WO2009076195A1 - Treatment of drug-induced nausea with opioid antagonists - Google Patents

Abstract

The invention provides methods of treating gastrointestinal side effects associated with administration of an antiretroviral agent by co-administration of an opioid antagonist.

Description

TREATMENT OF DRUG-INDUCED NAUSEA WITH OPIOID ANTAGONISTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/992,555 filed December 5, 2007, the contents of which are incorporated herein by reference in its entirety.

INTRODUCTION

Infection with the human immunodeficiency virus (HIV), which may progress to acquired immune deficiency syndrome (AIDS), is a deadly disease that affects many millions of people worldwide (Sande and Volberding, 1999; Selgelid, 2005). If patients are not treated in a timely fashion, the disease can cause morbidity and lead to death because of immune dysfunction and opportunistic infections. To reduce viral loads and improve life expectancy, treatment guidelines require that patients comply with often complicated drug regimens for an extended period of time (Proctor et al., 1999; Bartlett, 2004). The main obstacles to such compliance are treatment-induced adverse effects. Adverse effects not only deteriorate quality of life, but negatively impact compliance (Carr, 2002). Nausea and emesis are examples of drug-induced adverse effects that may affect compliance (Gartland, 2001 ; Lichterfeld et al., 2002; Barlett 2004). Such side effects often limit the usefulness of a drug and may even render the drug unacceptable for use.

While numerous antinausea and antiemetic compositions exist, these compositions often produce their own undesired patient side-effects. The result is often that the patient is put in the position of choosing between the condition she or he seeks to alleviate and the side effect of the therapy. It is desirable to have effective antinausea and antiemetic formulations which have few side effects and permit use of therapeutic agents to effectively treat a condition or illness.

BRIEF DESCRIPTION OF THE INVENTION

Methods, embodying the principles the invention, include relieving gastrointestinal side effects associated with the administration of certain drugs, specifically antiretroviral drugs, for the treatment of viral infections, particularly retroviral infections, e.g., HIV. Among the various classes of antiretroviral drugs used to treat, e.g., HIV are protease inhibitors. Protease inhibitors induce adverse gastrointestinal side effects, such as nausea and vomiting (Elperin and Sax 1996). Specifically, in accordance with the invention, methods are provided for relieving, e.g., alleviating, inhibiting, attenuating, or reducing, nausea, emesis and other adverse gastrointestinal effects, including those associated with treatment of retroviral infections, e.g., HIV, using antiretroviral agents, including protease inhibitors, by administering opioid antagonists, including but not limited to peripherally restricted opioid antagonists, e.g., methylnaltrexone.

In one embodiment, the invention provides methods of treating a gastrointestinal disorder by co-administering an antiretroviral agent, especially a protease inhibitor, and an opioid antagonist. In an illustrated embodiment, nausea and emesis, induced by treatment of HIV with ritonavir, are alleviated by administering an opioid antagonist, such as the peripheral opioid antagonist methylnaltrexone.

Opioid antagonists for use in the methods in accordance with the invention generally include heterocyclic amine compounds that belong to several different classes of compounds. For example, one suitable class is tertiary derivatives of morphinan, and in particular, tertiary derivatives of noroxymorphone. In one embodiment, the tertiary derivative of noroxymorphone is, e.g., naloxone or naltrexone.

In some embodiments, the opioid antagonist is a peripheral opioid antagonist. Suitable peripheral opioid antagonists are generally heterocyclic amine compounds that belong to several different classes of compounds. For example, one suitable class is quaternary derivatives of morphinan, and in particular, quaternary derivatives of noroxymorphone. In one embodiment, the quaternary derivative of noroxymorphone is, e.g., N-methylnaltrexone (or simply methylnaltrexone), N- methylnaloxone, N-methylnalorphine, N-diallylnormorphine, N-allyllevallorphan, or N- methylnalmefene. Another class is N-substituted piperidines. In one embodiment, the N-piperidine is a piperidine-N-alkylcarbonylate, such as, e.g., alvimopan. Yet another class of compounds which may be of value in the methods of the invention is quaternary derivatives of benzomorphans.

In some embodiments of the invention, the opioid antagonist may be a μ-opioid antagonist. In other embodiments, the opioid antagonist may be a κ-opioid antagonist. The invention also encompasses administration of more than one opioid antagonist, including combinations of μ-antagonists, combinations of κ-antagonists, and combinations of μ- and κ-antagonists, for example, a combination of methylnaltrexone and alvimopan, or a combination of naltrexone and methylnaltrexone. BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood and appreciated by reference to the detailed description of specific embodiments presented herein in conjunction with the accompanying drawings of which:

Fig. 1 depicts the chemical structures of naltrexone and methylnaltrexone, and the conversion reaction of naltrexone to methylnaltrexone.

Fig. 2 is a graph showing dose-related effects of treatment with ritonavir on kaolin intake in rats.

Fig. 3 is a graph depicting dose-related effects of pretreatment with naloxone on kaolin intake induced by ritonavir in rats. (NLX, naloxone; RIT, ritonavir)

Fig. 4 is a graph of an area under the curve (AUC) for kaolin intake from time 0 to 120 hr comparing naltrexone treatment v. control (no treatment.)

Fig. 5 is a graph of dose-related effects of pretreatment with methylnaltrexone on kaolin intake induced by ritonavir in rats. (MNTX, methylnaloxone; RIT, ritonavir)

Fig. 6 is a graph of an area under the curve (AUC) for kaolin intake from time 0 to 120 hr comparing methylnaltrexone treatment v control (no treatment.)

Fig. 7 shows HPLC chromatograms of methylnaltrexone and naltrexone in plasma samples, wherein (A) is a chromatogram of a standard plasma extract of methylnaltrexone and naltrexone; (B) is a chromatogram showing a high concentration of methylnaltrexone detected after administration; and (C) is a chromatogram showing methylnaltrexone level 120 minutes after administration. (MNTX, methylnaltrexone; NTX, naltrexone)

DETAILED DESCRIPTION

The invention provides compositions and methods utilizing a combination of an antiretroviral agent, particularly a protease inhibitor such a ritonavir, and opioid antagonists. Methods in accordance with the invention include treating gastrointestinal adverse side effects associated with treatment of, e.g., HIV with protease inhibitors. As explained in the Examples below, combinations of ritonavir and an opioid antagonist, such as methylnaltrexone (MNTX), unexpectedly treated the gastrointestinal side effects of ritonavir.

