WO2005097203A2 - Serum protein conjugates of ion channel modulating compounds and uses thereof - Google Patents
Serum protein conjugates of ion channel modulating compounds and uses thereof Download PDFInfo
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- WO2005097203A2 WO2005097203A2 PCT/US2005/011322 US2005011322W WO2005097203A2 WO 2005097203 A2 WO2005097203 A2 WO 2005097203A2 US 2005011322 W US2005011322 W US 2005011322W WO 2005097203 A2 WO2005097203 A2 WO 2005097203A2
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- 0 CS(SCCNCC(ON(C(CC1*)=O)C1=O)=O)(=O)=O Chemical compound CS(SCCNCC(ON(C(CC1*)=O)C1=O)=O)(=O)=O 0.000 description 4
- XDTMQSROBMDMFD-UHFFFAOYSA-N C1CCCCC1 Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- VBHQKCBVWWUUKN-OWZOALSMSA-N COc1ccc(CCOC(CCCC2)[C@@H]2N(CC2)C[C@@H]2O)cc1OC Chemical compound COc1ccc(CCOC(CCCC2)[C@@H]2N(CC2)C[C@@H]2O)cc1OC VBHQKCBVWWUUKN-OWZOALSMSA-N 0.000 description 1
- PPWNVROXFCHKCK-YPAWHYETSA-N COc1ccc(CCO[C@H](CCCC2)[C@@H]2N(CC2)C[C@@H]2OC(CNCCSS(C)(=O)=O)=O)cc1OC Chemical compound COc1ccc(CCO[C@H](CCCC2)[C@@H]2N(CC2)C[C@@H]2OC(CNCCSS(C)(=O)=O)=O)cc1OC PPWNVROXFCHKCK-YPAWHYETSA-N 0.000 description 1
- QPUYECUOLPXSFR-UHFFFAOYSA-N Cc1cccc2ccccc12 Chemical compound Cc1cccc2ccccc12 QPUYECUOLPXSFR-UHFFFAOYSA-N 0.000 description 1
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- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6445—Haemoglobin
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- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/12—Oxygen or sulfur atoms
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- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/36—Oxygen or sulfur atoms
- C07D207/40—2,5-Pyrrolidine-diones
- C07D207/404—2,5-Pyrrolidine-diones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
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- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/36—Oxygen or sulfur atoms
- C07D207/40—2,5-Pyrrolidine-diones
- C07D207/416—2,5-Pyrrolidine-diones with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to other ring carbon atoms
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- C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
- C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
- C07D295/096—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
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- C07F7/02—Silicon compounds
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Abstract
Serum protein conjugates of ion channeling modulating compounds, including, for example, serum protein conjugates of the ion channel modulating compound of the following formula (I) are described herein, as well as methods of making and using such serum protein conjugates and pharmaceutical compositions containing such serum protein conjugates.
Description
SERUM PROTEIN CONJUGATES OF ION CHANNEL MODULATING COMPOUNDS AND USES THEREOF
FIELD OF THE INVENTION The field of the compounds and methods described herein is generally serum protein conjugates of ion channel modulating compounds and their uses, and includes, but is not limited to serum protein conjugates of ion channel modulating compounds and their uses as antiarrhythmics, particularly for the treatment and/or prevention of atrial fibrillation (AF) and for the treatment and/or prevention of atrial flutter. Also described herein are methods for preparing serum protein conjugates of ion channel modulating compounds.
BACKGROUND OF THE INVENTION Ion channels are ubiquitous membrane proteins in the cells of warm-blooded animals such as mammals. Their critical physiological roles include control of the electrical potential across the membrane, mediation of ionic and fluid balance, facilitation of neuromuscular and neuronal transmission, rapid transmembrane signal transduction, and regulation of secretion and contractility. For example, cardiac ion channels are proteins that reside in the cell membrane and control the electrical activity of cardiac tissue. In response to external stimuli, such as changes in potential across the ceil membrane, these ion channels can form a pore through the cell membrane, and allow movement of specific ions into or out of the cell. The integrated behavior of thousands of ion channels in a single cell results in an ionic current, and the integrated behavior of many of these ionic currents makes up the characteristic cardiac action potential. Arrhythmia is a variation from the normal rhythm of the heart beat and generally represents the end product of abnormal ion-channel structure, number or function. Both atrial arrhythmias and ventricular arrhythmias are known. The major cause of fatalities resulting from cardiac arrhythmias is the subtype of ventricular arrhythmias known as ventricular fibrillation (VF). Conservative estimates indicate that, in the U.S. alone, each year over one million Americans will have a new or recurrent coronary attack (defined as myocardial infarction or fatal coronary heart disease). About
650,000 of these individuals will be first heart attacks and 450,000 of these individuals will be recurrent attacks. About one-third of individuals experiencing these attacks will die as a result. At least 250,000 people a year die of coronary heart disease within 1
hour of the onset of symptoms and before they reach adequate medical aid. These are sudden deaths caused by cardiac arrest, usually resulting from ventricular fibrillation. Atrial fibrillation (AF) is the most common arrhythmia seen in clinical practice and is a cause of morbidity in many individuals (Pritchett E.L., N. Engl. J. Med. 327(14):1031 Oct. 1, 1992, discussion 1031-2; Kannel and Wolf, Am. Heart J. 123(1):264-7 Jan. 1992). The prevalence of AF is likely to increase as the population ages and it is estimated that 3-5% of patients over the age of 60 years have AF (Kannel W.B., Abbot R.D., Savage D.D., McNamara P.M., N. Engl. J. Med. 306(17): 1018-22, 1982; Wolf P.A., Abbot R.D., Kannel W.B., Stroke 22(8):983-8,
1991). While AF is rarely fatal, it can impair cardiac function and is a major cause of stroke (Hinton R.C., Kistler J.P., Fallon J.T., Friedlich A.L., Fisher CM., Am. J. Cardiol. 40(4):509-13, 1977; Wolf P.A, Abbot R.D., Kannel W.B., Arch. Intern. Med. 147(9):1561-4, 1987; Wolf P.A, Abbot R.D., Kannel W.B., Stroke 22(8):983-8, 1991; Cabin H.S., Clubb K.S., Hall O, Perlmutter R.A., Feinstein A.R., Am. J. Cardiol. 65(16): 1112-6, 1990). Antiarrhythmic agents have been developed to prevent or alleviate cardiac arrhythmia. For example, Class I antiarrhythmic compounds have been used to treat supraventricular arrhythmias and ventricular arrhythmias. Treatment of ventricular arrhythmia is very important since such an arrhythmia can be fatal. Serious ventricular arrhythmias (ventricular tachycardia and ventricular fibrillation) occur most often in the presence of myocardial ischemia and/or infarction. Ventricular fibrillation often occurs in the setting of acute myocardial ischemia, before infarction fully develops. At present, there is no satisfactory pharmacotherapy for the treatment and/or prevention of ventricular fibrillation during acute ischemia. In fact, many Class I antiarrhythmic compounds may actually increase mortality in patients who have had a myocardial infarction. Class la, lc and III antiarrhythmic drugs have been used to convert recent onset AF to sinus rhythm and prevent recurrence of the arrhythmia (Fuch and Podrid, 1992; Nattel S., Hadjis T., Talajic M., Drugs 48(3):345-71 , 1994). However, drug therapy is often limited by adverse effects, including the possibility of increased mortality, and inadequate efficacy (Feld G.K., Circulation 83(6):2248-50, 1990; Coplen S.E., Antman E.M., Berlin J.A., Hewitt P., Chalmers T.C., Circulation 1991 ; 83(2):714 and Circulation 82(4): 1 06-16, 1990; Flaker G.O, Blackshear J.L., McBride R., Kronmal R.A., Halperin J.L., Hart R.G., J. Am. Coll. Cardiol. 20(3):527-32, 1992; CAST, N. Engl. J. Med.
321:406, 1989; Nattel S., Cardiovasc. Res. 37(3):567-77, 1998). Conversion rates for Class I antiarrhythmics range between 50-90% (Nattel S., Hadjis T., Talajic M., Drugs 48(3):345-71, 1994; Steinbeck G., Remp T., Hoffmann E., J. Cardiovasc. Electrophysiol. 9(8 Suppl):S104-8, 1998). Class III antiarrhythmics appear to be more effective for terminating atrial flutter than for AF and are generally regarded as less effective than Class I drugs for terminating of AF (Nattel S., Hadjis T., Talajic M., Drugs 48(3):345-71, 1994; Capucci A, Aschieri D., Villani G.Q., Drugs Aging 73(7):51-70, 1998). Examples of such drugs include ibutilide, dofetilide and sotalol. Conversion rates for these drugs range between 30-50% for recent onset AF (Capucci A., Aschieri D., Villani G.Q., Drugs Aging 73(7):51-70, 1998), and they are also associated with a risk of the induction of Torsades de Pointes ventricular tachyarrhythmias. For ibutilide, the risk of ventricular proarrhythmia is estimated at ~4.4%, with ~1.7% of patients requiring cardioversion for refractory ventricular arrhythmias (Kowey P.R., VanderLugt J.T., Luderer J.R., Am. J. Cardiol. 78(8A):46-52, 1996). Such events are particularly tragic in the case of AF as this arrhythmia is rarely a fatal in and of itself. Conjugation of serum proteins to compounds, such as therapeutic compounds, has been reported to effect biodistribution and extension of in vivo half life of the conjugated compounds, thus providing a method of extended release of bioactive molecules. There remains a need in the art to identify new antiarrhythmic treatments, for both ventricular arrhythmias as well as for atrial arrhythmias. The present invention fulfills this need, and further provides other related advantages.
Related Literature Certain ion channel modulating agents are disclosed in PCT Published Patent Application No. WO 1999/50225; PCT Published Patent Application No. WO 2000/047547; PCT Published Patent Application No. WO 2004/098525; PCT Published Patent Application No. WO 2004/099137; PCT Published Patent Application No. WO 2005/018635; and U.S. Published Patent Application No. US 2005002693.
SUMMARY OF THE INVENTION In one aspect, this invention is directed to serum protein conjugates of ion channel modulating compounds comprising a serum protein and an ion channel modulating compound. In another aspect, this invention is directed to reactive ion channel modulating
compounds which are useful in the preparation of the serum protein conjugates of the invention. In another aspect, this invention is directed to the preparation of serum protein conjugates comprising a serum protein and an ion channel modulating compound. In another aspect, this invention is directed to methods of using the serum protein conjugates of the invention and pharmaceutical compositions containing the serum protein conjugtes of the invention. These and other aspects of the invention are described in more detail below.
DETAILED DESCRIPTION OF THE INVENTION Described herein are serum protein conjugates of ion channel modulating compounds comprising a serum protein and an ion channel modulating compound. Methods of producing the serum protein conjugates, pharmaceutical compositions and therapeutic uses thereof are also described. As disclosed within the present invention, a variety of cardiac pathological conditions may be treated and/or prevented by the use of one or more of the compounds disclosed herein that, either singly or together with one or more additional therapeutic agents, are able to selectively inhibit certain combinations of cardiac ionic currents. More specifically, the cardiac currents referred to above are the sodium currents and early repolarising currents. Early repolarising currents correspond to those cardiac ionic currents which activate rapidly after depolarization of membrane voltage and which effect repolarisation of the cell. Many of these currents are potassium currents and may include, but are not limited to, the transient outward current l oι such as Kv4.2 and Kv4.3), and the ultrarapid delayed rectifier current (lKur) such as Kv1.5, Kv1.4 and Kv2.1). The ultrarapid delayed rectifier current (lKur) has also been described as lsus. A second calcium dependent transient outward current (lto2) has also been described. The cardiac pathological conditions that may be treated and/or prevented by the compounds of the present invention may include, but are not limited to, arrhythmias such as the various types of atrial and ventricular arrhythmias. Of particular interest to the present invention are the ion channel modulating compounds disclosed in PCT Published Patent Application No. WO 1999/50225; PCT Published Patent Application No. WO 2000/047547; PCT Published Patent Application No. WO 2004/098525; PCT Published Patent Application No. WO 2004/099137; PCT Published Patent Application No. WO 2005/018635; and U.S. Published Patent
Application No. US 2005002693; the disclosures of which are incorporated in full herein by reference in their entireties.
