~c' NH N~ ~OM:Ol(O l) M o 42 43 23 lTriphosgene US 9,566,341 Bl 41 42 -continued HO ('l ('l Cl~Cl O Cl~Cl NH N~ NH )l ~o~o o ~o~oyo u~ u ~ 0 N~ 9 0 10 0-mono benzyl protected dials ( 40) will be used as (41) on debenzylation gives composite ( 42) and on followed starting materials for the projected synthesis or materials 25 reaction with triphosgene treated benzyl protected naltrexol will be synthesized according to literature protocols (G. (Hamad, Kiptoo et al. 2006) (25) will give a new interme Venkateswar Reddy 2009), (Simas, Pais et al. 2003). DCC diate (43). Hydrogenolysis of (43) will give codrug-(9) mediated union between diclofenac acid (22) and 0-mono (Scheme 8). Codrug-(10) will also be synthesized by using benzyl dial ( 40) will give derivative-(41 ). This derivative naltrexone (23) and triphosgene reagent. Scheme-9 HO~o~o~o~o~~ 0 ,# - 0 OH N~ 0 ,,-...._ [,O~ J...... ,O~OH HO' '-f 01n -..._,r II 45 0 25 "" DCC/DMA~ BnO~I# 0 OH N~ HO~o~o~o~o - 0 46 US 9,566,341 Bl 43 44 -continued ~. 0 ~~ ~0Vyo~o I # o'&c' 00 OH N~ 11 12 Pegylated codrugs-(11) and -(12) are designed based on 30 1999), (Jiang and Yu 2008). PEG hydroxyl acid (44) on reported studies (Bonina, Puglia et al. 2001) and will be DCC coupling with naltrexone (23) will give peglylated synthesized as described in scheme-9. To avoid potential naltrexone compound (45) and on further DCC coupling toxicity, the peg spacers will be used from n=4. Mono with diclofenac acid (22) will give PEG linked codrug-(11 ). 0-benzyl PEG spacer will be synthesized with small adap By using benzyl protected naltrexol (25) PEG spacer linked tation per the reported protocols (Bellouard, Chuburu et al. diclofenac-naltrexol codrug-(12) will be synthesized. Scheme-10 1. 22 DCC DMAP 2. H+ 47 48 ~ ~ Cl~Cl Cl~Cl HN ~OyiyOH 23 V~oyrt'{o~ ~ 0 no I # DCC/DMAP u ~ 0 0 0 OH 49 N~ 0 13 DCC/DMAP25 l US 9,566,341 Bl 45 46 -continued 50 14 15 Anhydride codrugs are proposed based on research -continued articles of diclofenac anhydride prodrugs (Mizrahi and Domb 2009). Mono benzyl protected dicarboxylic acids (47) will be used for synthesis of codrugs-(13) and -(14) as outlined in scheme-10. Debenzylation and DCC mediated coupling with naltrexone (23) will give codrug-(13). If 20 starting materials are not commercially available they will be synthesized according to the following reference (Bal lard, Richards et al. 2008). Diclofenac derivative (49) on DCC coupling with benzyl protected naltrexol (25) will give intermediate (50). This compound on hydrogenolysis will give codrug-(14). 25 Scheme-11 HO N~ 30 Cl N N~ Dibenzyl phopspono acetic acid 35 Cc"Cl DCC- 15 DMAP 40 To increase the aqueous solubility, several codrugs are proposed in schemes-11-14. These codrugs are planned 45 based on the following published research (Du, Hong et al. 0 N'7 2007), (Rouquayrol, Gaucher et al. 2001), (Baba and Yosh ioka 2006). Water solubilizing phosphate, amino acid and ~~~o L-malic acid moieties will be attached to phenolic hydroxy 50 group of naltrexone (23) and aliphatic hydroxyl group of uc:O naltrexol (24). Naltrexol derivative in scheme-12 will be synthesized according to reported synthesis (Nelson, Davis 52 et al. 1994). Scheme-12 1. 22 DCCI DMAP 2. H+ N~ 53 us 9 ' 566 '341 Bl 47 48 -continued 56 tDibenzyl phospono !acetic acid QC c' ~oM "'~"~c Alpha-D-xylo u ~~N~ hexofuranose BnO 55 2 2-Dimethyl-.1 ,3-dioxolan- 'd 14~on-5-yl acetic ac1 58 US 9,566,341 Bl 49 50 -continued (YC! YNH Cl~O lJ g I N'7 0 HO..__Pr-( -9 \ 0 O OH 16 (YC! (YC! YNH YNH Cl~O Cl~O lJ g I lJ g I Acetonide 0-< 0 deprotection /~C~o > 57 17 (YC! YNH Cl~O lJ g I 18 US 9,566,341 Bl 51 52 Scheme-13 HO Dibenzyl phosphono acetic acid Y:c'NH c'()Ao N~ ~ I o 0 0 2 OH 0 Bno,~...... _)l BnO/ 0 HOyVlo Acetonide 0 deprotection c;:('NH N~ c'Cno ~(' N~ ~ I o c'~o ~ I o 59 20 0 0 H1/Pd-~ HO,~...... _)l HO/ 0 (YCl YNH N~ Cl~Oug 19 Final codrugs and all intermediate compounds will be verted to hydrochloride, glycolate and lactate salts according completely characterized by 1 HNMR, 13CNMR spectros to mentioned reference (B. S. Somashekar 2005). copy and mass spectrometry. All the codrugs will be con- Scheme-14 ~ Cyclic /N anhydrides ¢N O=S-NH2 II 0 60 US 9,566,341 Bl 53 54 23 DCCI -DMAP N~ 0 61 21 DCC/DMAP25 l 3 CF BnO~ N I # ¢ O 0 OH N\; O=S-N ,O 11 H-JLM"n ';;" I( 0 0 62 22 Celecoxib (60) will be reacted with different cyclic anhy 0.42±0.12 nmol/(cm2 /hr) (p<0.005). The skin concentration drides to make intermediate (61), similar to some published for both naltrexone and the compound also increased. n=4 protocols (Mantarosie, Coman et al. 2008), (Paun, Stere et 40 for the flux calculation. FIG. 15. So the flux increased by al. 2008). Terminal carboxylic acid free compound (61) will about 13 times using the salt form of the compound over the attach to naltrexone (23) or benzyl protected naltrexol (25) free base form. Precipitation issues were observed with purely aqueous to get target codrugs-21 and 22 (Scheme-14). donor, so an in vitro formulation development experiment was carried out by varying the amount of PG in the donor. Example 2 45 10%, 25% and 50% PG was used with the salt form and it was observed that while there was no significant difference The compound of FIG. 2U (Compound 2U) is studied. in flux between the aqueous formulation and 10% PG Stability: The chemical stability of the compound in the containing formulation (p<0.05), but flux decreased signifi pH 5.0 acetate buffer (0.3M) was 3.9±0.56 days and the cantly for higher concentrations of PG. The data was com- enzymatic stability in the hairless guinea pig (HGP) plasma 50 pared using one way ANOVA and Tukey's posthoc analysis was 6.4±0.84 min (n=3) indicating that the compound would for pairwise multiple comparisons. The 10% PG containing be degraded to regenerate the parent drugs as soon as it formulation was hence chosen for in vivo studies. All enters the body. In both the studies regeneration of the experiments were carried out with a saturated donor solu parents was observed with the degradation of the compound. tion. In vitro: The first in vitro experiments showed that the 55 In vivo experiments. The pores can be clearly visualized compound degraded in the skin to regenerate back the parent with gentian violet in FIG. 15 (A) immediately following drugs even at 24 hours and amount of parent drugs from the MN treatment. In panel (B) the pores cannot be visualized compound increased with time. Steady state flux was at day 4 (96 h) pores treatment with MN followed by obtained both from naltrexone and diclofenac of 0.42±0.12 application of NTX HCl gel under occlusion. Pores can be 2 2 nmol/(cm /hr) and 0.19±0.06 nmol/(cm /hr) respectively 60 visualized by India ink staining in panel C where the (FIG. 14A). The naltrexone flux was significantly higher hydrochloride salt form of the compound was used follow- compared to diclofenac (p<0.05). The skin concentration ing MN treatment indicating that the pores can stay open up data is shown below in FIG. 14B. n=3 for flux calculation; to a 7 day time point. n=2 for skin concentration studies at 36 and 48 hr timepoint The compound of FIG. 2V (Compound 2V) is studied. and n=3 for 12 and 24 hr. (FIG. 14B). 