Before explaining at least one embodiment of the invention, it is to be understood that the invention is not limited in its application to the details set forth in the following description as exemplified by the Examples. Such description and

Examples are not intended to limit the scope of the invention as set forth in the appended claims. The invention is capable of other embodiments or of being practiced or carried out in various ways. While the following detailed description describes the invention through reference to embodiments utilizing ritonavir as the drug, it should be understood that other protease inhibitors or combinations thereof where nausea and other gastrointestinal effects are adverse side effects, may also be suitable for use in accordance with the principles of the invention.

Further, no admission is made that any reference, including any patent or patent document, cited in this specification constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents form part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and applicant reserves the right to challenge the accuracy and pertinency of any of the documents cited herein.

Throughout this disclosure, various aspects of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, as will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, as well as all integral and fractional numerical values within that range. As only one example, a range of 20% to 40% can be broken down into ranges of 20% to 32.5% and 32.5% to 40%, 20% to 27.5% and 27.5% to 40%, etc. Further, any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. Also, as will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than" and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. In the same manner, all ratios disclosed herein also include all subratios falling within the broader ratio. These are only examples of what is specifically intended. Moreover, the phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably.

It is also understood that the use of "comprising," "including," "having," and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items, e.g., other steps, ingredients or elements that do not affect the final result can be added. These terms also encompass the terms "consisting of" and "consisting essentially of." The use of "consisting essentially of means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.

Unless otherwise defined, all scientific and technical terms are used herein according to conventional usage and have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. However, as used herein, the following definitions may be useful in aiding the skilled practitioner in understanding the invention:

"Subject" refers to mammals, e.g., humans, mice, dogs, cats, rats.

"Alkyl" refers to a univalent aliphatic hydrocarbon group which is saturated and which may be straight, branched, or cyclic having from 1 to about 10 carbon atoms in the chain, and all combinations and subcombinations of chains therein. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. "Lower alkyl" refers to an alkyl group having 1 to about 6 carbon atoms.

"Alkenyl" refers to a univalent aliphatic hydrocarbon group containing at least one carbon-carbon double bond and having from 2 to about 10 carbon atoms in the chain, and all combinations and subcombinations of chains therein. Exemplary alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.

"Alkynyl" refers to a univalent aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and having from 2 to about 10 carbon atoms in the chain, and combinations and subcombinations of chains therein. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

"Alkylene" refers to a divalent aliphatic hydrocarbon group having from 1 to about 6 carbon atoms, e.g., -CH₂-, methylene, and all combinations and subcombinations of chains therein. The alkylene group may be straight, branched, or cyclic. There may be optionally inserted along the alkylene group one or more oxygen, sulfur, or optionally substituted nitrogen atoms, wherein the nitrogen substituent has an alkyl group as described previously.

"Alkenylene" refers to a divalent alkylene group containing at least one carbon- carbon double bond, which may be straight, branched, or cyclic. Exemplary alkenylene groups include, but are not limited to, ethenylene (-CH=CH-) and propenylene (-CH=CHCH₂-).

"Cycloalkyl" refers to a saturated monocyclic or bicyclic hydrocarbon ring having from about 3 to about 10 carbons, and all combinations and subcombinations of rings therein. The cycloalkyl group may be optionally substituted with one or more cycloalkyl-group substituents. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

"Acyl" means an alkyl-CO group wherein alkyl is as previously described. Exemplary acyl groups include, but are not limited to, acetyl, propanoyl, 2- methylpropanoyl, butanoyl, and palmitoyl.

"Aryl" refers to an aromatic carbocyclic radical containing from about 6 to about

10 carbons, and all combinations and subcombinations of rings therein. The aryl group may be optionally substituted with one or two or more aryl group substituents. Exemplary aryl groups include, but are not limited to, phenyl and naphthyl.

"Aryl-substituted alkyl" refers to a linear alkyl group, preferably a lower alkyl group, substituted at a terminal carbon with an optionally substituted aryl group, preferably an optionally substituted phenyl ring. Exemplary aryl-substituted alkyl groups include, for example, phenylmethyl, phenylethyl, and 3(4-methylphenyl)propyl.

"Heterocyclic" refers to a monocyclic or multicyclic ring system carbocyclic radical containing from about 4 to about 10 members, and all combinations and subcombinations of rings therein, wherein one or more of the members of the ring is an element other than carbon, for example, nitrogen, oxygen, or sulfur. The heterocyclic group may be aromatic or nonaromatic. Exemplary heterocyclic groups include, for example, pyrrole and piperidine groups.

"Halo" refers to fluoro, chloro, bromo, or iodo.

"Peripheral," in reference to opioid antagonists, designates opioid antagonists that act primarily on physiological systems and components external to the central nervous system. In other words, they exhibit reduced or substantially no central nervous system (CNS) activity. For example, they do not readily cross the blood-brain barrier in an amount effective to inhibit the central effects of opioids, i.e., they do not effectively inhibit the analgesic effects of opioids when administered peripherally, that is, they do not reduce the analgesic effect of the opioids. The peripheral opioid antagonist compounds employed in the methods of the invention suitably exhibit less than about 5-15% of their pharmacological activity in the CNS, with about 0% (i.e., no)

CNS activity being most suitable. The non-centrally acting characteristic of a peripheral opioid antagonist is often related to charge, polarity, and/or size of the molecule or species. For example, peripherally-acting quaternary amine opioid antagonists as described herein are positively charged while the central-acting tertiary amine opioid antagonists are neutral molecules. The peripheral opioid antagonists useful in the present invention are typically μ- and κ-opioid antagonists.

The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" are meant to refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject such as a human. The opioid antagonists in accordance with the invention may be formulated as a free base, neutral or in a salt form. A "pharmaceutically acceptable salt" refers to a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts are non-toxic conventional salts and quaternary ammonium salts of the compounds in accordance with the invention that have properties acceptable for therapeutic use. Such salts may be prepared, e.g., from inorganic or organic bases or acids. For example, acid addition salts may include acetate, ascorbate, benzoate, bisulfate, chloride, citrate, lactate, maleate, oxalate, sulfonate, tartrate and the like. Base salts may include alkali metal salts such as potassium and sodium salts, alkaline earth metals such as calcium and magnesium salts and ammonium salts with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. Additionally, basic nitrogen containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides, as well as long chain halides, and dialkyl or diamyl sulfates. Unless the context clearly indicates to the contrary, terms such as "compounds of the invention", "a compound of the invention" or "compounds in accordance with the invention" and the like, as used herein, are intended to include the chemically feasible pharmaceutically acceptable salts of the referenced compounds. Additional information on suitable pharmaceutically acceptable salts can be found in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams and Wilkens, Philadelphia, PA, which is incorporated herein by reference.