A. Definitions In accordance with the present invention and as used herein, the following terms are defined to have the following meanings, unless explicitly stated otherwise. Terms not specifically defined herein are understood to have their common meaning. Certain chemical groups named herein are preceded by a shorthand notation indicating the total number of carbon atoms that are to be found in the indicated chemical group. For example; C-ι-C20alkyl describes an alkyl group, as defined below, having a total of 1 to 20 carbon atoms, and CrC20alkoxy describes an alkoxy group, as defined below, having a total of 1 to 20 carbon atoms. The total number of carbons in the shorthand notation does not include carbons that may exist in substituents of the group described. "Acyl" refers to branched or unbranched hydrocarbon fragments terminated by a carbonyl -(C=O)- group containing the specified number of carbon atoms. Examples include acetyl [CH3(C=O)-, a C2acyl] and propionyl [CH3CH2(C=O)~, a C3acyl]. "Alkanoyloxy" refers to an ester substituent wherein the ether oxygen is the point of attachment to the molecule. Examples include propanoyloxy [(CH3CH2(C=O)-O-, a C3alkanoyloxy] and ethanoyloxy [CH3(C=O)-O-, a C2alkanoyloxy]. "Alkoxy" refers to an O-atom substituted by an alkyl group, for example, methoxy [-OCH3, a Cialkoxy]. "Alkoxyalkyl" refers to an alkylene group substituted with an alkoxy group. For example, methoxyethyl [CH3OCH2CH2-] and ethoxymethyl (CH3CH2OCH2-] are both C alkoxyalkyl groups. "Alkoxycarbonyl" refers to an ester substituent wherein the carbonyl carbon is the point of attachment to the molecule. Examples include ethoxycarbonyl [CH3CH2O(C=O)-, a C3alkoxycarbonyl] and methoxycarbonyl [CH3O(C=O)-, a C2alkoxycarbonyl]. "Alkyl" refers to a branched or unbranched hydrocarbon fragment containing the specified number of carbon atoms and having one point of attachment. Examples include n-propyl (a C3alkyl), /so-propyl (also a C3alkyl), and f-butyl (a C4alkyl).
"Alkylene" refers to a divalent radical which is a branched or unbranched hydrocarbon fragment containing the specified number of carbon atoms, and having two points of attachment. An example is propylene [-CH2CH2CH2-, a C3alkylene]. "Alkylcarboxy" refers to a branched or unbranched hydrocarbon fragment terminated by a carboxylic acid group [-COOH]. Examples include carboxymethyl [HOOC-CH2-, a C2alkylcarboxy] and carboxyethyl [HOOC-CH2CH2-, a C3alkylcarboxy]. "Aryl" refers to aromatic groups which have at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl (also known as heteroaryl groups) and biaryl groups, all of which may be optionally substituted. Carbocyclic aryl groups are generally preferred in the compounds, where phenyl and naphthyl groups are preferred carbocyclic aryl groups. "Aralkyl" refers to an alkylene group wherein one of the points of attachment is to an aryl group. An example of an aralkyl group is the benzyl group [C6H5CH2-, a C7aralkyl group]. "Cycloalky refers to a ring, which may be saturated or unsaturated and monocyclic, bicyclic, or tricyclic formed entirely from carbon atoms. An example of a cycloalkyl group is the cyclopentenyl group (C5H7-), which is a five carbon (C5) unsaturated cycloalkyl group. "Carbocyclic" refers to a ring which may be either an aryl ring or a cycloalkyl ring, both as defined above. "Carbocyclic aryl" refers to aromatic groups wherein the atoms which form the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups such as phenyl, and bicyclic carbocyclic aryl groups such as naphthyl, all of which may be optionally substituted. "Heteroatom" refers to a non-carbon atom, where boron, nitrogen, oxygen, sulfur and phosphorus are preferred heteroatoms, with nitrogen, oxygen and sulfur being particularly preferred heteroatoms. "Heteroaryl" refers to aryl groups having from 1 to 9 carbon atoms and the remainder of the atoms are heteroatoms, and includes those heterocyclic systems described in "Handbook of Chemistry and Physics," 49th edition, 1968, R.C. Weast, editor; The Chemical Rubber Co., Cleveland, OH. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental Heterocyclic Systems. Suitable heteroaryls include furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, and the like.
"Hydroxyalkyl" refers to a branched or unbranched hydrocarbon fragment bearing a hydroxy (-OH) group. Examples include hydroxymethyl (-CH2OH, a Ci hydroxyalkyl) and 1 -hydroxyethyl (-CHOHCH3, a C2hydroxyalkyl). "Thioalkyl" refers to a sulfur atom substituted by an alkyl group, for example thiomethyl (CH3S-, a dthioalkyl). "Modulating" in connection with the activity of an ion channel means that the activity of the ion channel may be either increased or decreased in response to administration of a compound or composition or method described herein. Thus, the ion channel may be activated, so as to transport more ions, or may be blocked, so that fewer or no ions are transported by the channel. As used herein, a "subject" may generally be any human or non-human animal that would benefit from the methods described in this application. In one version of the methods, a subject is a human subject. In some versions of the methods, a subject is a warm-blooded animal. In some versions of the methods, a subject is a mammal. In some versions, the subject is any domestic animal, including, but not limited to dogs and cats. In some versions, the subject is any livestock animal, including but not limited to horses, pigs and cattle. In some versions, the subject is any zoo animal, including but not limited to Bengal tigers. As used herein, unless the context makes clear otherwise, "treatment," and similar words such as "treated," "treating" etc., is an approach for obtaining beneficial or desired results, including and preferably clinical results. Treatment can involve optionally either the amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. As used herein, unless the context makes clear otherwise, "prevention," and similar words such as "prevented," "preventing" etc., is an approach for preventing the onset of a disease or condition or preventing the occurrence of the symptoms of a disease or condition, or optionally an approach for delaying the onset of a disease or condition or delaying the occurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset of the disease or condition. As used herein, an "effective amount" or a "therapeutically effective amount" of a substance is that amount sufficient to affect a desired biological effect, such as beneficial results, including clinical results. As used herein, unless the context makes clear otherwise, "inhibition" and
similar words such as "inhibit" of any ion channel means any decrease in current through that channel. When "inhibition" is used in the context of a specified concentration, it is determined by the IC50. For example, an ion channel modulating compound which inhibits an ion channel at a concentration of 1 μM, the ion channel may be said to have an IC50 of 1 μM for that ion channel modulating compound. This example is for illustrative purposes only and is in no way intended to be limiting. As used herein, unless the context makes clear otherwise, "IC50" or "IC50 concentration" means a drug concentration at which the specified current amplitude (peak or steady-state, or integrated current) is inhibited by 50%. As used herein, unless the context makes clear otherwise, "blocking" or "block" of an ion channel means any block or inhibition of current through that ion channel. As used herein, unless the context makes clear otherwise, "recovery time constant of inhibition" refers to a time constant at which recovery of current amplitude occurs, presumed to reflect dissociation of a drug from its binding site, as for example, a sodium channel when the stimulus rate is decreased from 10 Hz to 1 Hz. "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences. Mack Publishing Co. (current edition). For example, sterile saline and phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used. "Pharmaceutically acceptable salt" refers to salts of a compound of the invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts) which retain the biological effectiveness and properties of the compounds of the present invention and which are not biologically or otherwise undesirable. The compounds of the invention described herein may be used in either the free base or salt forms, with both forms being considered as being within the scope intended herein.
Pharmaceutically-acceptable salts of the compounds of the invention include, but are not limited to, amine salts, such as but not limited to /V,Λ/'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, Λ/-methylgIucamine, procaine, Λ/-benzylphenethylamine, 1-para- chloro- benzyl-2-pyrrolidin-1'-ylmethylbenzimidazole, diethylamine and other
alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc, aluminum, and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochloride and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. Other examples of pharmaceutically acceptable salts include but not limited to those described in for example: "Handbook of Pharmaceutical Salts, Properties, Selection, and Use", P.
Heinrich Stahl and Camille G. Wermuth (Eds.), Published by VHCA (Switzerland) and Wiley-VCH (FRG), 2002. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. It is also to be understood that the compounds described herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. In the case of amino acid residues, such residues may be of either the L- or D-form. The configuration for naturally occurring amino acid residues is generally L. When not specified the residue is the L form. As used herein, the term "amino acid" refers to α-amino acids which are racemic, or of either the D- or L-configuration. The designation "d" preceding an amino acid designation (e.g., dAla, dSer, dVal, etc.) refers to the D-isomer of the amino acid. The designation "dl" preceding an amino acid designation (e.g., dlPip) refers to a mixture of the L- and D-isomers of the amino acid. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form. For purposes of this invention, when a bond is indicated in a formula as a wavy line, such as the bond between the oxygen atom and cyclohexyl moiety in compound of formula (IA), it is meant to indicate a bond which can give rise to either R or S stereochemistry.
Following the standard chemical literature description practice and as used herein, a full wedge bond means above the ring plane, and a dashed wedge bond means below the ring plane; one full bond and one dashed bond (i.e., — ) means a trans configuration, whereas two full bonds or two dashed bonds means a cis configuration. In the formulae depicted herein, a bond to a substituent and/or a bond that links a molecular fragment to the remainder of a compound may be shown as intersecting one or more bonds in a ring structure. This indicates that the bond may be attached to any one of the atoms that constitutes the ring structure, so long as a hydrogen atom could otherwise be present at that atom. Where no particular substituent(s) is identified for a particular position in a structure, then hydrogen(s) is present at that position. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. Thus, in the description of the compounds of formulae (I), (IA) and (IX) and Compound A, as described herein, all enantiomeric and diastereomeric forms of the compounds are intended. Pure stereoisomers, mixtures of enantiomers and/or diastereomers, and mixtures of different ion channel modulating compounds are described. The compounds of of formulae (I), (IA) and (IX) may therefore occur as racemates, racemic mixtures and as individual diastereomers or enantiomers with all isomeric forms being included in the present invention. A racemate or racemic mixture does not imply a 50:50 mixture of stereoisomers. Where a given structural formula or chemical name is presented for a compound of formulae (I), (IA) and (IX) it is intended that all possible solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors of the compound are also separately described by the chemical structural formula or chemical name. As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce
substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound. The serum protein conjugates of the invention may contain an "aminocycioalkyl ether moiety", i.e., the following moiety:
B. Compounds of formula (I). (IA), (IX) and Compound A In one aspect, the serum protein conjugates of the invention comprise an ion channel modulating compound and a serum protein. Generally, any compound that modulates ion channel activity may by an ion channel modulating compound. A compound that modulates ion channel activity may be a compound that increases or
decreases ion channel activity. An ion channel modulating compound that decreases ion channel activity may be a compound that blocks ion channel activity completely or partially. In another version, any compound that either singly or together with one or more additional compounds selectively inhibit certain combinations of cardiac ionic currents is an ion channel modulating compound. The cardiac currents may be the sodium currents and early repolarizing currents. Ion channel modulating compounds may block cardiac currents from extracellular loci. Such compounds act on an extemal locus of the ion channel that is accessible from the extracellular surface. This facilitates access to the ion channel and provides rapid onset kinetics and exhibits frequency dependent blockade of currents. Such properties are all beneficial for compounds used to treat arrhythmias. An ion channel modulating compound may selectively inhibit cardiac early repolarizing currents and cardiac sodium currents. Ion channel modulating compounds may be used to selectively inhibit cardiac early repolarizing currents and cardiac sodium currents under conditions where an
"arrhythmogenic substrate" is present in the heart. An "arrhythmogenic substrate" is characterized by a reduction in cardiac action potential duration and/or changes in action potential morphology, premature action potentials, high heart rates and may also include increased variability in the time between action potentials and an increase in cardiac milieu acidity due to ischaemia or inflammation. Changes such as these are observed during conditions of myocardial ischaemia or inflammation and those conditions that precede the onset of arrhythmias such as atrial fibrillation. An ion channel modulating compound may be an atrial selective agent. An ion channel modulating compound may treat or prevent ventricular arrhythmia. An ion channel modulating compound may block cardiac sodium currents or cardiac early repolarizing currents. An ion channel modulating compound may inhibit multiple cardiac ionic currents. An ion channel modulating compound may be used to treat or prevent arrhythmic, including ventricular or atrial arrhythmia, particularly atrial fibrillation. The ion channel modulating compounds may block the cardiac ion channels responsible for early repolarizing currents and sodium currents; and/or block cardiac early repolarizing currents and cardiac sodium currents under conditions where an arrhythmogenic substrate is present in the heart; and/or block the cardiac ion channels responsible for early repolarizing currents and sodium currents under conditions where an arrhythmogenic substrate is present in the heart; and/or block cardiac early repolarizing currents and cardiac sodium currents from extracellular loci in cardiac
cells. In one variation, the cardiac early repolarizing currents referred to above comprise ionic currents which activate rapidly after depolarization of membrane voltage and which effect repolarization of the cell. The early repolarizing currents may comprise the cardiac transient outward potassium current (lt0) and/or the ultrarapid delay rectifier current (lKUr)- The cardiac transient outward potassium current (lt0) and/or the ultrarapid delay rectifier current (iKur) may comprise at least one of the Kv4.2, Kv4.3, Kv2.1 , Kv1.4 and Kv1.5 currents. Ion channel modulating compounds may generally have any pKa, however ion channel modulating compounds typically have pKa values of between 4-9, and may have pKa values that are less than 8, including pKa values between 5-7.5. Methods to determine pKa values are well known in the art (see, e.g., Perrin, "Dissociation Constants of Organic Bases in Aqueous Solution", Butterworth, London, 1972). For ion channel modulating compounds with the specific ranges of pKa described above, the fraction of the charged (protonated) species will be increased under the pathological conditions such as cardiac arrhythmias and the presence of an arrhythmogenic substrate in the heart as described above due to the increase in cardiac milieu acidity. Where the charged form of a compound is active, its potency increases under conditions associated with an increase in cardiac milieu acidity. Particular ion channel modulating compounds have structural characteristics that may be determined by various physical methods, such as single crystal X-ray crystallography. For instance, some ion channel modulating compounds comprise a cycloalkane ring and substituents J and K as shown below in structure T, wherein the relative positions of J and K provide a "C" shaped angle and wherein n = 1, 2, 3 or 4.