65 Stability A chemical stability study was done for the The flux of naltrexone from the hydrochloride salt form of compound 2 in 0.3M acetate buffer pH 5.0 at 32° C. The half the compound was 5.42±1.5 nmol/(cm2 /hr) compared to life was approximately 56.3±9.5 days US 9,566,341 Bl 55 56 In vitro experiments The compound was tested in a 10% bottom of the rubber ringed barrier to maintain mt1mate PG containing saturated donor formulation for in vitro flux contact with the skin and prevent evaporation of the 300 µL and a naltrexol flux of approximately 0.29±0.09 nmol/cm2/h gel formulations. was obtained from the compound compared to 3.07±0.27 Gel Formulations nmol/cm2 /h naltrexol flux from 10% PG containing satu 4% w/w gels were prepared with the hydrochloride salt rated naltrexol formulation. See FIG. 18. form of the compound for in vivo studies. Hydrochloride salt The compound of FIG. 2W (Compound 2W) is studied. gels were formulated by dissolving 4% compound salt in A chemical stability study was done for the compound propylene glycol: acetate buffer at pH 4.8-10:90 and 3.0% 2W in 0.3M acetate buffer hydroxyethylcellulose polymer. As the polymer was dis- 10 solved over a 30 minute period, the solution was continu ously stirred to ensure salvation as well as to initiate Compound 2A Compound 2B Compound 2C polymerization. The solution was stirred still a viscous gel resulted and then left at room temperature for 11/2 to 2 hours Chemical stability 3.9 ± 0.56 56.3 ± 9.5 37.08 ± 1.22 to remove bubbles and ensure complete polymerization. The (days) 15 gel was saturated with excess solid and therefore was a Enzymatic stability 6.4 ± 0.84 min (minutes) cloudy gel. Pharmacokinetic Animal Studies Hairless IAF guinea pigs (Charles River) weighing 350- Methods: 650 g were used for these studies. All animal studies were For all in vitro experiments female Yucatan minipig skin 20 in accordance with the institutional guidelines and approved (6 months old) were used. Skin sections were obtained by by the University of Kentucky IACUC. Surgical procedure removing the subcutaneous fatty tissue by scalpel dissection, was performed to canulate the jugular vein. Blood samples and were stored at -20° C. A PermeGear flow-through (0.3 mL) were drawn from the jugular vein at regular time (In-Line, Riegelsville, Pa., USA) diffusion cell system was intervals for the time studied. MN treated animals were first used for the skin permeation studies. Skin used for 25 cleaned topically with isopropyl alcohol, and two 50 MN microneedle treatment was placed on a wafer of polydim arrays were then manually applied to each dose site. For MN ethylsiloxane polymer, which mimicked the underlying treated GPs, four 0.3 mL gel doses were applied to the dorsal mechanical support of tissue because of its comparable region of the hairless guinea pigs at 4 sites to obtain steady structural flexibility and elasticity. state plasma levels. After application of the gel the skin was The skin was pierced 20 times with an array containing 5 30 occluded with the patch and occlusive tape was applied to MN (i.e., to make a total of 100 individual and non-over prevent evaporation of the formulation Blood samples were lapping piercings) before mounting the skin in the diffusion obtained for 7 days while the patch was changed with new cell for the treatment group. The insertion of MN into skin formulation at 3.5 days. The blood samples were immedi was carried out manually by applying gentle finger pressure ately centrifuged at 10,000xg for 3 min; plasma was sepa- 35 rated and stored at - 70° C. until analysis. followed by instantaneous removal. MN's were distributed Staining evenly within the 0.95 cm2 area of skin and could easily be The patches were removed at predetermined time points visualized after each 5 MN insertion, as to prevent reappli and the site was cleaned with sterile gauze. Gentian violet/ cation in the same area. Single MN array of 5 were used India ink was applied as the dyeing agent at the MN treated If simply out of ease for the experimental procedure. any 40 sites and allowed to dry before excess dye was removed with damage to an MN section was observed the section was ethanol and gauze. Both the dyes stain the viable epidermis replaced. Cells containing MN treated skin showed no but not the intact SC. Hence, a grid is observed only in the presence of receiver solution back flow into the donor presence of micropores in the skin, no staining is observed compartment. Untreated skin samples were simply placed in in its absence immersing the skin pieces in ACN overnight the diffusion cells. Diffusion cells were kept at 32° C. using 45 in a water bath shaker at 32° C. The donor was removed a circulating water bath. Data were collected using skin from from the top of the skin at the end of the study and analyzed a single pig 3-4 cells for the MN treated formulations. by direct injection onto HPLC after suitable dilution using The physiological receiver solution was water adjusted to ACN. pH 7.4 containing 20% ethanol, and the flow rate was HPLC analysis. Quantitative analysis of naltrexone, adjusted to 1.5 ml/h. Each cell was charged with 0.25 ml of 50 diclofenac and the compound concentrations in all samples the donor in the donor compartment of the chamber. The were carried out using a HPLC assay. The receiver samples diffusion cells were covered with a stopper to prevent were concentrated 10 fold with acetonitrile by evaporating 1 evaporation. Samples were collected from the receiver com ml of the