The terms "treating" or "treatment" used herein include any means of control of a medical or pathological condition such as prevention, care, relief of the condition, attenuation, alleviation or a reduction of the condition, and inhibition or arrest of progression of the condition.

As used herein, the term "side effect" is meant to refer to an effect other than the purpose or desired effect of a drug when administered to a subject. Side effects may be beneficial or undesirable, i.e., adverse. In the instant case, undesirable effects often occur after the administration of a nti retroviral agents, such as ritonavir. Such side effects include gastrointestinal side effects such as nausea, emesis, diarrhea and abdominal pain.

As used herein, the term "potency" is meant to refer to the ability or capacity of an antiretroviral agent to treat a retroviral infection, e.g., HIV, in a subject suffering from such infection. Potency may also be expressed as the dose of a drug required to produce a specific effect of a given intensity. In the following description of the methods of the invention, process steps are carried out at room temperature and atmospheric pressure unless otherwise specified.

In accordance with the invention, antiretroviral agents of particular interest for treating infections by retroviruses, primarily HIV, are protease inhibitors. Proteases (also known as peptidases) are enzymes that cleave proteins at specific peptide bonds. Many biological functions are controlled or mediated by proteases and their complementary protease inhibitors.

The genomes of retroviruses encode a protease that is responsible for the proteolytic processing of one or more polyprotein precursors such as the pol and gag gene products. (See, Wellink, Arch. Virol. 98 1 (1988)). Retroviral proteases most commonly process the gag precursor into core proteins, and also process the pol precursor into reverse transcriptase and retroviral protease. The correct processing of these precursor polyproteins by the retroviral protease is necessary for the assembly of new infectious virions. It has been shown that in vitro mutagenesis which produces protease-defective virus leads to the production of immature core forms which lack infectivity. (See, Crawford, J. Virol. 53 899 (1985); Katoh, et al., Virology 145 280 (1985)). Thus, retroviral protease inhibition provides an attractive target for antiviral therapy. (See, Mitsuya, Nature 325 775 (1987)).

Several protease inhibitors are used as efficacious antiretroviral agents; however, these agents produce several adverse effects, especially gastrointestinal side effects. Because compliance with treatment protocols is a prerequisite for effective antiviral therapy in patients with HIV and AIDS, drug-induced adverse effects can prevent compliance. To date, the mechanism by which protease inhibitors cause gastrointestinal effects, e.g., nausea and/or vomiting, has not been investigated. Use of antiemetics has had some effectiveness in reducing nausea and vomiting but these adverse gastrointestinal effects remain a problem for protease inhibitor treatment protocols.

Protease inhibitors are classified as peptidic and nonpeptidic. Peptidic protease inhibitors include amprenavir, atazanavir, fosamprenavir (which is metabolized to the active amprenavir), indinavir, lopinavir, nelfinavir, ritonavir and sequinavir. Nonpeptidic protease inhibitors include darunavir and tipranavir. Of particular interest is ritonavir, which is a potent protease inhibitor alone and is also used in low dose to boost to the activity of other protease inhibitors, e.g., the combination of ritonavir and lopinavir. Ritonavir is a tripeptide, as shown in formula (A) below.

(A)

Ritonavir was originally designed as an inhibitor of HIV protease but was found to also boost the potency of other protease inhibitors. Specifically, ritonavir inhibits cytochrome P450-3A4 (CYP3A4), a liver enzyme involved in the metabolism of protease inhibitors. Inhibition of HIV protease by ritonavir and/or other protease inhibitors potentiated by ritnovir leads to an increase in non-infectious viral particles, resulting in the alleviation of the symptoms of HIV. A side effect of ritonavir, however, is nausea and emesis.

As explained in the Examples below, the inventor evaluated the effects of opioid receptor antagonists on protease-inhibitor induced, such as ritonavir-induced, nausea and vomiting. Both naloxone (a centrally acting opioid antagonist) and methylnaltrexone (a peripherally acting opioid antagonist) were found to reduce ritonavir-induced nausea and vomiting.

The opioid antagonists in accordance with the invention thus include both centrally and peripherally acting opioid antagonists. It is contemplated that those antagonists of particular value are suitably the peripherally restricted opioid antagonists. Especially suitable may be μ-opioid antagonists, especially peripheral μ- opioid antagonists.

Opioid antagonists form a class of compounds that can vary in structure while maintaining their antagonist properties. These compounds include tertiary and quaternary morphinans, in particular noroxymorphone derivatives, N-substituted piperidines, and in particular, piperidine-N-alkylcarboxylates, and tertiary and quaternary benzomorphans, and normorphinan derivatives, in particular 6-carboxy- normorphinan derivatives. Tertiary compound antagonists are fairly lipid soluble and cross the blood-brain barrier easily.

Examples of opioid antagonists, which cross the blood-brain barrier and are centrally (and peripherally) active, include, e.g., naloxone, naltrexone (each of which is commercially available from Baxter Pharmaceutical Products, Inc.), and nalmefene (available, e.g., from DuPont Pharma). Peripherally restricted antagonists, on the other hand, are typically charged, polar, and/or of high molecular weight, and such properties impede their crossing the blood-brain barrier. For example, methylnaltrexone is a quaternary derivative of the tertiary opioid antagonist, naltrexone (Fig. 1 ). Addition of the methyl group to naltrexone forms a compound with greater polarity and lower lipid solubility. Thus, methylnaltrexone does not cross the blood- brain barrier and has the potential for blocking the undesired gastrointestinal adverse effects which are typically mediated by peripherally located receptors.