•C" angle
R- ^"K where J and K are as described above and groups P and R are moieties such that there is not substantial rotation about the L-M bond. In one example P and R are taken together form a cyclic moiety that prevents substantial rotation about the L-M bond. In one version, the ion channel modulating compound comprises an amino substituted 5, 6, 7 or 8-membered ring, which may be a 5, 6, 7, or 8-membered substituted or unsubstituted cycloalkyl ring. The amino substituted cycloalkane ring may be an aminocyclohexyl ring and may be further substituted with one or more additional moieties. In one version, the amino substituted cycloalkane ring is further substituted with an ether moiety. In some instances, the ion channel modulating compound comprises an aminocyclohexyl ring that is further substituted with an ether moiety. In another, the ion channel modulating compound is a protonated version of any of the ion channel modulating compounds described herein. That is, for each ion channel modulating compound described herein, the quaternary protonated amine form of the compound may also be considered as an amino ion channel modulating compound. These quaternary protonated amine forms of the compounds may be present in the solid phase, for example in crystalline or amorphous form, and may be
present in solution. These quaternary protonated amine forms of the compounds may be associated with pharmaceutically acceptable anionic counter ions, including but not limited to those described in for example: "Handbook of Pharmaceutical Salts, Properties, Selection, and Use", P. Heinrich Stahl and Camille G. Wermuth (Eds.), Published by VHCA (Switzerland) and Wiley-VCH (FRG), 2002. Of particular interest to the present invention are the compounds disclosed in PCT Published Patent Application No. WO 1999/50225; PCT Published Patent Application No. WO 2000/047547; PCT Published Patent Application No. WO 2004/098525; PCT Published Patent Application No. WO 2004/099137; PCT Published Patent Application No. WO 2005/018635; and U.S. Published Patent Application No. US 2005002693; the disclosures of which are incorporated in full herein by reference in their entireties. Accordingly, one preferred embodiment of this invention are those serum protein conjugates wherein the ion channel modulating compound is a compound of formula (I), or solvates or pharmaceutically acceptable salts thereof:
C. Serum Protein Conjugates of Ion Channel Modulating Compounds A serum protein conjugate comprising a serum protein ("a protein") and an ion channel modulating compound is described. Unless indicated otherwise, the term "serum protein conjugate" is used herein to describe a conjugate between at least one ion channel modulating compound and a serum protein. Any serum protein may be employed in the serum protein conjugate, and an ion channel modulating compound may be any compound exhibiting ion channel modulating activity. The serum protein conjugate comprises at least one protein, and one or more than one ion channel modulating compound. If the serum protein conjugate comprises more than one ion channel
modulating compound, the serum protein conjugate may comprise one or more than one type of ion channel modulating compound. An ion channel modulating compound type is determined by the chemical structure, such that ion channel modulating compounds with different chemical structures are of different types. As such, high loading of a single ion channel modulating compound in a serum protein conjugate may be achieved by attaching more than one of the same ion channel modulating compound to a protein. Alternatively, different ion channel modulating compounds may be attached to the same protein, such that the serum protein conjugate may simultaneously comprise different ion channel modulating compounds. The serum protein conjugate may contain ion channel modulating compounds in any amount that is allowed by the loading capacity of the protein. The loading capacity of the protein refers to the number of ion channel modulating compounds that may be attached to the protein. The loading capacity may be determined by the amino acid composition of the protein. That is, amino acid residues whose side chains are amenable to further substitution are typically present in the protein and provide attachment sites for an ion channel modulating compound. Although usually present in the side chain moiety of an amino acid, an attachment site may be any functional group on the protein that allows attachment of an additional compound, such as an ion channel modulating compound or a linker. A serum protein conjugate as described herein contains at least one ion channel modulating compound and may contain any number of additional ion channel modulating compounds up to and including the maximum number that is determined by the loading capacity of the protein. In some variations, the serum protein conjugate comprises an ion channel modulating compound and a protein in a 1:1 molar ratio of ion channel modulating compound : protein. Such serum protein conjugates exhibit excellent batch-to-batch reproducibility and do not exhibit the range of serum protein conjugate molecular weight distributions that may be present in serum protein conjugates comprising a higher ion channel modulating compound : protein molar ratio. For instance, a serum protein conjugate comprising a protein having numerous lysine residues, wherein the lysine residues may be used as attachment sites for additional compounds such as ion channel modulating compounds or linkers, will have a high loading capacity. However, not all of the available attachment sites on a protein are necessarily occupied by an ion channel modulating compound in a serum protein conjugate. For instance, a protein with a loading capacity of 10 may form a composition comprising serum protein conjugates of varying molecular weights, which may occur when serum protein
conjugates form by occupation of all and fewer than all of the available attachment sites, for instance, when 1 , 5, 8 and 10 attachment sites are used for the attachment of an ion channel modulating compound. In contrast, a protein having only one attachment site will result in a serum protein conjugate with a specific chemistry that will provide a 1 :1 molar ratio of ion channel modulating compound : protein. For a serum protein conjugate comprising more than one ion channel modulating compound, the ion channel modulating compounds may be interspersed in the protein at any location amenable to their attachment. A serum protein conjugate comprising two or more types of ion channel modulating compounds may have the ion channel modulating compounds present in the serum protein conjugate in any molar ratio of a first ion channel modulating compound to a second ion channel modulating compound, wherein the first and second ion channel modulating compounds are not the same. In some instances, the serum protein conjugate will comprise two different ion channel modulating compounds in a 1 :1 molar ratio of a first ion channel modulating compound to a second ion channel modulating compound. In other instances, a serum protein conjugate may comprise two different ion channel modulating compounds, wherein a first ion channel modulating compound is about twice as abundant in the serum protein conjugate as the second ion channel modulating compound. The protein of the serum protein conjugate may be any size or length. As used herein, the size of the protein refers to the overall molecular weight of the protein, typically measured in Daltons. As used herein, the length of the protein refers to the number of amino acid monomer units from which the protein is derived. Examples of serum protein conjugate of ion channel modulating compounds, such as compounds of formula (I), (IA), (IX), (XV) and Compound A, described above, with proteins, such as human serum albumin, and the methods for their preparation are illustrated in the reaction schemes, in particular, Schemes 5-9.
Serum Proteins The serum protein of the conjugate may be any protein that is amenable to the attachment of an ion channel modulating compound and is available in serum. Attachment of an ion channel modulating compound may be by any covalent means, including but not limited to a direct bond or via a linker as detailed in the "linker" section below. By available in serum, it is meant that the protein may exist in a serum environment. Any naturally occurring protein, non-naturally occurring protein, or
modified form of any of the foregoing may be used in the conjugate. The serum protein of the conjugate may be a serum mobile protein. Serum mobile proteins are proteins that do not have a fixed position for any extended period of time, generally not exceeding 5 minutes, and more usually not exceeding one minute. These proteins are present in the serum for extended periods of time and typically have a half-life of at least about 12 hours. Examples of serum mobile proteins include albumin, globulins, ceuroplasmin, transferrin, ferritin, immunoglobulin, thyroxin binding protein, steroid binding protein, and a/pΛa-2-macroglobulin. In one variation, the serum mobile protein is human serum albumin (HSA). The serum protein of the conjugate may be attached to the ion channel modulating compound, either directly or via a linker, such as those described in the section entitled "linkers" below, at any site on the protein that is amenable to such attachment (i.e. at an attachment site). Typically, the attachment sites on a protein will be a reactive side chain of an amino acid residue. Examples of reactive side chains include the amino functionality of the lysine side chain or the thiol functionality of the cysteine side chain. For a protein comprising a lysine or cysteine amino acid residue, an ion channel modulating compound may be conjugated thereto, provided that the ion channel modulating compound or a linker comprises an "aminophilic" or "thiophilic" group, respectively. By "aminophilic" or "thiophilic", it is meant that the group has a complementary functionality to an amino group of the lysine side chain or the thiol group of the cysteine side chain, such that a bond will form. For instance, the amino functionality of lysine is reactive with a complementary electrophilic group, such as carbonyl-containing functionalities, including but not limited to esters. Examples of aminophilic groups include ester, anhydride, acid halide sulfonyl halide, imiidate ester, isocyanate, isothiocyanate, aldehyde and ketone groups. Specific examples of carbonyl-containing functionalities that may be present on an ion channel modulating compound or linker to affect attachment to a lysine residue include Λ/-hydroxy- succinimide, maleimide, maleimide acids, and maleimide esters. A conjugate may comprise an ion channel modulating compound attached to a protein via a cysteine residue. Typically, if a conjugate comprises an ion channel modulating compound attached to the protein via a cysteine, the attachment takes place by a reaction between a thiol on the protein and a thiophilic group on the ion channel modulating compound or linker. Examples of thiophilic groups include haloacetyl, alkyl halide, alkyl sulfonate, maleimide, α,β-unsaturated carbonyl, alkyl mercurial, sulfhydryl, and ,β-unsaturated sulfone. Specific thiophilic groups include
maleimido, iodoacetyl, and methyl thiyl groups. If the serum protein of the conjugate is serum albumin, conjugation to an ion channel modulating compound typically takes place via a thiol functionality on the albumin. The single free thiol group of albumin, highly conserved among species, is located at amino acid residue 34 (Cys3 ). It has been demonstrated that the Cys3 of albumin has increased reactivity relative to free thiols on other free thiol-containing proteins. This is attributed in part to the very low pK value of 5.5 for the Cys34 of albumin. This is much lower than typical pK values for cysteine residues in general, which are typically about 8. Because of this low pK, under normal physiological conditions, Cys3 of albumin is predominantly in the ionized form, which dramatically increases its reactivity. In addition to the low pK value of Cys34, another factor which enhances the reactivity of Cys34 is its location, which is in a crevice close to the surface of one loop of the region V of albumin. These properties make Cys34 highly reactive with thiophilic groups, including maleimide, and make HSA an excellent protein for use in the conjugates described herein. If the serum protein is naturally occurring, the protein may be conjugated or attached to the ion channel modulating compound in vitro, in vivo or ex vivo. If the protein is conjugated to the ion channel modulating compound in vitro, the protein is contacted with the ion channel modulating compound under appropriate conditions to effect attachment. If the ion channel modulating compound is conjugated to the protein in vivo, the ion channel modulating compound is delivered to the serum, whereby the ion channel modulating compound is delivered in an amount to effect conjugation with the protein in vivo. If the ion channel modulating compound is conjugated ex vivo, the serum of an individual is removed, and the ion channel modulating compound is contacted with the removed serum, thereby effecting conjugation. The ex vivo conjugate may then be administered. If the protein is not naturally occurring, the protein may be conjugated or attached to the ion channel modulating compound in vitro as described above. An ion channel modulating compound may be attached to the protein or a linker from any site on the ion channel modulating compound that is amenable to such attachment. That is, any atom or atoms on the ion channel modulating compound may be replaced with a covalent bond to a protein or a linker. For instance, a hydroxyl group or an amino group on the ion channel modulating compound may be modified to attach the ion channel modulating compound to the protein or linker. In another variation, an alkoxy group on the ion channel modulating compound is modified such
that the oxygen containing functionality is used in the attachment of the ion channel modulating compound to the protein. The same and additional ion channel modulating compounds that may be used in the conjugates described herein are provided herein. Any of the ion channel modulating compounds described herein may be attached to a serum protein to form a conjugate by the substitution of any valency in the ion channel modulating compound with a bond to the serum protein or to a linker that in turn is bound to the serum protein. Typically, a valency occupied by a hydrogen atom is substituted with a bond to the serum protein or to a linker, although any valency amenable to substitution may be used. The chemical terms recited in the section below are applicable to identical terms as used throughout this patent. Attachment of Ion Channel Modulating Compounds to Serum Proteins An ion channel modulating compound may be attached to a serum protein by any method that is amenable thereto. By attached to the serum protein, it is meant that the ion channel modulating compound is attached either via a direct bond to the protein or via a linker that is in turn bound to the protein. An ion channel modulating compound may be attached to a linker or to the protein by any bond, including but not limited to a covalent, ionic, hydrogen, dative, van der Waals, or other chemical bonding or any combination of chemical bonding. In a particular version, the ion channel modulating compound is attached to the protein via a covalent bond. Mutually reactive groups on the protein and the ion channel modulating may be used to effect conjugation. Mutually reactive groups are recognized by those of skill in the art and examples of mutually reactive groups are described herein. Linkers The ion channel modulating compound may be attached to the protein either directly (i.e. by a direct bond) or via a linker. Typically, the ion channel modulating compound is bound to the serum protein via a linkage bond, such as an ester, amide, carbamate, urea or boronate linkage. If additional atoms are required to form the linkage bond, a linker may be used, wherein the linker may be used to facilitate the formation of the linkage bond. The linker may be of any size, from a small moiety that is only used to facilitate the formation of the linkage bond, to a larger group which is employed as a connector and/or spacer group. These groups are collectively referred to as "linkers." The linkers may be used as a spacer molecule to create a separation between the ion channel modulating compound and the serum protein, and/or to avoid
undesired steric interactions. The spatial separation may be desired for modified, enhanced, or optimal function of the serum protein conjugate. The linkers may also facilitate the preparation or use of the serum protein conjugate. The linker may be primarily hydrophobic in nature or may be primarily hydrophilic in nature and may thus contribute to the overall hydrophobicity or hydrophilicity of the serum protein conjugate. The linker may be cleavable or noncleavable. A cleavable linker comprises a bond that may be cleaved in vivo including but not limited to cleavage via enzymatic, non-enzymatic, or hydrolytic cleavage. An example of a cleavable linker includes a linker that includes an ester bond. In synthesizing a serum protein conjugate comprising a linker, it may be useful to employ a linker that has at least two functional groups, one for bonding of the linker to the ion channel modulating compound and one for bonding of the linker to the serum protein. A linker functional group will usually be chosen depending on the chemistry of the ion channel modulating compound and the serum protein, such as described above for aminophilic or thiophilic functional groups. In one variation, the linker molecule is a bifunctional linker molecule. A bifunctional linker molecule comprises two reactive termini, one of which is available for linkage to the ion channel modulating compound and one of which is available for linkage to the serum protein. The functional groups on the reactive termini may be the same or different. Typically, a conjugate comprises a serum protein and an ion channel modulating compound, wherein the ion channel modulating compound is attached to the serum protein via a linker, such as a bifunctional linker. Often, the linker comprises a first linkage group, a spacer group, and a second linkage group, as shown in the schematic below. The first linkage group may be the same or different than the second linkage group.