solution under nitrogen and reconstituting in 100 partment in six-hour increments over 48 h or for the length µl of ACN and injected into the HPLC. The skin samples of the treatment. All samples were stored at 4 ° C. until 55 were directly injected onto the HPLC. The HPLC system analyzed by HPLC. consisted of a Waters 717 plus autosampler, a Waters 600 The skin samples at the end of the treatment period was quaternary pump, and a Waters 2487 dual wavelength absor removed, the treatment area was cut out with a scalpel and bance detector with Waters Empower™ software. A Brown diced into small pieces. Extraction of drug was done by The lee (Wellesley, Mass., USA) C-18 reversed phase Spheri-5 transdermal occlusive protective covering patches were fab- 60 µm column (220x4.6 mm) with a C-18 reversed phase guard ricated by sandwiching a rubber ringed barrier to create a column of the same type (15x3.2 mm) by Perkin Elmer® reservoir between a drug-impermeable backing membrane was used with the UV detector set at a wavelength of 215 nm (Scotchpak™ #1109 SPAK 1.34 MIL Heat Sealable Poly or 280 nm. The mobile phase consisted of 70:30 (v/v) ester Film) and anARcare® 7396 adhesive around the edge ACN:(0.1 % TFA with 0.065% I-octane sulfonic acid of the rubber spacer. The impermeable backing laminate was 65 sodium salt, adjusted to pH 3.0 with TEA aqueous phase). adhered to the rubber retaining ringed barrier with 3M™ Samples were run at a flow rate of 1.5 ml/min with a run double sided tape. Finally, ARcare® 7396 was placed on the time of 8.5 min. The injection volume used was 25 µl for US 9,566,341 Bl 57 58 receiver samples and 100 µl for the skin samples. The but not the intact SC. Hence, a grid is observed only in the retention time of naltrexone was 2.3 minutes, diclofenac was presence of micropores in the skin, no staining is observed 3.5 minutes and compound was 6.5 minutes. Samples were in its absence. analyzed within a linear range of standard curve for all the three compounds from 100-10000 ng/ml. The standard solu Example 3 tions exhibited excellent linearity over the entire concentra tion range employed in the assays. For the plasma samples Microneedle enhanced transdermal drug delivery enables the mobile phase consisted of 50:50 (v/v) ACN:(0.1 % TFA the transport of a host of molecules that cannot be delivered with 0.065% I-octane sulfonic acid sodium salt, adjusted to across the skin by passive diffusion alone. However, the skin pH 3.0 with TEA aqueous phase). Samples were run at a 10 being a self-regenerating organ heals itself and thus prevents flow rate of 1.0 ml/min with a run time of 37 minutes and delivery of molecules across micropores for a seven day injection volume of 10 µ!.The retention time for naltrexone time period, the ideal transdermal delivery goal. Hence, it is was 3.3 minutes, diclofenac was 16 minutes and compound necessary to employ a second drug molecule, a cyclooxy was 33 minutes. Compound 2B and Compound 2C were genase inhibitor to enhance pore lifetime by decreasing detected using the same assay. The retention times were 5.8 15 inflammatory response following microneedle treatment. A and 4.3 minutes respectively. codrug approach using a 3-0-ester codrug of naltrexone, the In vivo experiments. Fabrication of transdermal patches model drug with diclofenac, a cyclooxygenase inhibitor was Transdermal Patches. tested in vitro as well as in vivo to look at stability, The transdermal occlusive protective covering patches bioconversion as well as permeation. The results indicate were fabricated by sandwiching a rubber ringed barrier to 20 that the approach could be useful for transdermal drug create a reservoir between a drug-impermeable backing delivery of naltrexone from a single patch for a week, but membrane (Scotchpak™ #1109 SPAK 1.34 MIL Heat Seal stability and solubility enhancement will be required before able Polyester Film) and anARcare® 7396 adhesive around it can deliver therapeutically relevant levels of the drug. The the edge of the rubber spacer. The impermeable backing skin concentration of diclofenac was high enough to keep laminate was adhered to the rubber retaining ringed barrier 25 the pores open in vivo in a hairless guinea pig model as with 3M™ double sided tape. Finally, ARcare® 7396 was evidenced by pore visualization studies. placed on the bottom of the rubber ringed barrier to maintain intimate contact with the skin and prevent evaporation of the INTRODUCTION 300 µL gel formulations. Gel Formulations 30 Transdermal drug delivery is a potential alternative route 4% w/w gels were prepared with the hydrochloride salt for a host of drug molecules that are difficult to deliver via form of the compound for in vivo studies. Hydrochloride salt the oral or parenteral route. It avoids first pass effects, gels were formulated by dissolving 4% compound salt in hepatotoxicity, gastric irritation issues as well as needlepho propylene glycol: acetate buffer at pH 4.8-10:90 and 3.0% bia. However, only small, lipophilic molecules can be deliv hydroxyethylcellulose polymer. As the polymer was dis- 35 ered by passive diffusion alone across the stratum comeum solved over a 30 minute period, the solution was continu (SC), the main barrier to transdermal drug delivery (Praus ously stirred to ensure salvation as well as to initiate nitz, et al. (2004), Prausnitz and Langer (2008)). Hence, polymerization. The solution was stirred still a viscous gel physical and chemical enhancement techniques are being resulted and then left at room temperature for 11/2 to 2 hours used to enhance the number the molecules that can be to remove bubbles and ensure complete polymerization. The 40 delivered via this route. Physical enhancement techniques gel was saturated with excess solid and therefore was a include iontophoresis, electroporation, low frequency ultra cloudy gel. sound and microneedle (MN) enhanced delivery. Chemical Pharmacokinetic Animal Studies enhancement techniques include chemical enhancers and the Hairless IAF guinea pigs (Charles River) weighing 350- prodrugs/codrugs approach. In this study MN enhanced 650 g were used for these studies. All animal studies were 45 delivery was combined with a codrug approach to develop in accordance with the institutional guidelines and approved a seven day sustained release patch system. MN is a mini by the University