A peripheral opioid antagonist suitable for use in the invention may be a compound which is a quaternary morphinan derivative, and in particular, a quaternary noroxymorphone of formula (I):

wherein R is alkyl, alkenyl, alkynyl, aryl, cycloalkyl-substituted alkyl, or arylsubstituted alkyl, and X^" is the anion, especially a chloride, bromide, iodide, or methylsulfate anion. The noroxymorphone derivatives of formula (I) can be prepared, for example, according to the procedure in U.S. Patent No. 4,176,186, which is incorporated herein by reference; see also, U.S. Patent Nos. 4,719,215; 4,861 ,781 ; 5,102,887; 5,972,954; and 6,274,591 ; U.S. Patent Application Nos. 2002/0028825 and 2003/0022909; and PCT publication Nos. WO 99/22737 and WO 98/25613, all of which are hereby incorporated by reference.

A compound of formula (I) of particular value is N-methylnaltrexone (or simply methylnaltrexone), wherein R is cyclopropylmethyl as represented in formula (II):

wherein X^" is as described above. As noted above, methylnaltrexone is a quaternary derivative of the opioid antagonist naltrexone. Methylnaltrexone exists as a salt (e.g., N-methylnaltrexone bromide) and the terms "methylnaltrexone" or "MNTX", as used herein, therefore embrace such salts. "Methylnaltrexone" or "MNTX" thus specifically includes, but is not limited to, bromide salts, chloride salts, iodide salts, carbonate salts, and sulfate salts of methylnaltrexone. A variety of names used for the bromide salt of MNTX in the literature, for example, include: methylnaltrexone bromide; N- methylnaltrexone bromide; naltrexone methobromide; naltrexone methyl bromide; SC- 37359; MRZ-2663-BR; and N-cyclopropylmethylnoroxy-morphine-methobromide.

Methylnaltrexone is commercially available from, e.g., Mallinckrodt

Pharmaceuticals, St. Louis, Mo. Methylnaltrexone is provided as a white crystalline powder, freely soluble in water, typically as the bromide salt. The compound as provided is 99.4% pure by reverse phase HPLC, and contains less than 0.011 % unquaternized naltrexone by the same method. Methylnaltrexone can be prepared as a sterile solution at a concentration of, e.g., about 5 mg/mL.

Other suitable peripheral opioid antagonists may include N-substituted piperidines, and in particular, piperidine-N-alkylcarboxylates as represented by formula (III):

wherein R¹ is hydrogen or alkyl; R² is hydrogen, alkyl, or alkenyl; R³ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl- substituted alkyl, or aryl-substituted alkyl; R⁴ is hydrogen, alkyl, or alkenyl; A is OR⁵ or NR⁶R⁷; wherein R⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl- substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl; R⁶ is hydrogen or alkyl; R⁷ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl- substituted alkyl, cycloalkenyl-substituted alkyl or aryl-substituted alkyl, or alkylene- substituted B or together with the nitrogen atom to which they are attached, R⁶ and R⁷ form a heterocyclic ring selected from pyrrole and piperidine; B is

wherein R⁸ is hydrogen or alkyl; R⁹ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl or aryl- substituted alkyl or together with the nitrogen atom to which they are attached, R⁸ and R⁹ form a heterocyclic ring selected from pyrrole and piperidine; W is OR¹⁰, NR¹¹R¹², or OE; wherein R¹⁰ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl- substituted alkyl, cycloalkenyl-substituted alkenyl, or aryl-substituted alkyl; R¹¹ is hydrogen or alkyl; R¹² is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, aryl-substituted alkyl, or alkylene-substituted C(=O)Y or, together with the nitrogen atom to which they are attached, R¹¹ and R¹² form a heterocyclic ring selected from pyrrole and piperidine; E is

alkylene-substituted (C=O)D, or -R¹³OC(=O)R¹⁴; wherein R¹³ is alkyl-substituted alkylene; R¹⁴ is alkyl; D is OR¹⁵ or NR¹⁶R¹⁷; wherein R¹⁵ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl substituted alkyl, or aryl-substituted alkyl; R¹⁶ is hydrogen, alkyl, alkenyl, aryl, aryl-substituted alkyl, cycloalkyl, cycloalkenyl, cycloalkyl substituted alkyl, or cycloalkenyl-substituted alkyl;

R¹⁷ is hydrogen or alkyl or, together with the nitrogen atom to which they are attached,

R¹⁶ and R¹⁷ form a heterocyclic ring selected from the group consisting of pyrrole or piperidine;

Y is OR¹⁸ or NR¹⁹R²⁰; wherein R¹⁸ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl- substituted alkyl; R¹⁹ is hydrogen or alkyl; R²⁰ is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkylsubstituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl or, together with the nitrogen atom to which they are attached, R¹⁹ and R²⁰ form a heterocyclic ring selected from pyrrole and piperidine; R²¹ is hydrogen or alkyl; and n is 0 to 4.

Particular piperidine-N-alkylcarbonylates which may be of value are N- alkylamino-3,4,4 substituted piperidines, such as alvimopan represented below as formula (IV):

Suitable N-substituted piperidines may be prepared as disclosed in U.S. Patent

Nos. 5,270,328; 6,451 ,806; 6,469,030, all of which are hereby incorporated by reference. Alvimopan is available from Adolor Corp., Exton, PA. Such compounds have moderately high molecular weights, a zwitterion form, and a polarity that prevent penetration of the blood-brain barrier.

Still other suitable peripheral opioid antagonist compounds may include quaternary benzomorphan compounds. Quaternary benzomorphan compounds which may be employed in the methods of the present invention have the following formula (V):

wherein R¹ is hydrogen, acyl, or acetoxy; and R² is alkyl or alkenyl; R is alkyl, alkenyl, or alkynyl and X^" is an anion, especially a chloride, bromide, iodide, or methylsulfate anion.

Specific quaternary derivatives of benzomorphan compounds that may be employed in the methods of the invention include the following compounds of formula (V): 2'-hydroxy-5,9-dimethyl-2,2-diallyl-6,7-benzomorphanium-bromide; 2'-hydroxy-5,9- dimethyl-2-n-propyl-2-allyl-6,7-benzomorphanium-bromide; 2'-hydroxy-5,9-dimethyl-2- n-propyl-2-propargyl-6,7-benzomorphanium-bromide; and 2'-acetoxy-5,9-dimethyl-2-n- propyl-2-allyl-6,7-benzomorphanium-bromide.

Other quaternary benzomorphan compounds that may be employed in methods of the invention are described, for example, in U.S. Pat. No. 3,723,440, the entire disclosure of which is incorporated herein by reference.