First Linkage Group Second Linkage Group Space tr Group The first linkage group usually provides an attachment from the linker to the ion channel modulating compound via a linkage bond such as an ester, amide, carbamate,
urea or boronate linkage. The first linkage group is typically selected from a functional group selected from the group consisting of ester, amide, carbamate, urea, boronate and maleimido groups. The second linkage group usually provides an attachment from the linker to the serum protein. In one variation, the second linkage group is a thiol-reactive group or an amino-reactive group. In one variation, the second linkage group is a thiol-reactive group selected from the group consisting of pyrrolidin-yl-2,5-dione-3-yl, acetyl-2-yl, and methyl-thiyl. The spacer group may be a group of any elemental composition or size. In one variation, the spacer group may be selected from the group consisting of a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, alkaryl, alkoxy, and polyethylene glycol. In another variation, the spacer group is selected from the group consisting of a substituted or unsubstituted: CrC20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C5-C20 aryl, C6- C30 alkaryl, C2-C20 alkoxy, and polyethylene glycol comprising 2 to 200 ethylene glycol monomer units. In another variation, the spacer group may be a substituted or unsubstituted alkaryl group, wherein one of the hydrogen atoms bonded to the terminal carbon of a straight-chain d -C20alkyl, C2 -C20alkenyl or C2 -C20alkynyl group is replaced with a C -C20aryl moiety. In another variation, the spacer group is a substituted or unsubstituted aryl group, which may be optionally substituted with up to 6 additional functionalities, including an amino group. An additional functionality is any organic functionality, including but not limited to an alkane, alkene, alkyne, arene, halide, alcohol, ether, amine, nitrite, nitro, sulfide, sulfoxide, sulfone, thiol, carbonyl, aldehyde, ketone, carboxylic acid, ester, amide, acid halide and anhydride. In a particular variation, the spacer group is phenyl-prop-1-yI and the second linkage group is attached to the spacer group by the substitution of the hydrogen at the para position of the phenyl ring with a bond to the second linkage group. In another variation, the second linkage group is a maleimido group, and the bond from the spacer group to the second linkage group is a bond from the spacer group to the nitrogen of the maleimido group. In yet another variation, the second linkage group comprises an acetyl moiety, and the second linkage group is attached to the spacer group via an amide bond between the carbonyl functionality of the acetyl moiety and an amino moiety on the spacer group.
P. Reactive Ion Channel Modulating Compounds Also described herein are thiol-reactive ion channel modulating compounds.
Reactive ion channel modulating compounds are modified ion channel modulating compounds, wherein the compounds have been modified to enhance the reactivity of the compounds to form serum protein conjugates with serum proteins. Reactive ion channel modulating compounds include any modified ion channel modulating compound that is capable of forming a conjugate with a protein or linker. The reactive ion channel modulating compounds may be thiol-reactive or amino-reactive. If the reactive ion channel modulating compound is a thiol-reactive compound, the ion channel modulating compound has been modified such that a protein with a thiol functionality may form a conjugate with the thiol-reactive ion channel modulating compound. In one variation, a thiol-reactive ion channel compound of the formula (IV-R), or a solvate or pharmaceutically acceptable salt thereof, is provided:
(V-R) wherein the ring of formula (V-R) is formed from the nitrogen as shown as well as three to nine additional ring atoms independently selected from carbon, nitrogen, oxygen, and sulfur; where any two adjacent ring atoms may be joined together by single or
double bonds, and where any one or more of the additional carbon ring atoms may be substituted with one or two substituents selected from hydrogen, hydroxy, d-C3hydroxyalkyl, oxo, C2-C4acyl, d-C3alkyl, C2-C4alkylcarboxy, C C3alkoxy, C-ι-C20alkanoyloxy, or may be substituted to form a spiro five- or six-membered heterocyclic ring containing one or two heteroatoms selected from oxygen and sulfur; and any two adjacent additional carbon ring atoms may be fused to a C3-C8carbocyclic ring, and any one or more of the additional nitrogen ring atoms may be substituted with substituents selected from hydrogen, d-C6alkyl, C2-C4acyl, C2-C4hydroxyalkyl and C3-C8alkoxyalkyl; or Ri and R2, when taken together with the nitrogen atom to which they are directly attached in formula (IV-R), may form a bicyclic ring system selected from 3-azabicyclo[3.2.2]nonan-3-yl, 2-azabicyclo[2.2.2]octan-2-yl,
3-azabicyclo[3.1.0]hexan-3-yl and 3-azabicyclo[3.2.0]heptan-3-yl; wherein the bicyclic ring may be substituted with a Z'-Z"-Z'" moiety; Z' is a first linkage group; Z" is a spacer group; Z"1 is a thiol-reactive group; R3 and R4 are independently attached to the cyclohexane ring shown in formula (IV-R) at the 3-, 4-, 5- or 6- positions and are independently selected from hydrogen, hydroxy, d-Cealkyl and d-C6alkoxy, and, when both R3 and R4 are attached to the same cyclohexane ring atom, may together form a spiro five- or six-membered heterocyclic ring containing one or two heteroatoms selected from oxygen and sulfur; R5, R6 and R1 are independently selected from hydrogen, d-Cβalkyl, aryl and benzyl, or R6 and R14, when taken together with the carbon to which they are attached, may form a spiro C3-C3cycloalkyl; A is selected from C5-C12alkyl, a C3-C13carbocyclic ring, and ring systems selected from formulae (III), (IV), (V), (VI), (VII) and (VIII):
C3-C8alkoxyalkyl, d-C8hydroxyalkyl, and C7-C12aralkyl; or Ri and R2, when taken together with the nitrogen atom to which they are directly attached in formula (Al), form a ring denoted by formula (All):
(All) wherein the ring of formula (All) is formed from the nitrogen as shown as well as three to nine additional ring atoms independently selected from carbon, nitrogen, oxygen, and sulfur; where any two adjacent ring atoms may be joined together by single or double bonds, and where any one or more of the additional carbon ring atoms may be substituted with one or two substituents selected from hydrogen, hydroxy, d-C3hydroxyalkyl, oxo, C2-C4acyl, C C3alkyl, C2-C4alkylcarboxy, d-C3alkoxy, CrC20alkanoyloxy, or may be substituted to form a spiro five- or six-membered heterocyclic ring containing one or two heteroatoms selected from oxygen and sulfur; and any two adjacent additional carbon ring atoms may be fused to a C3-C8carbocyclic ring, and any one or more of the additional nitrogen ring atoms may be substituted with substituents selected from hydrogen, CrC6alkyl, C2-C4acyl, C2-C4hydroxyalkyl and C3-C8alkoxyalkyl; or RT and R2, when taken together with the nitrogen atom to which they are directly attached in formula (Al), may form a bicyclic ring system selected from 3-azabicyclo[3.2.2]nonan-3-yl, 2-azabicyclo[2.2.2]octan-2-yl,
3-azabicyclo[3.1.0]hexan-3-yl and 3-azabicyclo[3.2.0]heptan-3-yl; wherein the bicyclic ring may be substituted with a Z'-Z"-Z'" moiety; R3 and R4 are independently attached to the cyclohexane ring shown in formula (Al) at the 3-, 4-, 5- or 6- positions and are independently selected from hydrogen, hydroxy, d-C6alkyl and CrC6alkoxy, and, when both R3 and R4 are attached to the same cyclohexane ring atom, may together form a spiro five- or six-membered heterocyclic ring containing one or two heteroatoms selected from oxygen and sulfur; R5, R6 and R14 are independently selected from hydrogen, CrC6alkyl, aryl and benzyl, or R6 and Rι4, when taken together with the carbon to which they are attached, may form a spiro C3-C5cycloalkyl; A is selected from formula (AMI):
(IV-C), or a solvate or pharmaceutically acceptable salt thereof, is provided:
E. Methods of Making Reactive Ion Channel Modulating Compounds In another aspect, the present invention provides methods for preparing reactive ion channel modulating compounds, such as those of formulae (IV-R), (IV-R- a), (IVC) and (Al). In general, one method comprises attaching a bifunctional linker to a site on the ion channel modulating compound that is available for such attachment. The method may comprise conjugation of an aminocyclohexyl ether compound to a heterobifunctional linker which contains at least one thiol-reactive functionality, as shown below, whererin the variables listed below are as previously defined for formula (IV-R).