of Kentucky IACUC. Surgical procedure mally invasive physical enhancement technique. The was performed to canulate the jugular vein. Blood samples method adopted is the "poke and patch" approach3 where (0.3 mL) were drawn from the jugular vein at regular time solid stainless steel MN arrays are used to create micropores intervals for the time studied. MN treated animals were first 50 (also referred to as micro channels) across the SC. The MN cleaned topically with isopropyl alcohol, and two 50 MN is then removed and a matrix/reservoir type patch is applied arrays were then manually applied to each dose site. For MN to deliver drugs through the micropores. Pain associated treated GPs, four 0.3 mL gel doses were applied to the dorsal with the application of these MNs is also negligible when region of the hairless guinea pigs at 4 sites to obtain steady compared to a hypodermic needle (Kaushik, et al., (2001), state plasma levels. After application of the gel the skin was 55 Gill, et al. (2008)) which could potentially lead to better occluded with the patch and occlusive tape was applied to compliance, one of the major drawbacks of addiction prevent evaporation of the formulation Blood samples were therapy (O'Malley, et al. (1992)). obtained for 7 days while the patch was changed with new Naltrexone (NTX), the model compound (M.W of341.41, formulation at 3.5 days. The blood samples were immedi log P of 1.8) is one of the leading FDA approved pharma ately centrifuged at 10,000xg for 3 min; plasma was sepa- 60 cotherapy for alcohol addiction. It is a µ-opioid receptor rated and stored at 70 g C until analysis. antagonist and is known to decrease alcohol craving in Staining recovering addicts. The drug exerts its mechanism of action The patches were removed at predetermined time points by attenuating the ethanol induced stimulation of the and the site was cleaned with sterile gauze. Gentian violet/ mesolimbic-dopaminergic pathway (Lee, et al. (2005), Swift India ink was applied as the dyeing agent at the MN treated 65 (2010)). Currently, NTX is marketed as a 50 mg once daily sites and allowed to dry before excess dye was removed with oral formulation, Revia® (Naltrexone Hydrochloride). This ethanol and gauze. Both the dyes stain the viable epidermis has compliance issues in addicts Hulse and Basso (2000), US 9,566,341 Bl 59 60 Volpicelli (1997). It also has an oral bioavaibility ranging Oesch, et al. (2007). Once in the skin the DIC will act locally from 5-40% and is associated with side effects like hepato to keep the pores open while NTX is delivered systemically. toxicity, nausea and vomiting (1996, PDR. 2d Ed, New The overall objective of this paper is to use a codrug Jersey, Medical Economics). The intramuscular injectable approach to reduce the inflammatory response in combina 28 day controlled release formulation of NTX; Vivitrol™ 5 tion with occlusion for delayed barrier recovery following was approved by the FDA in 2006. Injection site reactions MN treatment to reach the ultimate goal of a seven day patch and difficulty of removal in case of emergency opiate system. treatment in addition to the above are some of the major Materials and Methods drawbacks of this dosage form (Alkermes, Vivitrol Alert). Chemicals 6-~ naltrexol is the major metabolite of NTX and has a 10 Naltrexone HCl was purchased from Mallinckrodt (St. higher half-life of 13 hours in plasma compared to that of 4 Louis, Mo.), diclofenac acid from AK Scientific, Inc. hours for NTX. It has been shown to be an effective therapy (Mountain view, CA) and diclofenac sodium salt from TCI for alcohol abuse as well (McCaul, et al., (2000)). NTX cannot be delivered in relevant therapeutic concen America (Portland, Oreg.). Water was purified using NANOpure Diamond™, Barnstead water filtration system. trations transdermally by passive delivery alone. Some of 15 the earlier efforts in the laboratory have looked at delivery Hanks' balanced salts modified powder, propylene glycol ofNTX via the prodrugs and codrugs approach. The results and ethanol, 200 proof were purchased from Sigma (St. indicated that although flux enhancement was achieved in Louis, Mo.). 4-(2-Hydroxyethyl)-1-piperazineethanesulfo vitro in some cases, the flux enhancement was not enough to nic acid (HEPES), sodium bicarbonate, sodium acetate, deliver the molecule in therapeutically relevant concentra- 20 acetic acid and gentamicin sulfate and were obtained from tions in vivo (Hammell, et al. (2004), Haranath, et al. (2005), Fis her Scientific (Fairlawn, N .J.). 1-0ctanesulfonate, Kiptoo, et al. (2008)). Using MN it has been shown both in sodium salt was obtained from Regis Technologies, Inc vitro as well as in vivo that NTX can be delivered in the (Morton Grove, Ill.). Trifluroacetic acid (TFA), triethylam lower therapeutic range of 2 ng/ml Banks, et al. (2008), ine (TEA) and acetonitrile (ACN) were obtained from EMD Wermeling, et al. (2008), Verebey, et al. (1976)). However, 25 chemicals (Gibbstown, N.J.). Natrosol® (hydroxyethlycel one of the issues with this approach is that the micropores lulose) was obtained from Hercules. Chemicals for codrug begin to close between 48-72 hour (Wermeling, et al. (2008), synthesis were purchased from Alfa Aessar. Banks, et al. (2010), Kalluri and Banga (2011 ). Therefore, in Synthetic Procedure for Codrug order to develop a 7 day patch system, pore lifetime Diclofenac sodium salt was dissolved in 1:1 (Ethyl enhancement techniques need to be employed along with 30 acetate:Water) mixture and acidified with concentrated MN for successful therapy. hydrochloric acid. Organic layer was separated and aqueous Different techniques can be used to delay barrier recovery layer treated with ethyl acetate two times. Combined organic after MN treatment. The skin is a self-regulatory organ and layer was dried on anhydrous sodium sulfate and rotary employs different methods for recovery after assault. Cuta evaporated to remove reaction solvents. Diclofenac free acid neous wound healing itself consists of multiple phases, 35 inflammatory response being the first step. Increased syn was activated with DCC (N, N'-Dicyclohexyl carbodiim thesis and secretion of lamellar body contents and transfor ide)) and DMAP (4-Dimethylaminopyridine) in anhydrous mation into bilayer is also a part of the recovery process tetrahydrofuran. After