Other peripheral opioid anatagonists may include 6-carboxy-normorphinan derivatives, particularly N-methy-C-normorphinan derivatives, as described in U.S. Application Serial No. 11/888,955, entitled "6-Carboxy-Normorphinan Derivatives, Synthesis and Uses Thereof," hereby incorporated in its entirety herein by reference. As described above, compounds of the invention may suitably exist and be formulated as pharmaceutically acceptable salts.

The compounds employed in methods of the invention may exist in prodrug form. As used herein, "prodrug" is intended to include any agent which releases or is converted to the active parent drug according to formulas (I) to (V) or other formulas or compounds that are metabolized in vivo to an active drug or other compounds employed in the methods of the invention in vivo when such prodrug is administered to a mammalian subject. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.

Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or carboxylic acid, respectively. Other examples include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups; and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, iso- propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and the like.

As noted, the compounds employed in the methods of the invention may be prepared in a number of ways well known to those skilled in the art. All preparations disclosed in association with the invention are contemplated to be practiced on any scale, including milligram, gram, multigram, kilogram, multikilogram, or commercial pharmaceutical scale.

Compounds employed in methods of the invention may contain one or more asymmetrically-substituted carbon atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. It is well known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic form, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers.

In some embodiments of the invention, the opioid antagonist may be a μ-opioid antagonist. In other embodiments, the opioid antagonist may be a κ-opioid antagonist. The invention also encompasses administration of more than one opioid antagonist, including combinations of μ-opioid antagonists, combinations of κ-opioid antagonists, and combinations of μ- and κ-opioid antagonists, for example, a combination of methylnaltrexone and alvimopan.

In an illustrated embodiment, the inventor found that methylnaltrexone and naltrexone significantly attenuated protease inhibitor-induced nausea and vomiting. Methylnaltrexone and other peripherally restricted opioid antagonists generally may have clinical value in treating protease inhibitor-induced nausea and emesis, especially without affecting analgesia from opioid treatment, if such treatment is given.

As explained in the Examples below, a rat pica model was used to evaluate the symptoms of nausea and emesis. Rats exposed to a variety of emetic stimuli feed on non-nutritive substances like clay, a phenomenon called pica behavior. Pica behavior in rats is thus analogous to nausea and vomiting in humans and other species (Mitchell et al., 1976; Takeda et al., 1993). Pica behavior in rats is mediated by mechanisms and receptors involving serotonin and dopamine, similar to those in humans and other species (Takeda et al., 1993; Takeda et al., 1995). The rat pica model has been used extensively and validated in several studies researching antiemetic drugs (Takeda et al., 1995; Aung et al., 2004). A dose-dependent pica response induced by ritonavir has already been demonstrated (Aung et al., 2005). As demonstrated below, the inventor used the pica model to demonstrate that treatment with methylnaltrexone and naltrexone significantly reduced ritonavir-induced pica.

In the treatment of rodents with methylnaltrexone, it has been suggested that methylnaltrexone may be partially metabolized via demethylation as measured by the exhaled ¹⁴Cθ₂ breath test (Kotake et al., 1989). However, systemic methylnaltrexone administration did not antagonize morphine-induced analgesia in rats subjected to the radiant-heat tail-flick assay, and morphine-induced reduction in gastrointestinal tract movement was antagonized by methylnaltrexone in a dose-related manner (Gmerek et al., 1986). HPLC was used to measure plasma naltrexone levels after methylnaltrexone administration. No detectable naltrexone level was found with the highest methylnaltrexone dose, indicating that at the doses studied, methylnaltrexone was not demethylated.

The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, e.g., any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical (as by powder, ointment, drops, transdermal patch, or iontophoretic device), transdermal, sublingual, intramuscular, infusion, intravenous, pulmonary, intramuscular, intracavity, as an aerosol, aural (e.g., via eardrops), intranasal, inhalation, intraocular, or subcutaneous.

Additionally, the opioid antagonists may be administered as an enterically coated tablet or capsule. In some embodiments, the opioid antagonist is administered by a slow infusion method or by a time-release or controlled-release method or as a lyophilized powder.

When administered, the compounds of the invention are given in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions or preparations. Such preparations may routinely contain salts, buffering agents, preservatives, and optionally other therapeutic ingredients.

When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, succinic, naphthalene-2-sulfonic, pamoic, 3-hydroxy-2- naphthalenecarboxylic, and benzene sulfonic.

Suitable buffering agents include, but are not limited to, acetic acid and salts thereof (1-2% w/v); citric acid and salts thereof (1-3% w/v); boric acid and salts thereof (0.5-2.5% w/v); and phosphoric acid and salts thereof (0.8-2% w/v).

Suitable preservatives include, but are not limited to, benzalkonium chloride

(0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v); and thimerosal (0.004-0.02% w/v). For ease of administration, a pharmaceutical composition of the opioid antagonist may also contain one or more pharmaceutically acceptable excipients, such as lubricants, diluents, binders, carriers, and disintegrants. Other auxiliary agents may include, e.g., stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, coloring, flavoring, and/or aromatic active compounds.

A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. For example, suitable pharmaceutically acceptable carriers, diluents, solvents, or vehicles include, but are not limited to, water, salt (buffer) solutions, alcohols, gum arabic, mineral and vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, vegetable oils, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxyl methylcellulose, polyvinyl pyrrolidone, etc. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms may be ensured by the inclusion of various antimicrobial, e.g., antibacterial and antifungal, agents such as paraben, chlorobutanol, phenol, sorbic acid and the like.

If a pharmaceutically acceptable solid carrier is used, the dosage form of the compounds suitable for use in methods of the invention may be tablets, capsules, powders, suppositories, or lozenges. If a liquid carrier is used, soft gelatin capsules, transdermal patches, aerosol sprays, topical cream, syrups or liquid suspensions, emulsions, or solutions may be the dosage form.

For parenteral application, particularly suitable are injectable, sterile solutions, preferably nonaqueous or aqueous solutions, as well as dispersions, suspensions, emulsions, or implants, including suppositories. Ampoules are often convenient unit dosages. Injectable depot-form may also be suitable and may be made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide- polyglycolide, poly(orthoesters), and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled.

For enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules such as soft gelatin capsules. A syrup, elixir, or the like can be used wherein a sweetened vehicle is employed. As noted, other delivery systems may include time-release, delayed-release, or sustained-release delivery systems. Such systems can avoid repeated administrations of the compounds of the invention, increasing convenience to the patient and the physician and maintaining sustained plasma levels of compounds. Many types of controlled-release delivery systems are available and known to those of ordinary skill in the art. Sustained- or controlled-release compositions can be formulated, e.g., as liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc. Thus, the opioid antagonists in accordance with the invention may be administered as an enterically coated tablet or capsule. In some embodiments, the opioid antagonist is administered by a slow infusion method or by a time-release or controlled-release method or as a lyophilized powder.