F. Administration of the Serum Protein Conjugates of the Invention The present invention provides a composition or medicament that includes one or more serum protein conjugates of the invention, selected from any of the compounds, or a solvate, pharmaceutically acceptable salt, ester, amide, complex, chelate, stereoisomer, stereoisomeric mixture, geometric isomer, crystalline or amorphous form, metabolite, metabolic precursor or serum protein conjugate thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures
thereof, described above, in combination with a pharmaceutically acceptable carrier, diluent or excipient, and further provides a method for the manufacture of such a composition or medicament. The present invention further provides a composition or medicament that includes one or more serum protein conjugates of the invention, selected from any of the serum protein conjugates, or a solvate, pharmaceutically acceptable salt, ester, amide, complex, chelate, stereoisomer, stereoisomeric mixture, geometric isomer, crystalline or amorphous form, metabolite, metabolic precursor or serum protein conjugate thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof, described above, in combination with appropriate amounts of sodium chloride USP, citric acid USP, sodium hydroxide NF and water for injection USP, and further provides a method for the manufacture of such a composition or medicament. In other embodiments, the present invention provides a composition or medicament that includes a compound which is (1 R,2R)-2-[(3R)-hydroxypyrrolidinyl]-1- (3,4-dimethoxyphenethoxy)-cyclohexane monohydrochloride, or any solvate thereof; in combination with appropriate amounts of sodium chloride USP, citric acid USP, sodium hydroxide NF and water for injection USP; and further provides a method for the manufacture of such a composition or medicament. The present invention further provides a composition or medicament that includes one or more serum protein conjugates of the invention, selected from any of the serum protein conjugates, or a solvate, pharmaceutically acceptable salt, ester, amide, complex, chelate, stereoisomer, stereoisomeric mixture, geometric isomer, crystalline or amorphous form, metabolite, metabolic precursor or serum protein conjugate thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof, described above, in combination with appropriate amounts of sodium chloride USP, citric acid USP, sodium hydroxide NF and water for injection USP, that resulted in an isotonic intravenous solution of said compound at a concentration of about 0.1 mg/mL to 100mg/mL in sodium citrate of about 1 to 400 nM at a pH of about 7.5 to 4.0; and further provides a method for the manufacture of such a composition or medicament. The present invention further provides a composition or medicament that includes one or more serum protein conjugates of the invention, selected from any of the serum protein conjugates, or a solvate, pharmaceutically acceptable salt, ester, amide, complex, chelate, stereoisomer, stereoisomeric mixture, geometric isomer,
crystalline or amorphous form, metabolite, or metabolic precursor, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof, described above, in combination with appropriate amounts of sodium chloride USP, citric acid USP, sodium hydroxide NF and water for injection USP, that resulted in an isotonic intravenous solution of said compound at a concentration of about 5mg/mL to 80mg/mL in sodium citrate of about 10 to 80 nM at a pH of about 6.5 to 4.5; and further provides a method for the manufacture of such a composition or medicament. The present invention further provides a composition or medicament that includes one or more serum protein conjugates of the invention, selected from any of the serum protein conjugates, or a solvate, pharmaceutically acceptable salt, ester, amide, complex, chelate, stereoisomer, stereoisomeric mixture, geometric isomer, crystalline or amorphous form, metabolite, or metabolic precursor thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof, described above, in combination with appropriate amounts of sodium chloride USP, citric acid USP, sodium hydroxide NF and water for injection USP, that resulted in an isotonic intravenous solution of said compound at a concentration of about 10mg/mL to 40mg/mL in sodium citrate of about 20 to 60 nM at a pH of about 6.0 to 5.0; and further provides a method for the manufacture of such a composition or medicament. The present invention further provides a composition or medicament that includes one or more serum protein conjugates of the invention, selected from any of the serum protein conjugates, or a solvate, pharmaceutically acceptable salt, ester, amide, complex, chelate, stereoisomer, stereoisomeric mixture, geometric isomer, crystalline or amorphous form, metabolite, or metabolic precursor thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof, described above, in combination with appropriate amounts of sodium chloride USP, citric acid USP, sodium hydroxide NF and water for injection USP, that resulted in an isotonic intravenous solution of said compound at a concentration of about 20mg/mL in sodium citrate of about 40 nM at a pH of about 5.5; and further provides a method for the manufacture of such a composition or medicament. In another embodiment, the present invention provides compositions which include a compound of the present invention in admixture or otherwise in association with one or more inert carriers, excipients and diluents, as well as optional ingredients if desired. These compositions are useful as, for example, assay standards, convenient means of making bulk shipments, or pharmaceutical compositions. An assayable amount of a compound of the invention is an amount which is readily
measurable by standard assay procedures and techniques as are well known and appreciated by those skilled in the art. Assayable amounts of a compound of the invention will generally vary from about 0.001 wt% to about 75 wt% of the entire weight of the composition. Inert carriers include any material which does not degrade or otherwise covalently react with a compound of the invention. Examples of suitable inert carriers are water; aqueous buffers, such as those which are generally useful in High Performance Liquid Chromatography (HPLC) analysis; organic solvents such as acetonitrile, ethyl acetate, hexane and the like (which are suitable for use in in vitro diagnostics or assays, but typically are not suitable for administration to a warm- blooded animal); and pharmaceutically acceptable carriers, such as physiological saline. Thus, the present invention provides a pharmaceutical or veterinary composition (hereinafter, simply referred to as a pharmaceutical composition) containing a compound of the present invention, in admixture with a pharmaceutically acceptable carrier, excipient or diluent. The invention further provides a pharmaceutical composition containing an effective amount of compound of the present invention, in association with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may be in any form which allows for the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, epidural, intrasternal injection or infusion techniques. Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet, capsule or cachet may be a single dosage unit, and a container of the compound in aerosol form may hold a plurality of dosage units. Materials used in preparing the pharmaceutical compositions should be pharmaceutically pure and non-toxic in the amounts used. The inventive compositions may include one or more compounds (active ingredients) known for a particularly desirable effect. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g.,
human), the particular form of the active ingredient, the manner of administration and the composition employed. In general, the pharmaceutical composition includes a compound of the present invention as described herein, in admixture with one or more carriers. The carrier(s) may be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) may be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration. When intended for oral administration, the composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. As a solid composition for oral administration, the composition may be formulated into a powder, granule, compressed tablet, pill, capsule, cachet, chewing gum, wafer, lozenges, or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following adjuvants may be present: binders such as syrups, acacia, sorbitol, polyvinylpyrrolidone, carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin, and mixtures thereof; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; fillers such as lactose, mannitols, starch, calcium phosphate, sorbitol, methylcellulose, and mixtures thereof; lubricants such as magnesium stearate, high molecular weight polymers such as polyethylene glycol, high molecular weight fatty acids such as stearic acid, silica, wetting agents such as sodium lauryl sulfate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent. When the composition is in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil. The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, aqueous or oily emulsion or suspension, or even dry powders which may be reconstituted with water and/or other liquid media prior to use. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the present compounds, one or more of a sweetening agent, thickening agent, preservative (e.g.,
alkyl p-hydroxybenzoate), dye/colorant and flavor enhancer (flavorant). In a composition intended to be administered by injection, one or more of a surfactant, preservative (e.g., alkyl p-hydroxybenzoate), wetting agent, dispersing agent, suspending agent (e.g., sorbitol, glucose, or other sugar syrups), buffer, stabilizer and isotonic agent may be included. The emulsifying agent may be selected from lecithin or sorbitol monooleate. The liquid pharmaceutical compositions of the invention, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile. A liquid composition intended for either parenteral or oral administration should contain an amount of the inventive compound such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a compound of the invention in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Preferred oral compositions contain between about 4% and about 50% of the active aminocyclohexyl ether compound. Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of active compound. The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment, cream or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis
device. Topical formulations may contain a concentration of the inventive compound of from about 0.1 to about 25% w/v (weight per unit volume). The composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol. Low-melting waxes are preferred for the preparation of a suppository, where mixtures of fatty acid glycerides and/or cocoa butter are suitable waxes. The waxes may be melted, and the aminocyclohexyl ether compound is dispersed homogeneously therein by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify. The composition may include various materials which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials which form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule or cachet. The composition in solid or liquid form may include an agent which binds to the aminocyclohexyl ether compound and thereby assists in the delivery of the active components. Suitable agents which may act in this capacity include a monoclonal or polyclonal antibody, a protein or a liposome. The pharmaceutical composition of the present invention may consist of gaseous dosage units, e.g., it may be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system which dispenses the active ingredients. Aerosols of serum protein conjugates of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. Preferred aerosols may be determined by one skilled in the art, without undue experimentation. Whether in solid, liquid or gaseous form, the pharmaceutical composition of the present invention may contain one or more known pharmacological agents used in methods for either modulating ion channel activity in a warm-blooded animal or for modulating ion channel activity in vitro, or used in the treatment and/or prevention of
arrhythmia including atrial/supraventricular arrhythmia and ventricular arrhythmia, atrial fibrillation, ventricular fibrillation, atrial flutter, ventricular flutter, diseases of the central nervous system, convulsion, cardiovascular diseases (e.g., diseases caused by elevated blood cholesterol or triglyceride levels), cerebral or myocardial ischemias, hypertension, long-QT syndrome, stroke, migraine, ophthalmic diseases, diabetes mellitus, myopathies, Becker's myotonia, myasthenia gravis, paramyotonia congenita, malignant hyperthermia, hyperkalemic periodic paralysis, Thomsen's myotonia, autoimmune disorders, graft rejection in organ transplantation or bone marrow transplantation, heart failure, atrial contractile dysfunction, hypotension, Alzheimer's disease, dementia and other mental disorders, alopecia, sexual dysfunction, impotence, demyelinating diseases, multiple sclerosis, amyotrophic lateral sclerosis, epileptic spasms, depression, anxiety, schizophrenia, Parkinson's disease, respiratory disorders, cystic fibrosis, asthma, cough, inflammation, arthritis, allergies, urinary incontinence, irritable bowel syndrome, and gastrointestinal disorders such as gastrointestinal inflammation and ulcer or other diseases. Other agents known to cause libido enhancement, analgesia or local anesthesia may be combined with compounds of the present invention. The compositions may be prepared by methodology well known in the pharmaceutical art. The aminocyclohexyl ether compounds of the present invention may be in the form of a solvate in a pharmaceutically acceptable solvent such as water or physiological saline. Alternatively, the compounds may be in the form of the free base or in the form of a pharmaceutically acceptable salt such as the hydrochloride, sulfate, phosphate, citrate, fumarate, methanesulfonate, acetate, tartrate, maleate, lactate, mandelate, salicylate, succinate and other salts known in the art. The appropriate salt would be chosen to enhance bioavailability or stability of the compound for the appropriate mode of employment (e.g., oral or parenteral routes of administration). A composition intended to be administered by injection can be prepared by combining the aminocyclohexyl ether compound of the present invention with water, and preferably buffering agents, so as to form a solution. The water is preferably sterile pyrogen-free water. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the aminocyclohexyl ether compound so as to facilitate dissolution or homogeneous suspension of the aminocyclohexyl ether compound in the aqueous delivery system. Surfactants are desirably present in aqueous compositions of the
invention because the aminocyclohexyl ether compounds according to the present invention may be hydrophobic. Other carriers for injection include, without limitation, sterile peroxide-free ethyl oleate, dehydrated alcohols, propylene glycol, as well as mixtures thereof. Suitable pharmaceutical adjuvants for the injecting solutions include stabilizing agents, solubilizing agents, buffers, and viscosity regulators. Examples of these adjuvants include ethanol, ethylenediaminetetraacetic acid (EDTA), tartrate buffers, citrate buffers, and high molecular weight polyethylene oxide viscosity regulators. These pharmaceutical formulations may be injected intramuscularly, epidurally, intraperitoneally, or intravenously. As used herein, "treating arrhythmia" refers to therapy for arrhythmia. An effective amount of a composition of the present invention is used to treat arrhythmia in a warm-blooded animal, such as a human. Methods of administering effective amounts of antiarrhythmic agents are well known in the art and include the administration of an oral or parenteral dosage form. Such dosage forms include, but are not limited to, parenteral dosage form. Such dosage forms include, but are not limited to, parenteral solutions, tablets, capsules, sustained release implants, and transdermal delivery systems. Generally, oral or intravenous administration is preferred for some treatments. The dosage amount and frequency are selected to create an effective level of the agent without harmful effects. It will generally range from a dosage of from about 0.01 to about 100 mg/kg/day, and typically from about 0.1 to 10 mg/kg where administered orally or intravenously for antiarrhythmic effect or other therapeutic application. Administration of compositions of the present invention may be carried out in combination with the administration of other agents. For example, it may be desired to administer an opioid antagonist, such as naloxone, if a compound exhibits opioid activity where such activity may not be desired. The naloxone may antagonize opioid activity of the administered compound without adverse interference with the antiarrhythmic activity. As another example, an aminocyclohexyl ether compound of the invention may be co-administered with epinephrine in order to induce local anesthesia.
G. Utility and Testing of the Serum protein conjugates of the Invention The present invention provides one or more serum protein conjugates of ion channel modulating compounds, or solvates, pharmaceutically acceptable salts,
esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above, for use in methods for modulating ion channel activity in a warm-blooded animal or for modulating ion channel activity in vitro. In one version of this embodiment, the warm-blooded animal in which the ion channel activity is modulated is a mammal; in one version, the warmblooded animal is a human; in one version, the warm-blooded animal is a farm animal. As disclosed within the present invention, a variety of cardiac pathological conditions may be treated and/or prevented by the use of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. These compounds of the present invention are ion channel modulating compounds that either singly or together with one or more additional compounds are able to selectively modulate certain ionic currents. The ion currents referred to herein are generally cardiac currents and more specifically, are the sodium currents and early repolarising currents. Early repolarising currents correspond to those cardiac ionic currents which activate rapidly after depolarization of membrane voltage and which effect repolarisation of the cell. Many of these currents are potassium currents and may include, but are not limited to, the transient outward current ltoι such as Kv4.2 and Kv4.3), and the ultrarapid delayed rectifier current (lKur) such as Kv1.5, Kv1.4 and Kv2.1). The ultrarapid delayed rectifier current (lKur) has also been described as lsus. A second calcium dependent transient outward current (lto2) has also been described. The pathological conditions that may be treated and/or prevented by the present invention may include, but are not limited to, various cardiovascular diseases. The cardiac pathological conditions that may be treated and/or prevented by the present invention may include, but are not limited to, arrhythmias such as the various types of atrial and ventricular arrhythmias, e.g., atrial fibrillation, atrial flutter, ventricular fibrillation and ventricular flutter. In one embodiment, the present invention provides serum protein conjugates of
ion channel modulating compounds that can be used to selectively inhibit cardiac early repolarising currents and cardiac sodium currents. In another embodiment, the present invention provides serum protein conjugates of ion channel modulating compounds that can be used to selectively inhibit cardiac early repolarising currents and cardiac sodium currents under conditions where an "arrhythmogenic substrate" is present in the heart. An "arrhythmogenic substrate" is characterized by a reduction in cardiac action potential duration and/or changes in action potential morphology, premature action potentials, high heart rates and may also include increased variability in the time between action potentials and an increase in cardiac milieu acidity resulting from ischaemia or inflammation. Changes such as these are observed during conditions of myocardial ischaemia or inflammation and those conditions that precede the onset of arrhythmias such as atrial fibrillation. In other embodiments, the present invention provides a method for modulating ion channel activity in a warm-blooded animal comprising administering to a warm- blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for modulating ion channel activity in an in vitro setting comprising administering in vitro an effective amount of one or more serum protein conjugates of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said serum protein conjugate or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for blocking/inhibiting the activity/conductance of ion channel in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more serum protein conjugates of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates,
stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said serum protein conjugate or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for blocking/inhibiting the activity/conductance of ion channel in an in vitro setting comprising administering in vitro an effective amount of one or more serum protein conjugates of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for modulating potassium ion channel activity in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for modulating voltage-gated potassium ion channel activity in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for modulating cardiac sodium currents activity in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts,
esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for modulating cardiac early repolarising currents and cardiac sodium currents ion channel activity in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for blocking/inhibiting cardiac early repolarising currents and cardiac sodium currents ion channel activity in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for blocking/inhibiting the cardiac ion channels responsible for cardiac early repolarising currents and cardiac sodium currents ion channel activity in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above.