two hours, equivalent naltrexone was (Feingold (2002), Feingold, et al. (2000), Menon, et al. added in dry tetrahydrofuran. Combined reactants were (1992). Occlusion alone can also delay the recovery process 40 allowed to stir over night under nitrogen atmosphere. After by decreasing the water flux across the barrier, one of the completion of the reaction, solvents were removed under major signals for recovery of structure and function reduced pressure and reaction crude mixture purified by (Grubauer et al. (1989)). flash silica gel column chromatography eluting with 80% There are several literature reports indicating the increase ethyl acetate and 20% cyclohexane and codrug-1 was in expression of both COX-I and COX-2 following damage 45 obtained in 56% (Scheme-I) yield. The codrug-1 was dis to the skin and the role of, both the enzymes in cutaneous solved in I, 4-dioxane and an equimolar amount of 2M repair (Pecker, et al. (2007), Davidson and Breyer (2003), hydrochloric acid in dioxane was added and refluxed for 1.5 Muller-Decker, et al. (2002), Futagami, et al. (2000)). hrs, and precipitated with hexanes to get hydrochloride salt Diclofenac (DIC) is a NSAID that nonselectively inhibits of codrug-24 (Scheme-I) 1 both COX-I and COX-2 thereby preventing formation of 50 HNMR (CDC1 3 , 300 MHz): o 7.29 (m, 2H), 7.19 (t, lH), downstream products like prostaglandins and thromboxane, 6.93 (m, 2H), 6.75 (d, lH), 6.60 (d, 2H), 6.57 (d, lH), 4.78 thus decreasing inflammation. It is currently available on the (s, lH), 3.67 (s, 2H), 3.12 (m, 2H), 2.79 (m, lH), 2.32 (t, market as an oral as well as topical formulation and is the 2H), 2.27 (m, 2H), 2.05 (m, 2H), 1.69 (m, 2H), 1.62, (m, first choice ofNSAID for this project. Previous experiments 2H), 0.46 (m, lH), 0.31 (m, 2H), 0.30 (m, 2H) 13 showed that by daily application of Solaraze Gel® (3% 55 CNMR(CDC13 , (300 MHz): o 4.22, 4.41, 9.77, 23.31, diclofenac sodium, 2.5% hyaluronic acid) after one time 31.09, 31.59, 36.45, 38.50, 43.75, 51.01, 59.48, 62.15, application of MN was able to deliver NTX over the seven 70.28, 90.93, 118.75, 119.52, 122.45, 123.05, 124.19, day time period in hairless guinea pig (HGP) (Banks, et al. 124.31, 128.30, 128.97, 129.73, 130.38, 130.64, 131.31, (2011 ). Daily application is irrational for a patch system and 132.63, 138.09, 142.92, 147.77, 169.98, 207.67 co formulating the drugs in high concentration at a pH 60 HRMS: Calculated for C34H320 5 N2 Cl2 618.1688 and optimum of 5 (pH of the outer layer of the skin is 4.5-5.5)3 2 found 618.1697. is difficult because the drugs have different physicochemical Hydrochloride Salt of Codrug-24 1 properties (FIG. 11). This leads to the codrug approach. The HNMR (CDC1 3 , 300 MHz): 09.98 (bs, NW) 7.33 (m, 3-0-ester-codrug contains a biolabile ester linkage that can 2H), 7.13 (t, 111), 6.99 (m, 3H), 6.61 (d, lH), 6.57 (d, lH), be cleaved by hydrolytic enzymes like esterase as soon as it 65 4.97 (s, lH), 4.73 (s, lH), 4.10 (s, 2H), 3.60 (m, 2H), 3.14 reaches the epidermal region of the skin to release the parent (m, 4H), 2.92 (m, lH), 2.65 (m, 2H), 2.41 (d, lH), 1.82 (m, compounds (Vaddi, et al. (2009), Prusakiewicz, et al. (2006), 3H), 1.25 (m 2H), 0.84 (m, 4H), 0.62 (m, lH) US 9,566,341 Bl 61 62 Quantitative Analysis stopper to prevent evaporation of formulation. Samples were NTX, DIC and the codrug concentrations in all samples collected at regular increments over 48 hours or for the were quantified using a HPLC assay which is a modified length of the treatment. Drug was concentrated I 0 fold by version of the previously reported NTX assay (Milewski and evaporating I ml of the collected sample under nitrogen and Stinchcomb (2011)). The HPLC system consisted of a 5 reconstituting in I 00 µl of ACN for quantification. All Waters 717 plus auto sampler, a Waters 600 quaternary samples were stored at 4 ° C. until analyzed by HPLC. The pump, and a Waters 2487 dual wavelength absorbance skin was removed at the end of the study and the treatment detector with Waters Empower™ software. A Perkin Elmer area was diced into small pieces using a scalpel after Spheri5 VL Cl8 column (5µ, 220x4.6 mm), a Cl8 guard removing all excess drugs from top of the skin. Drug was column (15x3.2 mm) was used with the UV detector set at 10 extracted out by immersing the skin pieces in ACN over a wavelength of 280 nm. The mobile phase consisted of night in a shaker water bath at 32° C. Samples were injected 70:30 (v/v) ACN:(0.1% TFA with 0.065% I-octane sulfonic into the HPLC after appropriate dilution for quantitative acid sodium salt, adjusted to pH 3.0 with TEA aqueous analysis. The donor was also removed and quantified at the phase). Samples were run at a flow rate of 1.5 ml/min with end of the study by direct injection onto HPLC after suitable a run time of 8.5 min. The injection volume used was 25 µl 15 dilution using ACN. All data was analyzed using Fick's first for receiver samples and I 00 µl for the skin samples. For the law of diffusion. J ss =P* li.C where J ss is the steady state flux, plasma samples the mobile phase consisted of 50:50 (v/v) P is the permeability coefficient and li.C is the concentration ACN:(O.I % TFA with 0.065% I-octane sulfonic acid difference between the donor and the receiver. Since the sodium salt, adjusted to pH 3.0 with TEA aqueous phase). receiver is maintained at sink conditions li.C is essentially Samples were run at a flow rate of 1.0 ml/min with a run 20 the concentration of the donor. Steady state flux is obtained time of 37 minutes and injection volume of 25 µ!. from a plot of cumulative amounts permeated per time. Chemical and Enzymatic Stability Permeability coefficients can be calculated using the known Chemical and enzymatic stability was determined for the donor concentration. codrugs. For chemical stability studies, the codrug was put In Vivo Studies in prewarmed 0.3M acetate buffer pH 5.0 after solubilization 25 Fabrication of Transdermal Patches in <5% ACN, if required, at 32° C. in an incubator. Samples The transdermal patches were fabricated by sandwiching were withdrawn at regular intervals and injected into the a blend of nitrile rubber and polyvinyl chloride plastic ring HPLC after appropriate dilutions. Enzymatic stability stud- barrier to create a reservoir between the skin and the ies are important for estimation of conversion of codrugs in impermeable backing membrane (Scotchpak™ #1109 the plasma. For plasma stability studies prewarmed HGP 30 SPAK 1.34 MIL Heat Sealable Polyester Film) and an plasma at 37° C. was spiked with codrug solution and ARcare® 7396 adhesive around the edge of the rubber samples were withdrawn at regular intervals. 