For example, compounds of this invention may be combined with pharmaceutically acceptable sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. A sustained-release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix may be desirably chosen from biocompatible materials such as liposomes; polymer-based system such as polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polysaccharides, polyamino acids, hyaluronic acid, collagen, chondroitin sulfate, polynucleotides, polyvinyl propylene, polyvinyl pyrrolidone, and silicone; nonpolymer system such as carboxylic acids, fatty acids, phospholipids, amino acids, and lipids such as sterols; hydrogel release systems; silastic systems; peptide-based systems; implants and the like. Specific examples include, but are not limited to: (a) erosional system in which the polysaccharide is contained in a form within a matrix, found in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152 (herein incorporated by reference in their entireties), and (b) diffusional system in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974, and 5,407,686 (herein incorporated by reference in their entireties). In addition, pump-based hard-wired delivery system can be used, some of which are adapted for implantation. Suitable enteric coatings are described in PCT publication No. WO 98/25613 and U.S. Patent No. 6,274,591 , both incorporated herein by reference. Sustained- or controlled-release compositions may also be formulated as those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc.

Respecting methylnaltrexone specifically, aqueous formulations may include a chelating agent, a buffering agent, an anti-oxidant and, optionally, an isotonicity agent, preferably pH adjusted to between 3.0 and 3.5. Formulations that are stable to autoclaving and long term storage are described in U.S. Patent Application Serial No. 10/821811 , published as 2004/0266806, entitled "Pharmaceutical Formulation," the disclosure of which is incorporated herein by reference. Formulations of methylnaltrexone with increased shelf-life are also described in International Patent Publication No. WO 2008/191 15, entitled "Formulations for Parenteral Delivery of Compounds and Uses Thereof," hereby incorporated by reference. Lyophilized formulations of methylnaltrexone are described in U.S. Patent Application Serial No. 11/899,724 and formulations comprising particles containing methylnaltrexone are described in U.S. Patent No. 6,419,959, each of which is incorporated herein by reference. Formulations suitable for transdermal delivery of methylnaltrexone are described in International Patent Publication No. 2007/41544, hereby incorporated by reference.

In one embodiment, compounds of the invention are administered in a dosing regimen which provides a continuous dosing regimen of the compound to a subject, e.g., a regimen that maintains minimum plasma levels of the opioid antagonist, and preferably eliminates the spikes and troughs of a drug level with conventional regimens. Suitably, a continuous dose may be achieved by administering the compound to a subject on a daily basis or controlled release basis using any of the delivery methods disclosed herein. In one embodiment, the continuous dose may be achieved using continuous infusion to the subject, or via a mechanism that facilitates the release of the compound over time, for example, a transdermal patch, or a sustained release formulation. Suitably, compounds of the invention are continuously released to the subject in amounts sufficient to maintain a concentration of the compound in the plasma of the subject effective to inhibit or reduce nausea, emesis and other adverse gastrointestinal effects induced by treatment with a protease inhibitor such as ritonavir.

Compounds in accordance with the invention, whether provided alone or in combination with other therapeutic agents, are provided in an antinausea and antiemetic effective amount. It will be understood, however, that the total dosage of the compounds and compositions of the invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically- effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, one technique is to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

If desired, an effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up a daily dose. As noted, those of ordinary skill in the art can readily optimize effective doses and co-administration regimens (as described herein) as determined by good medical practice and the clinical condition of the individual patient.

Generally, oral doses of the opioid antagonists, particularly peripheral antagonists, will range from about 0.01 to about 80 mg/kg body weight per day. It is expected that oral doses in the range from 1 to 20 mg/kg body weight will yield the desired results. Generally, parenteral administration, including intravenous and subcutaneous administration, will range from about 0.001 to 5 mg/kg body weight. It is expected that doses ranging from 0.05 to 0.5 mg/kg body weight will yield the desired results. Dosages may be adjusted appropriately to achieve desired drug levels, local or systemic, depending on the mode of administration. For example, it is expected that the dosage for oral administration of the opioid antagonists in an enterically coated formulation would be from 10 to 30% of the non-coated oral dose. In the event that the response in a patient is insufficient with such doses, even higher doses (or effectively higher than 30% dosage by a different, more localized delivery route) may be employed to the extent that the patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds. Appropriate system levels can be determined by, for example, measurement of the patient's plasma level of the drug using routine HPLC methods known to those skilled in the art.

In illustrated embodiments of the invention, the opioid antagonists are co- administered with an antiretroviral agent, e.g., ritonavir, or a combination of antiretroviral agents. The term "co-administration" is meant to refer to a combination therapy by any administration route in which two or more agents are administered to a patient or subject. Co-administration of agents may also be referred to as combination therapy or combination treatment. The agents may be in the same dosage formulations or separate formulations. For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times. The agents may be administered simultaneously or sequentially (e.g., one agent may directly follow administration of the other or the agents may be given episodically, e.g., one can be given at one time followed by the other at a later time, e.g., within a week), as long as they are given in a manner sufficient to allow both agents to achieve effective concentrations in the body. The agents may also be administered by different routes, e.g., one agent may be administered intravenously while a second agent is administered intramuscularly, intravenously, or orally. In other words, the co-administration of the opioid antagonist compound in accordance with the invention with, e.g., ritonavir, or a combination of ritonavir and one or more other protease inhibitors is suitably considered a combined pharmaceutical preparation, which contains an opioid antagonist and a protease inhibitor, e.g., ritonavir, or a combination of protease inhibitors such as ritonavir and another protease inhibitor, the preparation being adapted for the administration of the peripheral opioid antagonist on a daily or intermittent basis, and the administration of protease inhibitor, e.g., ritonavir, on a daily or intermittent basis. Thus, the opioid antagonists may be administered prior to, concomitant with, or after administration of the protease inhibitor, e.g., ritonavir. In an illustrated embodiment, particularly suitable is administration of the opioid antagonist prior to administration of ritonavir.

Co-administrable agents also may be formulated as an admixture, as, for example, in a single formulation or single tablet. These formulations may be parenteral or oral, such as the formulations described, e.g., in U.S. Patent Nos.