In other embodiments, the present invention provides a method for blocking/inhibiting cardiac early repolarising currents and cardiac sodium currents ion channel activity in a warm-blooded animal under conditions where an arrhythmogenic substrate is present in the heart of said warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for blocking/inhibiting the cardiac ion channels responsible for cardiac early repolarising currents and cardiac sodium currents ion channel activity in a warm-blooded animal under conditions where an arrhythmogenic substrate is present in the heart of said warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the cardiac early repolarising currents referred to in the present invention comprise ionic currents which activate rapidly after depolarisation of membrane voltage and which effect repolarisation of the cell. In other embodiments, the cardiac early repolarising currents referred to in the present invention comprise the cardiac transient outward potassium current (Ito) and/or the ultrarapid delayed rectifier current (lKUr). In other embodiments, the cardiac transient outward potassium current (lto) and/or the ultrarapid delayed rectifier current (lKUr) referred to in the present invention comprise at least one of the Kv4.2, Kv4.3, Kv2.1 , Kv1.4 and Kv1.5 currents. In other embodiments, the present invention provides a method for treating and/or preventing arrhythmia in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers,
crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In another embodiments, the present invention provides a method for treating and/or preventing atrial arrhythmia in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for treating and/or preventing ventricular arrhythmia in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In another embodiments, the present invention provides a method for treating and/or preventing atrial fibrillation in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for treating and/or preventing ventricular fibrillation in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable
salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In another embodiments, the present invention provides a method for treating and/or preventing atrial flutter in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. In other embodiments, the present invention provides a method for treating and/or preventing ventricular flutter in a warm-blooded animal comprising administering to a warm-blooded animal in need thereof, an effective amount of one or more compounds of the present invention or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof, as isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof; or a composition or medicament that includes said compound or mixture comprising compounds as described above. As noted above, the present invention provides for utilizing the compounds described above in in vitro and in vivo methods. In one embodiment, ion channels, such as cardiac potassium channels, are blocked in vitro or in vivo. Ion channels are ubiquitous membrane proteins in the cells of warm-blooded animals such as mammals. Their critical physiological roles include control of the electrical potential across the membrane, mediation of ionic and fluid balance, facilitation of neuromuscular and neuronal transmission, rapid transmembrane signal transduction, and regulation of secretion and contractility. Accordingly, compounds that are capable of modulating the activity or function of the appropriate ion channels will be useful in treating and/or preventing a variety of diseases or disorders caused by defective or inadequate function of the ion channels. The serum protein conjugates of the invention are found to have significant activity in
modulating various ion channel activity both in vivo and in vitro. In one embodiment, the present invention provides a compound of the present invention or a composition containing said compound, for use in methods for either modulating ion channel activity in a warm-blooded animal or for modulating ion channel activity in vitro. Some of the ion channels to which the compounds, compositions and methods of the present invention have modulating effect are various potassium and sodium channels. These potassium and sodium ion channels may be voltage-activated (also known as voltage-gated) or ligand-activated (also known as ligand-gated), and may be present in cardiac and/or neuronal systems. In one embodiment, the invention provides a compound of the present invention, or composition containing said compound, for use in methods for either modulating activity of ion channel(s) in a warm-blooded animal or for modulating activity of ion channel(s) in vitro, wherein said ion channel(s) correspond to some of the cardiac and/or neuronal ion channels that are responsible for one or more early repolarising currents comprising those which activate rapidly after membrane depolarisation and which effect repolarisation of the cells. In another embodiment, of the present invention, the above-mentioned early repolarising currents comprise the transient outward potassium current (lt0 for cardiac or lA for neuronal) and/or the ultrarapid delayed rectifier current (lKUr); and include at least one of the Kv4.2, Kv4.3, Kv2.1 , Kv1.3, Kv1.4 and Kv1.5 currents. In another embodiment, the present invention provides a compound of the present invention, or composition containing said compound, for use in methods for either modulating activity of ion channel(s) in a warm-blooded animal or for modulating activity of ion channel(s) in vitro, wherein said ion channel(s) correspond to either the cardiac or neuronal ion channel(s) that are responsible for Kv1.5 current. In yet another embodiment, the present invention provides a compound of the present invention, or composition containing said compound, for use in methods for either modulating activity of ion channel(s) in a warm-blooded animal or for modulating activity of ion channel(s) in vitro, wherein said ion channel(s) correspond to the potassium channel that are responsible for Kv4.2 current. Furthermore, the voltage-activated sodium ion channels comprise the Nav1 , Nav2 or Nav3 series and may be present in cardiac, neuronal, skeletal muscle, central nervous and/or peripheral nervous systems (e.g., hH1Na). For cardiac sodium channels, in studies on ion channels in isolated human atrial myocytes, compounds of the present invention have been shown to produce
frequency-dependent blockade of cardiac sodium channels. In these studies enchanced blockade of cardiac sodium channels was observed at faster rates of stimulation with sodium block increasing several-fold during rapid stimulation rates. These protocols have been designed to mimic the short recovery intervals during fibrillation. As noted earlier, modulating the activity of an ion channel as used above may imply but does not limit to blocking or inhibiting the conductance of the current through the ion channel. Thus, the present invention provides for methods of treating a disease or condition in a warm-blooded animal suffering from or having the disease or condition, and/or preventing a disease or condition from arising in a warm-blooded animal, wherein a therapeutically effective amount of a compound of the present invention, or a composition containing a compound of the present invention is administered to a warm-blooded animal in need thereof. Some of the diseases and conditions to which the compounds, compositions and methods of the present invention may be applied are as follows: arrhythmia including atrial/supraventricular arrhythmia and ventricular arrhythmia, atrial fibrillation, ventricular fibrillation, atrial flutter, ventricular flutter, diseases of the central nervous system, convulsion, cardiovascular diseases (e.g., diseases caused by elevated blood cholesterol or triglyceride levels), cerebral or myocardial ischemias, hypertension, long-QT syndrome, stroke, migraine, ophthalmic diseases, diabetes mellitus, myopathies, Becker's myotonia, myasthenia gravis, paramyotonia congenita, malignant hyperthermia, hyperkalemic periodic paralysis, Thomsen's myotonia, autoimmune disorders, graft rejection in organ transplantation or bone marrow transplantation, heart failure, atrial contractile dysfunction, hypotension, Alzheimer's disease, dementia and other mental disorder, alopecia, sexual dysfunction, impotence, demyelinating diseases, multiple sclerosis, amyotrophic lateral sclerosis, epileptic spasms, depression, anxiety, schizophrenia, Parkinson's disease, respiratory disorders, cystic fibrosis, asthma, cough, inflammation, arthritis, allergies, urinary incontinence, irritable bowel syndrome, and gastrointestinal disorders such as gastrointestinal inflammation and ulcer. Furthermore, the present invention provides a method for producing analgesia or local anesthesia in a warm-blooded animal which includes administering to a warmblooded animal in need thereof an effective amount of a compound of the present invention or a pharmaceutical composition containing said compound. These methods may be used to relieve or forestall the sensation of pain in a warm-blooded animal.
The invention further provides a method for enhancing libido in a warm-blooded animal which includes administering to a warm-blooded animal in need thereof an effective amount of a compound of the present invention or a pharmaceutical composition containing said compound. These compositions and methods may be used, for example, to treat a sexual dysfunction, e.g., impotence in males, and/or to enhance the sexual desire of a patient without a sexual dysfunction. As another example, the therapeutically effective amount may be administered to a bull (or other breeding stock), to promote increased semen ejaculation, where the ejaculated semen is collected and stored for use as it is needed to impregnate female cows in promotion of a breeding program. Furthermore, the present invention provides a method in an in vitro setting, wherein a preparation that contains ion channels is contacted with an effective amount of an aminocyclohexyl ether compound of the invention. Suitable preparations containing cardiac sodium channels and/or cardiac potassium channels include cells isolated from cardiac tissue as well as cultured cell lines. The step of contacting includes, for example, incubation of ion channels with a compound under conditions and for a time sufficient to permit modulation of the activity of the channels by the compound. Administration of compositions of the present invention may be carried out in combination with the administration of other agents. For example, it may be desired to administer an opioid antagonist, such as naloxone, if a compound exhibits opioid activity where such activity may not be desired. The naloxone may antagonize opioid activity of the administered compound without adverse interference with the antiarrhythmic activity. As another example, an aminocyclohexyl ether compound of the invention may be co-administered with epinephrine in order to induce local anesthesia. In order to assess whether a compound has a desired pharmacological activity with the present invention, it may be subjected to a series of tests. The precise test to employ will depend on the physiological response of interest. The published literature contains numerous protocols for testing the efficacy of a potential therapeutic agent, and these protocols may be employed with the present compounds and compositions (see, for example, PCT Published Patent Application No. WO 1999/50225; PCT Published Patent Application No. WO 2000/047547; PCT Published Patent Application No. WO 2004/098525; PCT Published Patent Application No. WO 2004/099137; PCT Published Patent Application No. WO 2005/018635; and U.S. Published Patent
Application No. 2005002693; and references cited therein). For example, in connection with treatment or prevention of arrhythmia, a series of four tests may be conducted. In the first of these tests, a compound of the present invention is given as increasing (doubling with each dose) intravenous infusion every 5 minutes to a conscious rat. The effects of the compound on blood pressure, heart rate and the ECG are measured continuously. Increasing doses are given until a severe adverse event occurs. The drug related adverse event is identified as being of respiratory, central nervous system or cardiovascular system origin. This test gives an indication as to whether the compound is modulating the activity of sodium channels and/or potassium channels, and in addition gives information about acute toxicity. The indices of sodium channel blockade are increasing P-R interval and QRS widening of the ECG. Potassium channel blockade results in Q-T interval prolongation of the ECG. A second test involves administration of a compound as an infusion to pentobarbital anesthetized rats in which the left ventricle is subjected to electrical square wave stimulation performed according to a preset protocol described in further detail below. This protocol includes the determination of thresholds for induction of extrasystoles and ventricular fibrillation. In addition, effects on electrical refractoriness are assessed by a single extra beat technique. In addition effects on blood pressure, heart rate and the ECG are recorded. In this test, sodium channel blockers produce the ECG changes expected from the first test. In addition, sodium channel blockers also raise the thresholds for induction of extrasystoles and ventricular fibrillation. Potassium channel blockade is revealed by increasing refractoriness and widening of the Q-T intervals of the ECG. A third test involves exposing isolated rat hearts to increasing concentrations of a compound. Ventricular pressures, heart rate, conduction velocity and ECG are recorded in the isolated heart in the presence of varying concentrations of the compound. The test provides evidence for direct toxic effects on the myocardium. Additionally, selectivity, potency and efficacy of action of a compound can be ascertained under conditions simulating ischemia. Concentrations found to be effective in this test are expected to be efficacious in the electrophysiological studies. A fourth test is estimation of the antiarrhythmic activity of a compound against the arrhythmias induced by coronary artery occlusion in anaesthetized rats. It is expected that a good antiarrhythmic compound will have antiarrhythmic activity at doses which have minimal effects on either the ECG, blood pressure or heart rate under normal conditions.