100 µl of spacer. 3MTM double sided tape was used on either side of plasma sample was extracted with I ml of I: I ACN:ethyl the rubber to attach the backing on one side and to maintain acetate, vortexed, centrifuged at I 0,000 rpm for 20 minutes contact with the skin on the other side and prevent evapo on a bench top Eppendorf Mini Spin®. The supernatant was 35 ration of the formulation. decanted into a glass tube and evaporated under nitrogen. Dose Preparation and Application Samples were reconstituted in I 00 µl ACN and injected onto All animal experiments were approved by the University the HPLC. All stability data was analyzed using pseudo first of Kentucky IACUC. A proof of concept in vivo experiment order kinetics. All stability studies were carried out at least, was carried out. 4% w/w gels were prepared with the in triplicate. 40 hydrochloride salt form of the codrug. Gels were formulated In Vitro Diffusion Studies by dissolving 4% codrug salt in PG: buffer at pH 5.0=10:90 The in vitro permeation experiments were carried out to and 3.0% hydroxyethylcellulose (HEC) polymer (required determine the transdermal flux of the parent drugs and the to have a gel formulation). Polymer was dissolved over a 30 codrug, permeation coefficients as well as the concentration minute period to obtain a homogenous gel. The solution was of the drug molecules in the skin at the end of the study. For 45 stirred until a viscous gel resulted and then left at room all in vitro experiments Yucatan miniature pig (YP) skin was temperature for I 1h to 2 hours to remove bubbles and ensure used. Full thickness skin was obtained by removing the complete polymerization. The gel was saturated with excess subcutaneous fatty tissue by scalpel dissection, and stored at solid and therefore was cloudy. MN treated animals were -20° C. A PermeGear flow-through (In-Line, Riegelsville, first cleaned topically with isopropyl alcohol, and 50 MN Pa., USA) diffusion cell system was used for the skin 50 array was applied twice mutually perpendicular to each permeation studies. Skin for MN treatment was placed on a other to give a total of 100 pores at each site. 0.3 mL gel wafer of polydimethylsiloxane polymer, which mimics the doses were applied to the dorsal region of the HGP. After underlying mechanical support of tissue because of its application of the gel the skin was occluded with the comparable structural flexibility and elasticity. The skin was occlusive patch and biocclusive tape was applied to prevent pierced 20 times with a 5 MN array (making 100 individual 55 evaporation of the formulation. The patch was changed with and non-over lapping piercings) before mounting the skin in new formulation at 3.5 days such that more than 50% intact the diffusion cell. MN pores were distributed evenly within codrug was always present in formulation. A control experi the 0.95 cm2 area of skin. Untreated skin samples were ment was also carried out to look at the effect of NTX HCl mounted directly onto the diffusion cells. The setup was alone in the same vehicle on pore closure kinetics. A 9% maintained at 32° C. using a circulating water bath. Data 60 solution ofNTX HCl was used to prepare a 3% HEC gel and collected using a minimum of 3-4 cells per treatment group. all other methods of application were constant with the The receiver solution was water alkalified to pH 7.4 con codrug gel. The patches were removed to obtain data at taining 20% ethanol at 37° C. Gentamycin sulfate (50 mg/L) successive 24 h time intervals. was added to the receiver for 7 day studies as an antibacterial Staining agent. The receiver flow rate was adjusted to 1.5 ml/h to 65 The patches were removed at predetermined time points maintain skin conditions. Each cell was charged with 0.25 and the site was cleaned with sterile gauze. Gentian violet/ ml of the donor and the diffusion cells were covered with a India ink was applied as the dyeing agent at the MN treated US 9,566,341 Bl 63 64 sites and allowed to dry before excess dye was removed with produce therapeutic efficacy. As also noted from the flux and ethanol and gauze. Both the dyes stain the viable epidermis skin concentration data, significantly higher amounts of but not the intact SC. Hence, a grid is observed only in the naltrexone was found in the receiver while diclofenac was presence of micropores in the skin, no staining is observed retained in the skin in higher concentration at 48 h. This in its absence. 5 difference in flux and skin concentration could be attributed Statistical Analysis to the difference in physicochemical properties of the drug Data for all experiments are reported as mean±standard molecules. While naltrexone is positively charged at the skin deviation. Statistical analysis of data will be carried out with surface pH of 5 (ref), at least 90% of the diclofenac will be Students' t-test and one way ANOVA with post hoc Tukey's negatively charged. Since skin is known to possess a nega- pairwise tests, ifrequired, using SIGMA-STAT (SPSS, Inc., 10 tive charge due to presence of negatively charged carboxylic Chicago, Ill., USA) software. P<0.05 will be considered to acid groups this might lead to slower migration and higher be statistically significant. retention of diclofenac molecules in the skin. Since the Results and Discussion hypothesis behind the project is based on the fact that Synthesis. The codrug and it's hydrochloride salt were diclofenac would work locally to reduce subclinical inflam- synthesized successfully. The structures were verified. 