6,277,384; 6,261 ,599; 5,958,452 and PCT Publication No. WO 98/25613, each of which is hereby incorporated by reference.

The present invention is further explained by the following examples, which should not be construed by way of limiting the scope of the present invention.

EXAMPLES

Example 1 : Animal model for nausea Rats react to emetic stimuli by altered feeding habits, manifested as increased consumption of non-nutritive substances such as kaolin (a type of clay), known as pica (Mitchell et al., 1976; Takeda et al., 1993; Takeda et al., 1995). The inventor has quantified kaolin consumption as a measure of nausea in experimental animals, and observed that drug-induced pica consumption can be reduced by selected pharmacological agents (Aung et al., 2003; Aung et al., 2005).

The experimental protocols described herein were approved by the Institutional Animal Care and Use Committee or IACUC of the University of Chicago.

Example 2: In vivo study

Adult male Wistar strain rats (Harlan Sprague Dawley, Indianapolis, IN) weighing between 150-300 g were used. All the animals were housed in standard isolation cages (45cm x 35cm x 25cm) in environmentally controlled conditions with a 12 hr light/12 hr dark cycle. Rats were allowed free access to water, standard laboratory rat chow (Harlan-Teklad, Madison, Wl), and kaolin (see below), placed in separated containers continuously available throughout the experiment.

Prior to the beginning of observation (Day 0), there was a 3-day adaptation period.

Preparation of kaolin

Kaolin pellets were prepared based on the method previously described (Mitchell et al., 1976; Takeda et al., 1995). In brief, 99 g of pharmacological grade kaolin (or hydrated aluminum silicate; Fisher, Fair Lawn, NJ) were mixed with 1 g of acacia (Fisher), i.e., in a 99:1 ratio, with distilled water to form a thick paste. The kaolin paste was rolled on a stainless steel tray and cut into pieces in the shape and size similar to regular rat chow pellets. The pellets were placed on steel trays, and completely dried at room temperature for 72 hr.

Measurement of pica (kaolin intake)

Ritonavir was the protease inhibitor used, and the rats were divided into four groups. There were 6-7 rats in each of the four groups: vehicle plus vehicle, vehicle plus ritonavir, naloxone plus ritonavir, and methylnaltrexone plus ritonavir. Rats received ritonavir, 20 mg/kg (Abbott Laboratories, North Chicago, IL), orally via a gavage tube in the morning on 2 consecutive days (0 hr and 24 hr) (Denissen et al., 1997; Yamaji et al., 1999; Shibata et al., 2002). Vehicle, naloxone 0.1 mg/kg or 0.3 mg/kg (Sigma, St. Louis, MO), or methylnaltrexone 0.3 mg/kg, 1.0 mg/kg, or 3.0 mg/kg (Mallinckrodt Chemicals, St. Louis, MO) pretreatment was administered intraperitoneally (Aung et al., 2003), 30 min before ritonavir administration of 20 mg/kg. Rats were observed immediately, at 15 min, 2 hr, and daily thereafter for signs of distress such as restlessness, respiratory distress, or diarrhea following test drug administrations.

During the experiment, kaolin and food pellets were weighed to the nearest 0.1 g and replaced in the containers every morning at the same time after collecting the remaining kaolin and food from the previous day. Kaolin and food intake was measured every 24 hr for 5 days. Kaolin and food pellets were weighed to the nearest 0.1 g, and placed in containers within the cage each morning. The kaolin and food remaining from the previous day was collected, dried for 72 hr, and weighed. Daily kaolin and food intake was measured for 5 days following the first ritonavir treatment.

Blood sample collection

In some experiments, blood samples were collected for the measurement of plasma naltrexone level, an indicator of possible demethylation of methylnaltrexone (Kotake et al., 1989). The rat was restrained and the tail vein was exposed. The tail was dipped in warm water to help dilate the vessel. A small rubber band was placed around the base of the tail. Blood samples were collected using a microhematocrit tube inserted into the hub of a small needle that was placed into the vein. Blood samples were collected at 0, 10, 20, 30, 60, 90, or 120 min after the first dose of methylnaltrexone.

Measurement of naltrexone and methylnaltrexone concentrations

Plasma naltrexone and methylnaltrexone levels were determined by high performance liquid chromatography (HPLC) adapted from a previously reported method (Osinski et al., 2002). Naltrexone and methylnaltrexone were separated from plasma by the solid phase extraction (SPE) technique. Plasma samples (100 to 200 μL) diluted in water were passed through SPE columns (Varian CBA columns, 100 mg,

Harbor City, CA) that had been conditioned with n-propanol and water. The analytes were eluted from the columns by the mixture of n-propanol and trifluoroacetic acid (25 mM) in an aqueous solution prepared in 2:1 proportion. The eluate was evaporated to dryness in a stream of nitrogen at 55°C. The residue was reconstituted in the mobile phase, filtered through the nylon HPLC syringe filter and subjected to analysis. In HPLC analysis, an electrochemical detector has high sensitivity for automated, analytical chromatography of electroactive compounds. The HPLC system (Shimadzu Corporation, Kyoto, Japan) and electrochemical detector (ESA Coulochem, model 5100A, Chelmsford, MA) consisted of an LC-10AD pump, SCL-10A system controller, and SIL-10A auto injector equipped with sample cooler. The electrochemical detector worked at the following settings: detector 1 , +360 mV, detector 2, +600 mV, guard cell +650 mV. Data were collected using EZChrom 2-2 HPLC software. In the mobile phase, sodium phosphate 30 mM, sodium acetate 20 mM, methanol 6%, tetrahydrofuran 1% at pH 4.2 were used. The system was calibrated daily in the range of 5 to 100 ng/mL. The practical limit of detection for plasma samples was approximately 2 ng/mL (100 pg/injection).

Statistical analysis

Data were expressed in mean ± standard error (S. E.). Area under the concentration curve (AUC) was calculated. Data were analyzed using a two-way analysis of variance (ANOVA) with group and time as the two factors. Statistical significance was considered at P < 0.05.

The results of the foregoing experiments are discussed below.