All of the foregoing tests may be performed using rat tissue. In order to ensure that a compound is not having effects which are only specific to rat tissue, further experiments may be performed in dogs and primates. In order to assess possible sodium channel and potassium channel blocking action in vivo in dogs, a compound is tested for effects on the ECG, ventricular epicardial conduction velocity and responses to electrical stimulation. An anesthetized dog is subjected to an open chest procedure to expose the left ventricular epicardium. After the pericardium is removed from the heart a recording/stimulation electrode is sewn onto the epicardial surface of the left ventricle. Using this array, and suitable stimulation protocols, conduction velocity across the epicardium as well as responsiveness to electrical stimulation can be assessed. This information coupled with measurements of the ECG allows one to assess whether sodium and/or potassium channel blockade occurs. As in the first test in rats, a compound is given as a series of increasing bolus doses. At the same time possible toxic effects of a compound on the dog's cardiovascular system is assessed. The effects of a compound on the ECG and responses to electrical stimulation are also assessed in intact, anesthetized monkeys (Macaca fascicularis). In this preparation, a blood pressure cannula and ECG electrodes are suitably placed in an anesthetized monkey. In addition, a stimulating electrode is placed onto the right atria and/or ventricle, together with monophasic action potential electrode. As in the tests described above, ECG and electrical stimulation response to a compound reveal the possible presence of sodium and/or potassium channel blockade. The monophasic action potential also reveals whether a compound widens the action potential, an action expected of a potassium channel blocker. As another example, in connection with the mitigation or prevention of the sensation of pain, the following test may be performed. To determine the effects of a compound of the present invention on an animal's response to a sharp pain sensation, the effects of a slight prick from a 7.5 g weighted syringe fitted with a 23G needle as applied to the shaved back of a guinea pig (Ca 'a porcellus) is assessed following subcutaneous administration of sufficient (50 μL, 10 mg/mL) solution in saline to raise a visible bleb on the skin. Each test is performed on the central area of the bleb and also on its periphery to check for diffusion of the test solution from the point of administration. If the test animal produces a flinch in response to the stimulus, this demonstrates the absence of blockade of pain sensation. Testing may be carried out at intervals for up to 8 hours or more post- administration. The sites of bleb formation are examined after 24 hours to check for skin abnormalities consequent to local
administration of test substances or of the vehicle used for preparation of the test solutions.
H. Preparation of the Compounds of Formula (I). (IA). (IX), (XV) and Compound A The ion channel modulating compounds of formulae (I), (IA), (IX) and/or (XV) and/or Compound A used in the present invention may be prepared as described in PCT Published Patent Application No. WO 1999/50225; PCT Published Patent Application No. WO 2000/047547; PCT Published Patent Application No. WO 2004/098525; PCT Published Patent Application No. WO 2004/099137; PCT Published Patent Application No. WO 2005/018635; and U.S. Published Patent Application No. US 2005002693; or may be prepared by methods known to one skilled in the art.
I. Preparation of Reactive Ion Channel Modulating Compounds and Serum Protein Conjugates of the Invention It is understood that in the following description, combinations of substituents and/or variables of any depicted formulae are permissible only if such contributions result in stable compounds. It will also be appreciated by those skilled in the art that in the processes described below the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., f-butyldimethylsilyl, butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include f-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R (where R is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are well-known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M.
Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. The protecting group may also be a polymer resin such as a Wang resin or a 2-chlorotrityl-chloride resin. It will also be appreciated by those skilled in the art, although such protected
derivatives of serum protein conjugates of this invention may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as "serum protein conjugates". All serum protein conjugates of compounds of this invention are included within the scope of the invention. The following reaction schemes and examples illustrate methods to make serum protein conjugates of this invention. It is understood that one of those skilled in the art would be able to make these compounds by similar methods or by methods known to one skilled in the art. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this invention. If applicable, the following parameters were determined: The reaction steps as described below may be used in the preparation of the conjugates, or alternate reaction steps may be used. Alternate reaction steps would be readily recognized by one of skill in the art and include the reaction steps described "Comprehensive Organic Transformations: A Guide to Functional Group Preparations", Richard C. Larock, Wiley-VCH: 1999 and in "March's Advanced Organic Chemistry: Reactions, Mechanisms and Structure", Jerry March & Michael Smith, John Wiley & Sons Inc: 2001. Commercially available heterobifunctional linkers may be obtained from lnterchim or Nektar Therapeutics and include examples such as but not limited to N- succinimidyl-3-(2-pyridylthio)proprionate and NHS PEG vinyl sulfone. Thiol-reactive linkers are also available from Biotium, Inc. Heterobifunctional linkers may also be synthesized by standard methods from commercially available starting materials. For example, 4-[4-(2,5-dioxo-2,5-dihydro- pyrrol-1-yl)-phenyl]-butyric acid is condensed from the commercially available 4-(4- aminophenyl)butyric acid and maleic anhydride (e.g., Booth CA and Philp D.
Tetrahedron Lett. 1998, 39, 6987 and Rich D.H., Gesellchen P.D., Tong A, Cheung A., Buckner C.K. J. Med. Chem. 1975, 18, 1004). The ion channel modulating compounds used in this invention may be prepared as described in PCT Published Patent Application No. WO 1999/50225; PCT Published Patent Application No. WO 2000/047547; PCT Published Patent Application
No. WO 2004/098525; PCT Published Patent Application No. WO 2004/099137; PCT Published Patent Application No. WO 2005/018635; and U.S. Published Patent Application No. US 2005002693; or may be prepared by methods described herein or by methods known to one skilled in the art. U.S. Published Patent Application Nos. 20030087826, 20030166512, and
20020193318 and PCT Published Patent Application No. WO 02/074246 are examples which disclose the conjugation of bioactive compounds to the Cys34 residue of human serum albumin. Conjugation of bioactive moieties to the Cys34 residue are also described in for example, Stamler J.S., Singel D.J., Loscalzo J. Science 1992, 258, 1898 report on complexation of nitric acid, Shockley P., Brown J.R. 1980, 39, 2069 report on Cys34 as a reactant with Λ/-danzylaziridine, which inhibits binding of stearate, palmitate and oleate, Shaw CF. Comments Inorg. Chem. 1989, 8, 233 report on coordination of gold complexes, Kratz F., Warnecke A., Scheuermann K., Stockmar C, Schwab J., Lazar P., Drϋckes P., Esser N., Drevs J., Rognan D., Bissantz C, Hinderling C, Folkers G., Fichtner I., Unger C. J. Med. Chem. 2002, 45, 5523 and references therein report on interaction of doxorubicin, Shaw III CF Chem. Rev. 1999, 99, 2589 reviews auranofin and auranofin analogues, Ivanov A.I., Christodoulou J., Parkinson J.A., Barnham K.J., Tucker A., Woodrow J., Sadler P.J. J. Biol. Chem. 1998, 273, 14721 report on interactions of cisplatin, Narazaki R., Otagiri M. Pharm. Res. 1997, 14, 351 determine covalent binding of a bucillamine derivative, Kratochwil N.A., Ivanov A.I. , Patriarca M., Parkinson J.A., Gouldsworthy A.M., Murdoch P. S., Sadler P.J. J. Am. Chem. Soc. 1999, 121, 8193 observe surprising Pt(ll) and Pt(IV) adducts, Beck J.L., Ambahera S., Yong S.R., Sheil M.M., Jersey J., Ralph S.F. Anal. Biochem. 2004, 325, 326 observed adducts with low molecular weight thiols and a gold(l) antiarthritic drug, and Espόsito B.P., Najjar R. Coord. Chem. Rev. 2002, 232, 137 review interactions of antitumoural platinum-group. Although anyone skilled in the art is capable of preparing the compounds of the invention according to the general techniques disclosed above, more specific details on synthetic techniques for compositions of the invention are provided elsewhere in this specification for convenience. Again, all reagents and reaction conditions employed in synthesis are known to those skilled in the art and are available from ordinary commercial sources.
SCHEME 1 : SYNTHESIS OF THIOL-REACTIVE ( R,2R)-2-[(3R)- HYDROXYPYRROLIDINYL]-1-(3,4-DIMETHOXYPHENETHOXY)CYCLOHEXANE (3).
SCHEME 2: SYNTHESIS OF THIOL-REACTIVE (1f?,2R)-2-[(3R)- HYDROXYPYRROLIDINYL]-1-(3,4-DIMETHOXYPHENETHOXY)CYCLOHEXANE (5).
1-(3,4-dimethoxyphenethoxy)cyclohexane Compound A with a bifunctional linker 4-(2- iodo-acetylamino)-benzoic acid 2,5-dioxo-pyrrolidin-1-yl ester (succinimidyl-4- iodoacetyl-aminobenzoate) (4) or a water-soluble sulfonate derivative (4a) of the bifunctional linker to form a thiol-reactive derivative (5). In a typical reaction, (1R,2ft)-2-[(3ft)-hydroxypyrrolidinyl]-1-(3,4- dimethoxyphenethoxy)cyclohexane (1 equiv) Compound A may be dissolved in a suitable solvent (e.g., DMF) and reacted with 4-(2-iodo-acetylamino)-benzoic acid 2,5- dioxo-pyrrolidin-1-yl ester (succinimidyl-4-iodoacetyl-aminobenzoate) (1 equiv) (4) or a water-soluble sulfonate derivative (4a) in a standard coupling reaction. The resultant thiol-reactive aminocyclohexyl ether (5) may be isolated and purified using standard work-up procedures.
SCHEME 3: SYNTHESIS OF THIOL-REACTIVE (1R,2R)-2-[(3R)- HYDROXYPYRROLIDINYL]-1-(3,4-DIMETHOXYPHENETHOXY)CYCLOHEXANE (7).
SCHEME 4: SYNTHESIS OF THIOL-REACTIVE (1R,2R)-2-[(3R)- HYDROXYPYRROLIDINYL]-1-(3,4-DIMETHOXYPHENETHOXY)CYCLOHEXANE (9).
Scheme 4 illustrates the derivatization of (1R,2R)-2-[(3R)-hydroxypyrrolidinyl]- 1-(3,4-dimethoxyphenethoxy)cyclohexane Compound A with PEG (8) to form the ester linked PEG-thiol-reactive adduct (9) (NHS vinyl sulfone NHS PEG VS) In a typical reaction, (1R,2R)-2-[(3R)-hydroxypyrrolidinyl]-1-(3,4- dimethoxyphenethoxy)cyclohexane (1 equiv) Compound A may be dissolved in a suitable solvent (e.g., DMF) and reacted with NHS PEG VS (1 equiv) (8) in a standard coupling reaction. The resultant thiol-reactive aminocyclohexyl ether (9) may be isolated and purified using standard work-up procedures.
SCHEME 5: SYNTHESIS OF THIOL-REACTIVE HETEROBIFUNCTIONAL LINKER (12).
Scheme 5 illustrates the functionalization of 2-aminoethylmethane thiosulfonate
hydrobromide (10) with Λ/-hydroxysuccinimide bromoacetate (11) or a water-soluble sulfonate derivative of the Λ/-hydroxysuccinimide (11a) to form the thiol-reactive heterobifunctional linker (12) or the water-soluble derivative (12a). In a typical reaction, 2-aminoethylmethane thiosulfonate hydrobromide (10) may be dissolved in a suitable solvent and reacted with Λ/-hydroxysuccinimide bromoacetate (11) or the optionally water soluble sulfonate derivative (11a) to form the thiol-reactive heterobifunctional linker (12) or the water-soluble derivative (12a). The resultant thiol-reactive heterobifunctional linker (12) or (12a) is isolated and purified using standard work-up procedures.
SCHEME 6: SYNTHESIS OF OF THIOL-REACTIVE (1 ,2R)-2-[(3R)- HYDROXYPYRROLIDINYL]-1-(3,4-DIMETHOXYPHENETHOXY)CYCLOHEXANE (13).