15 matory response thus enhancing the drug delivery window, HPLC method development. HPLC assay development this characteristic of the molecules would be beneficial in for the parent drugs and the codrug was the first step towards the long term. quantification of the data. The retention time of NTX was From literature it is known that a charged species has 2.3 minutes, DIC was 3.5 minutes and codrug was 6.5 higher flux through MN treated skin compared to a non- minutes. The retention time for the same compounds in 20 treated control. This property is attributed to the enhanced plasma samples were 3.3 minutes, 16 minutes and 33 solubility of a charged species in an aqueous donor rather minutes. All retention times reported are value±0.1 minutes. than the molecular species itself (Banks, et al. (2008). Samples were analyzed within a linear range of standard Viscosity of the donor formulation also has a predominant curve for all the three compounds from 100-10000 ng/ml. effect on the flux across MN treated skin where it has been The standard solutions exhibited excellent linearity over the 25 shown that 17 fold flux enhancement was obtained using a entire concentration range employed in the assays. low viscosity, aqueous donor compared to a high viscosity Stability studies. The chemical and enzymatic stability propylene glycol (PG) rich donor (concentration was main data indicated that the chemical half-life of the codrug in the tained at 110 mg/ml). No significant difference in flux was pH 5.0 buffer was 3.9±0.56 days and the enzymatic half-life observed across untreated intact skin using the above in the HGP plasma was 6.4±0.84 minutes. In both the studies 30 donors. Hence the hydrochloride salt form of the codrug was regeneration of the parents were observed with the degra tested for higher solubility and flux in aqueous donor for dation of the codrug. The chemical stability indicates that subsequent experiments. the codrug is not stable enough in an aqueous formulation In the next set of experiment hydrochloride salt of codrug for the development of a 7-day transdermal drug delivery (FIG. 12) was used as the donor solution with a concentra system. But still it could be utilized as a learning tool for 35 tion of39.3 mg/ml in 0.3M acetate buffer at pH 5.0. The flux understanding the bioconversion of the codrug in the epi of NTX from the codrug salt was 5.42±1.5 nmol/(cm2 /h) dermal region of the skin as well as for proof of concept that (FIG. 16) and the skin concentration for both NTX and the the codrug methodology could work to enhance the drug codrug increased (data not shown). Therefore a 13 fold flux delivery window following one time application of the MN. enhancement was obtained by using the salt form over the The plasma stability study indicates that even if intact 40 free base form (p<0.005) which can be attributed to the codrug permeates the skin and reaches the systemic circu solubility enhancement of the salt form in an aqueous donor. lation it would cleave back to the parent drugs almost Precipitation issues were observed with purely aqueous immediately thus contributing to the naltrexone flux. The donor, so in vitro formulation development experiment was systemic concentration of diclofenac should not be of con carried out by varying the amount of PG in the donor. 10%, cern since it would be orders of magnitude lower compared 45 25% and 50% PG was used with the salt form and it was to therapeutically relevant systemic doses. observed that while there was no significant difference in In vitro experiments. The goal of the first set of in vitro flux between the aqueous formulation and 10% PG contain experiments was to estimate flux and skin concentration of ing formulation (p<0.05), but flux decreased significantly the base form of the codrug. A saturated donor solution was for higher concentrations of PG. All experiments were used consisting of propylene glycol (PG):ethanol: water=43: 50 carried out with saturated donor solution. The data was 43:14 and had a concentration of 4.5 mg/ml. 4 treatment compared using one way ANOVA and Tukey's posthoc groups each having 3 diffusion cells (MN treatment) were analysis for pair wise multiple comparisons. The 10% PG dosed and then cells were removed every 12 hours to obtain containing formulation was hence chosen for in vivo studies. the skin concentration of the parent drugs as well as the In vivo studies. The pores can be clearly visualized with codrug at different timepoints. An intact skin control was 55 gentian violet in FIG. 17 (A) immediately following MN also present for the entire length of the study (48 hours). treatment. In panel (B) the pores cannot be visualized at day Steady state flux was obtained both from NTX and DIC of 4 (96 h) pores treatment with MN followed by application of 0.42±0.12 nmol/(cm2 /h) and 0.1910.06 nmol/(cm2 /h) NTX HCl gel under occlusion. This is in agreement with respectively (FIG. 14A). The flux ofNTX was significantly previously reported data in pharmacokinetic studies where it higher compared to the flux of DIC (p<0.05). The skin 60 has been shown that the micropores close anywhere between concentration data shows that the codrug degraded in the a 48-72 h timeline following MN treatment under occlusion skin to regenerate back the parent drugs even at 24 hours and (Stan PNAS ref). Pores can be visualized by India iuk amount of parent drugs from the codrug increased with time. staining in panel C where the hydrochloride salt form of the It also showed that the DIC increased in the skin with time codrug was used following MN treatment indicating that the and was higher compared to the NTX at 48 hours. 65 pores can stay open up to a 7 day time point. Therefore, it The codrug cleaved, and NTX flux was obtained from the can be concluded from the studies that the codrug is effective base form of the codrug, although it was not high enough to in keeping the pores open analogous to our hypothesis. US 9,566,341 Bl 65 66 From the studies so far it can be concluded that the codrug microneedle treatment in hairless guinea pigs Journal of cleaved back to parent drugs both in stability studies as well Pharmaceutical Sciences 99(7):3072-3080. as in vitro studies. The salt form of the codrug improved 2. Banks S L, Paudel K S, Brogden N K, Loftin C D and solubility and flux significantly in in vitro studies. Subse Stinchcomb A L (2011) Diclofenac Enables Prolonged quent in vivo studies with the salt form in an optimized 10% 5 Delivery of Naltrexone Through Microneedle-Treated PG containing formulation kept the pores open for a 7 day Skin. Pharm Res 28(5):1211-1219. target drug delivery window but the flux of NTX from the 3. Banks SL, Pinninti RR, Gill HS, Crooks PA, Prausnitz codrug was not high enough to quantify in an hairless guinea M R and Stinchcomb A L (2008) Flux across [corrected] pig model. This could be predicted from clearance estima microneedle-treated skin is increased by increasing tion from previous pharmacokinetic studies with NTX in the 10 charge of naltrexone and naltrexol in vitro. Pharm Res same animal model based on size of treated area since 25(7): 1677-1685. 