Effects of naloxone on ritonavir-induced nausea and vomiting

In rats treated with saline, less than 1.0 g kaolin was consumed daily during 5 consecutive days. After oral ritonavir doses of 10 or 20 mg/kg, kaolin consumption increased significantly at 24 to 48 hr in a dose-related manner (Fig. 2; P < 0.01 ). At 30 mg/kg, kaolin intake did not increase further suggesting possible toxic effects at the high dose, which was supported by the reduction of food intake. The ritonavir dose used for this study was 20 mg/kg.

Fig. 3 shows that the ritonavir-induced increase in kaolin intake was attenuated by 0.1 or 0.3 mg/kg pretreatment with naloxone [P < 0.01 ). The area under the curve (AUC) for kaolin intake from time 0 to 120 hr, was: vehicle plus vehicle, 87 ± 8.1 g^«hr; 0.3 mg/kg naloxone plus vehicle, 86 ± 9.2 g^«hr, vehicle plus ritonavir, 351 ± 18.2 g^«hr; 0.1 mg/kg naloxone plus ritonavir, 264 ± 16.7 g^«hr, and 0.3 mg/kg naloxone plus ritonavir, 205 ± 1 1.3 g*hr (Fig. 4). Naloxone significantly reduced kaolin intake induced by ritonavir (P < 0.01 compared to vehicle). Naloxone (0.3 mg/kg) alone did not significantly affect kaolin intake. Effects of methylnaltrexone on ritonavir-induced nausea and vomiting

Effects of pretreatment with methylnaltrexone on kaolin intake after ritonavir are shown in Fig. 5. Kaolin intake induced by ritonavir was attenuated by methylnaltrexone in a dose-dependent manner (P < 0.01 ). The AUC for kaolin intake from time 0 to 120 hr was: vehicle plus vehicle, 92 ± 8.6 g*hr; vehicle plus ritonavir, 360 ± 15.7 g^«hr; methylnaltrexone (0.3 mg/kg) plus ritonavir, 302 ± 13.2 g^«hr; methylnaltrexone (1.0 mg/kg) plus ritonavir, 242 ± 14.9 g*hr; and methylnaltrexone (3.0 mg/kg) plus ritonavir, 168 ± 11.5 g^«hr (Fig. 6).

With methylnaltrexone (3.0 mg/kg) alone, kaolin intake was not significantly affected. In all test groups, food intake was not significantly affected.

HPLC analysis of naltrexone

No detectable naltrexone level was found in association with methylnaltrexone (3.0 mg/kg). In contrast, methylnaltrexone levels were detected after it was administered. Chromatograms are shown in Fig. 7. But, at all measured time points, no naltrexone level was detected, as shown in (B) and (C).

In summary, the invention provides methods for treating drug-induced gastrointestinal side effects, such as nausea and emesis induced by an antiretroviral agent, e.g., a protease inhibitor, utilizing opioid antagonists, particularly, peripherally restricted opioid antagonists, such as methylnaltrexone.

The invention has now been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

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Claims

1. A method of treating adverse gastrointestinal side effects associated with treatment with an antiretroviral agent, comprising administering to a patient suffering therefrom, in combination with the antiretroviral agent, an amount of an opioid antagonist sufficient to treat drug-induced gastrointestinal side effects.

2. The method of claim 1 , wherein the side effect is nausea or emesis or both.

3. The method of claim 1 , wherein the opioid antagonist is administered prior to administering the antiretroviral agent.

4. The method of claim 1 , wherein the antiretroviral agent is a protease inhibitor.

5. The method of claim 4, wherein the protease inhibitor is ritonavir.

6. The method of claim 1 , wherein the opioid antagonist is naloxone.

7. The method of claim 1 , wherein the opioid antagonist is a peripheral opioid antagonist.

8. The method of claim 7, wherein the peripheral opioid antagonist is methylnaltrexone.

9. The method of claim 7, wherein the peripheral opioid antagonist is a piperidine- N-alkylcarboxylate compound.

10. The method of claim 7, wherein the peripheral opioid antagonist is a quaternary morphinan compound.

11. The method of claim 10, wherein the quaternary morphinan compound comprises a quaternary salt of a compound selected from the group consisting N- methylnaltrexone, N-methylnaloxone, N-methylnalorphine, N-diallylnormorphine, N- allyllevallorphan, and N-methylnalmefene.

12. The method of claim 1 , wherein the opioid antagonist is a μ-opioid antagonist, a K- opioid antagonist or combinations thereof.

13. A method of treating an adverse gastrointestinal side effect associated with administration of antiretroviral agents to a HIV-infected human subject, comprising coadministering to the human subject an antiretroviral effective amount of a protease inhibitor, which is ritonavir or a combination of ritonavir and another protease inhibitor, and an amount of an opioid antagonist effective to treat the adverse side effect.

14. The method of claim 13, wherein the adverse side effect is nausea, emesis, diarrhea, abdominal pain or combinations thereof.

15. The method of claim 13, wherein the opioid antagonist is a quaternary morphinan compound.

16. The method of claim 15, wherein the quaternary morphinan compound comprises a quaternary salt of a compound selected from the group consisting of N- methylnaltrexone, N-methylnaloxone, N-methylnalorphine, N-diallylnormorphine, N- allyllevallorphan, and N-methylnalmefene.

17. The method of claim 16, wherein the quaternary salt of a quaternary morphinan compound is N-methylnaltrexone.

18. A method of treating nausea and emesis following administration of an effective amount of ritonavir or a combination of ritonavir and another protease inhibitor to a HIV-infected subject in need thereof, comprising administering an effective amount of an opioid antagonist.

19. The method of claim 18, wherein the opioid antagonist is administered prior to administering the ritonavir or the combination with another protease inhibitor.

20. The method of claim 18, wherein the administration of the opioid antagonist is oral, sublingual, intramuscular, subcutaneous, intravenous, and transdermal.

21. The method of claim 20, wherein the administration is oral.

22. The method of claim 18, wherein the opioid antagonist is methylnaltrexone.

23. The method of claim 22, wherein the effective amount of methylnaltrexone is from about 0.001 mg/kg to about 80 mg/kg of body weight per day, from about 0.05 mg/kg to about 50 mg/kg of body weight per day or from about 1 mg/kg to about 20 mg/kg of body weight per day.

24. A method of treating a retrovirual infection in a human subject in need of such treatment, comprising co-administering an antiretroviral amount of ritonavir or a combination of ritonavir and another protease inhibitor and an amount of an opioid antagonist effective attenuate the frequency and/or severity of ritonavir or combination- induced gastrointestinal side effects in the subject.