4. Barr C S, Chen SA, Schwandt ML, Lindell S G, Sun H, Jss* A~Css*Cl Suomi S J and Heilig M (2010) Suppression of alcohol preference by naltrexone in the rhesus macaque: a critical In this case with 4 patches A is 400 MN treatments, flux 15 is 5.5 nmol/cm2 /h and Cl is 5.6 L/h which leads to a Css of role of genetic variation at the micro-opioid receptor gene around 1 ng/ml, the LLOQ on the LCMS assay. Hence locus. Biol Psychiatry 67(1):78-80. reliable pharmacokinetic data could not be obtained from 5. Breese GR, Chu K, Dayas CV, Funk D, Knapp DJ, Koob this codrug. Hence, physicochemical optimization is G F, Le DA, O'Dell LE, Overstreet DH, Roberts A J, required to obtain a molecule that would have higher sta- 20 Sinha R, Valdez G R and Weiss F (2005) Stress enhance bility and permeability in a range that can be clinically ment of craving during sobriety: a risk for relapse. Alco applicable, thus making the codrug approach applicable for hol Clin Exp Res 29(2):185-195. pore lifetime enhancement and NTX delivery. 6. Casswell S and Thamarangsi T (2009) Reducing harm Codrugs will possess a variety of different linking groups from alcohol: call to action. Lancet 373:2247-2257. of diverse susceptibility to hydrolysis including carboxylate 25 7. Cone E J, Gorodetzky C W and Yeh S Y (1974) The ester, carbamate ester and carbonate ester. A nontoxic linker urinary excretion profile of naltrexone and metabolites in moiety will be used in between, ifrequired (e.g. polyethyl man. Drug Metab Dispos 2(6):506-512. ene glycol, alkyl chain). It has been shown that the ionized 8. Cynkowski T, Cynkowska G and Walters K A (2008) form of the drug has a much higher flux compared to the Codrugs: potential therapies for dermatological diseases. unionized form via the MN pathway. This phenomenon is 30 Dermatologic, Cosmeceutic, and Cosmetic Development: mainly due to enhanced solubility of the ionized species in 255-266. formulation (Banks, et al. (2008). 9. Epperson C N, Toll B, Wu R, Amin Z, Czarkowski KA, The main rate limiting step of the codrug approach is the Jatlow P, Mazure CM and O'Malley S S (2010) Explor synthesis of the complex codrugs as well as the stability in ing the impact of gender and reproductive status on formulation. Solubility issues in an aqueous medium might 35 outcomes in a randomized clinical trial of naltrexone also be rate limiting since most of the codrugs have a very augmentation of nicotine patch. Drug Alcohol Depend high predicted log P value. 112(1-2): 1-8. The theoretical flux values required for NTX in vitro to 10. 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The role of apoptosis in therapy and enzyme inhibitor; prophylaxis of epithelial tumours by nonsteroidal anti wherein said first moiety is liuked to said second moiety inflammatory drugs (NSAIDs). British Journal of Derma to form a single chemical entity; tology 156:25-33. 15 wherein the compound has a structure selected from the 119. Davidson JM, Breyer MD 2003. Inflammatory Modu group consisting of: lation and Wound Repair. J Investig Dermatol 120(5):xi x11. 120. Muller-Decker K, Hirschner W, Marks F, Furstenberger G 2002. The Effects of Cyclooxygenase Isozyme Inhibi- 20 tion onlncisional Wound Healing in Mouse Skin. 119(5): rl 1189-1195. Cl~Cl 121. Futagami A, Ishizaki M, Fukuda Y, Kawana S, NH Yamanaka N 0000. Wound Healing Involves Induction of H H Cyclooxygenase-2 Expression in Rat Skin. Lab Invest 25 N'f.f,;"NYO 82(11):1503-1513. 0 122. Banks S, Paudel K, Brogden N, Loftin C, Stinchcomb A 2011. 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Vehicle Composition 45 Influence on the Microneedle-Enhanced Transdermal /'-..o~Nyo Flux of Naltrexone Hydrochloride. Pharmaceutical 0 Research 28(1):124-134. 128. U.S. Patent Application Publication No. 2012/0034293. 129. U.S. Patent Application Publication No. 2011/0245783. 50 130. U.S. Patent Application Publication No. 2011/0245288. 131. U.S. Patent Application Publication No. 2011/0052694. 132. U.S. Patent Application Publication No. 2010/0273895. 0 133. U.S. Patent Application Publication No. 2009/0291128. 134. U.S. Patent Application Publication No. 2009/0247619. 55 135. U.S. Patent Application Publication No. 2009/0246265. 2. The compound of claim 1, wherein the opioid and the 136. U.S. Patent Application Publication No. 2009/0156814. COX enzyme inhibitor are linked by a linker moiety. 137. U.S. Patent Application Publication No. 2009/0143762. 3. The compound of claim 2, wherein the linker moiety is 138. U.S. Patent Application Publication No. 2009/0036523. cleavable. 139. U.S. Patent Application Publication No. 2009/0017102. 60 4. The compound of claim 3, wherein the linker moiety is 140. U.S. Patent Application Publication No. 2008/0076789. cleavable by hydrolysis and/or enzymatic digestion. 141. U.S. Patent Application Publication No. 2008/0008745. 5. The compound of claim 2, wherein the linker moiety 142. U.S. Patent Application Publication No. 2005/0266061. confers increased aqueous solubility to the compound. 143. U.S. Patent Application Publication No. 2005/0154002. 6. A method for treating a subject, comprising 144. U.S. Patent Application Publication No. 2003/0032892. 65 identifying a subject in need of treatment for a condition 145. U.S. Patent Application Publication No. 2002/0111551. selected from the group consisting of narcotic depen 146. U.S. Patent Application Publication No. 2002/0111377. dence, alcohol dependence, amphetamine dependence, US 9,566,341 Bl 75 76 and smoking dependence; and delivering to the subject a therapeutically effective amount of the compound of claim 1. 7. The method of claim 6, wherein following delivery to the subject, the compound is transformed into at least two 5 active drug molecules. 8. The method of claim 7, wherein the transformation is by hydrolysis and/or enzymatic digestion. 9. The method of claim 6, wherein the compound is delivered transdermally. 10 10. The method of claim 9, wherein compound achieves improved duration of microporation-assisted delivery of the opioid as compared to the parent. * * * * *