Scheme 6 illustrates the derivatization of (1R,2R)-2-[(3R)-hydroxypyrrolidinyl]- 1-(3,4-dimethoxyphenethoxy)cycIohexane Compound A with (12) or a water-soluble sulfonate derivative (12a) of the heterobifunctional linker to form the ester linked thiol- reactive adduct (13). In a typical reaction, (1 R,2R)-2-[(3R)-hydroxypyrrolidinyl]-1-(3,4- dimethoxyphenethoxy)cyclohexane (1 equiv) Compound A may be dissolved in a suitable solvent (e.g. DMF) and reacted with (12) or a water soluble sulfonate derivative (12a) in a standard coupling reaction. The resultant thiol-reactive aminocyclohexyl ether (13) is isolated and purified using standard work-up procedures. Schemes 7-11 illustrate the conjugation of thiol-reactive derivative (3), (5), (7), (9) and (13) with the thiol functionality of albumin to generate the albumin conjugate (14)-(18). The single free thiol group of albumin, highly conserved among species, is located at amino acid residue Cys3 . It has been demonstrated recently that the Cys34 of albumin has an increased reactivity relative to free thiols on other free thiol-
containing proteins. This is due in part to the very low pKa value of 5.5 for the Cys34 of albumin. This is much lower than typical pK values for cysteine residues in general, which are typically about 8. Owing to this low pK, under normal physiological conditions, Cys34 of albumin is predominantly in the ionized form, which dramatically increases its reactivity. In addition to the low pK value of Cys34, another factor which enhances the reactivity of Cys34 is its location, which is in a crevice close to the surface of one loop of region V of albumin (Sugio S., Kashima A., Mochizuki S., Noda M., Kobayashi K. Prot. Eng. 1999, 12, 439 and references therein). This location makes Cys34 accessible to ligands of all kinds, and is an important factor in the biological role of Cys34 as free radical trap (e.g. Davies M.J., Gilbert B.C., Haywood R.M. Free Radic. Res. Commun. 1993, 18, 353) and free thiol scavenger (e.g. Soriani M., Pietraforte D., Minetti M. Arch. Biochem. Biophys. 1994, 312, 180). As a result, the reaction rate acceleration can be as much as 1000-fold relative to rates of reaction with other free- thiol containing proteins.
SCHEME 7: SYNTHESIS OF (1R,2R)-2-[(3R)-HYDROXYPYRROLIDINYL]-1-(3,4- DIMETHOXYPHENETHOXY)CYCLOHEXANE-ALBUMIN CONJUGATE (14)
SCHEME 8: SYNTHESIS OF (1R,2R)-2-[(3R)-HYDROXYPYRROLIDINYL]-1-(3,4- DIMETHOXYPHENETHOXY)CYCLOHEXANE-ALBUMIN CONJUGATE (15)
Scheme 8 illustrates the conjugation of thiol-reactive derivative (5) with thiol function of albumin (Cys34) to form the (1R,2R)-2-[(3R)-hydroxypyrrolidinyl]-1-(3,4- dimethoxyphenethoxy)cyclohexane-albumin conjugate (15). SCHEME 9: SYNTHESIS OF (1R,2R)-2-[(3R)-HYDROXYPYRROLIDINYL]-1-(3,4- DIMETHOXYPHENETHOXY)CYCLOHEXANE-ALBUMIN CONJUGATE (16)
Scheme 10 illustrates the conjugation of PEG-thiol-reactive derivative (9) with thiol function of albumin (Cys34) to form the PEG-(1R,2R)-2-[(3R)-hydroxypyrrolidinyl]- 1-(3,4-dimethoxyphenethoxy)cyclohexane-albumin conjugate (17). SCHEME 11: SYNTHESIS OF (1R,2R)-2-[(3R)-HYDROXYPYRROL!DINYL]-1-(3,4- DIMETHOXYPHENETHOXY)CYCLOHEXANE-ALBUMIN CONJUGATE (18)
Scheme 11 illustrates the conjugation of thiol-reactive derivative (13) with thiol function of albumin (Cys34) to form the (1 R,2R)-2-[(3R)-hydroxypyrrolidinyl]-1 -(3,4- dimethoxyphenethoxy)cyclohexane-albumin conjugate (18).
Aminocycioalkyl Ether Conjugation to Serum Protein via a Phenolic Functionality The synthetic procedures for the preparation of the conjugate ion channel modulating compounds to serum protein will depend on the bifunctional linker to link
the ion channel modulating agent with serum protein. The serum protein conjugate may be prepared by derivitization of an ion channel modulating compound, or by a modified synthesis of an ion channel modulating compound such that a serum protein conjugate is prepared. Illustrative examples of the synthetic protocols used in the synthesis of serum protein conjugate ion channel modulating compounds 34a,b-37a,b is depicted in the following synthetic schemes. The ion channel modulating compounds utilized herein may be prepared by following the general methods and specific experimental procedures described.
SCHEME 12: PREPARATION OF COMPOUNDS (28A) OR (28B)
Scheme 12 illustrates the preparation of compounds (28a) or (28b) via Williamson ether synthesis between compound (24) and the alkoxide of the appropriate phenethyl alcohol (26a) or (26b) followed by hydrogenolysis. The Williamson ether synthesis (Feuer, H.; Hooz, J. Methods of Formation of the Ether Linkage. In Patai, Wiley: New York, 1967; pp 445-492) between the chloride 24 with the alkoxide of the appropriate phenethyl alcohol (26a or 26b) in a polar solvent such as DME (Scheme 12) provided the corresponding aminoether in high yield. Subsequent hydrogenolysis of (27a or 27b) provides 28a or 28b. Reaction of benzyl alcohol with 2-bromoethyl isocyanate or 4-bromobutyl isocyanate (Minin, Patricia L. and Walton, John C. J. Org. Chem. 2003, 68, 2960-
2963) in dichloromethane at room temperature give intermediates 25a and 25b, respectively (Scheme 12). Subsequent reaction of homovanillyl alcohol with intermediates 25a and 25b in the presence of sodium carbonate and in a polar solvent such as DMF give precursors 26a and 26b, respectively.
SCHEME 13: PREPARATION OF AMINOALCOHOL (23)
Bn-. TBDMSCI/lmidazole Bn. N R>""OH DMF f?>""OTBDMS 20 21 H2/Pd-C/EtOH
OTBDMS
23 22
Scheme 13 illustrates the preparation of aminoalcohol (23) via cyclohexene oxide opening with secondary amine in the presence of water. Aminoalcohols were prepared by typical SN2 cyclohexene oxide opening with the secondary amine of choice in the presence of water which provides aminoalcohols with an anti relationship relative to the cyclohexane ring. More specifically, aminoalcohol 23 (Scheme 13) required the preparation of amine 22. Λ/-Benzyl-3-(R)- pyrrolidinol 20 was silylated with tert-butyldimethylsilyl chloride and imidazole in DMF to give 21 , hydrogenolysis of the benzyl protecting group provided 22. Cyclohexene oxide ring opening with 22 in water gave aminoalcohol 23.
SCHEME 14: DERIVATIZATION OF 28A,B WITH BIFUNCTIONAL LINKERS 6, 8, 12 AND 29
Standard work-up procedures well known in the art permit the isolation of thiol-reactive derivatives 30a,b-33a,b.
SCHEME 15: CONJUGATION OF THIOL-REACTIVE DERIVATIVES (30A.B-33A.B) WITH THIOL FUNCTIONALITY OF ALBUMIN TO GENERATE ALBUMIN CONJUGATE (34A,B-37A,B)
BIOLOGICAL EXAMPLE 1 ASSESSMENT OF ANTIARRHYTHMIC EFFICACY Antiarrhythmic efficacy may be assessed by investigating the effect of a serum protein conjugate of the invention on the incidence of cardiac arrhythmias in anesthetized rats subjected to coronary artery occlusion. Rats weighing 200-300 gms are subjected to preparative surgery and assigned to groups in a random block design. In each case, the animal is anesthetized with pentobarbital during surgical preparation. The left carotid artery is cannulated for measurement of mean arterial blood pressure and withdrawal of blood samples. The left jugular vein is also cannulated for injection of drugs. The thoracic cavity is opened and a polyethylene occluder loosely placed around the left anterior descending coronary artery. The thoracic cavity is then closed. An ECG is recorded by insertion of electrodes placed along the anatomical axis of the heart. In a random and double-blind manner, an infusion of vehicle or the compound to be tested is given about 15 min post-surgery. After 5 minutes infusion, the occluder is pulled so as to produce a coronary artery occlusion. ECG, arrhythmias, blood pressure, heart rate and mortality are monitored for 15 minutes after occlusion. Arrhythmias are recorded as ventricular tachycardia (VT) and ventricular fibrillation
(VF) and scored according to Curtis, M.J. and Walker, M.J.A., Cardiovasc. Res. 22:656 (1988). Rats are excluded from the study if they did not exhibit pre-occlusion serum potassium concentrations within the range of 2.9-3.9 mM. Occlusion is associated with increases in R-wave height and "S-T" segment elevation; and an occluded zone
(measured after death by cardiogreen dye perfusion) in the range of 25%-50% of total left-ventricular weight. Results of the test compounds may be expressed as values of a given infusion rate in micromol/kg/min. (ED50AA) which will reduce the arrhythmia score in treated animals to 50% of that shown by animals treated only with the vehicle in which the test
compound(s) is dissolved.
BIOLOGICAL EXAMPLE 2 MEASUREMENT OF CARDIOVASCULAR AND BEHAVIORAL EFFECTS Preparative surgery is performed in Sprague Dawley rats weighing 200-300 gm and anaesthetized with 65mg/kg (i.p.) pentobarbital. The femoral artery and vein are cannulated using polyethylene (PE)-10 tubing. Prior to surgery, this PE-10 tubing had been annealed to a wider gauge (PE-50) tubing for externalization. The cannulated PE-10/PE-50 tubing is passed through a trocar and exteriorised together with three (lead II) limb ECG leads (see below). The trocar is threaded under the skin of the back and out through a small incision at the mid-scapular region. A ground ECG electrode is inserted subcutaneously using a 20 gauge needle with the lead wire threaded through it. To place the other ECG electrodes, a small incision is made in the anterior chest region over the heart and ECG leads are inserted into the subcutaneous muscle layer in the region of the heart using a 20 guage needle. Other ECG leads are inserted into the subcutaneous muscle layer in the region near the base of the neck and shoulder (right side). The animal is returned to a clean recovery-cage with free access to food and water. The treatment and observational period for each animal commenced after a 24-hour recovery period. A 15 min observational period is recorded followed by the intravenous infusion regime of the test compound at an initial dose of 2.0μmol/kg/min (at 1 ml/hr). This rate is doubled every 5 minutes until one of the following effects is observed: a) partial or complete convulsions b) severe arrhythmias c) bradycardia below 120 beats/min d) hypotension below 50mmHg e) the dose exceeds 32 times the initial starting dose (i.e. 64 μmol/kg/min). Blood pressure (BP), heart rate (HR) and ECG variables are continuously recorded while behavioral responses are also monitored and the total accumulative drug dose and drug infusion rate at which the response (such as convulsion, piloerection, ataxia, restlessness, compulsive chewing, lip-smacking, wet dog shake etc.) occurred are recorded. Estimates of plasma concentrations of the test compound are determined by removing a 0.5 mL blood sample at the end of the experiment. Blood samples are centrifuged for 5 min at 4600 x g and the plasma decanted. Brain tissue samples are
also extracted and kept frozen (-20°C) along with the plasma samples for chemical analysis. Electrocardiograph (ECG) parameters: PR, QRS, QT-, (peak of T-wave), QT2 (midpoint of T-wave deflection) and hemodynamic parameters: BP and HR are analyzed using the automated analysis function in LabView (National Instruments) with a customized autoanalysis software (Nortran Pharmaceuticals). The infused dose producing 25% from control (D25) for all recorded ECG variables is determined. Results of the tests can be expressed as D25 (micromol/kg) which are the doses required to produce a 25% increase in the ECG parameter measured. The increases in P-R interval and QRS interval indicate cardiac sodium channel blockade while the increase in Q-T interval indicates cardiac potassium channel blockade.
BIOLOGICAL EXAMPLE 3 ELECTROPHYSIOLOGICAL TEST (IN VIVO) Male Sprague-Dawley rats weighing between 250-350g are used. They are randomly selected from a single group and anesthetized with pentobarbital (65mg/kg, ip.) with additional anesthetic given if necessary. The trachea is cannulated and the rat is artificially ventilated at a stroke volume of 10 mlJkg, 60 strokes/minute. The right external jugular vein and the left carotid artery are cannulated for intravenous injections of compounds and blood pressure (BP) recording, respectively. Needle electrodes are subcutaneously inserted along the suspected anatomical axis (right atrium to apex) of the heart for ECG measurement. The superior electrode is placed at the level of the right clavicle about 0.5 cm from the midline, while the inferior electrode is placed on the left side of the thorax, 0.5 cm from the midline and at the level of the ninth rib. Two Teflon-coated silver electrodes are inserted through the chest wall using 27G needles as guides and implanted in the epicardium of left ventricle (4-5 mm apart). Square pulse stimulation is provided by a stimulator controlled by a computer. In-house programmed software is used to determine the following: threshold current (\T) for induction of extra systoles, maximum following frequency (MFF), effective refractory period (ERP) and ventricular flutter threshold (VTt). Briefly, iT is measured as the minimal current (in μA) of a square wave stimulus required to capture and pace the heart at a frequency of 7.5 Hz and a pulse width of 0.5msec; ERP is the minimum delay (in msec) for a second stimulus required to cause an extra systole with the heart
entrained at a frequency of 7.5 Hz (1.5 x iT and 0.2msec pulse width), MFF is the maximum stimulation frequency (in Hz) at which the heart is unable to follow stimulation (1.5x iT and 0.2msec pulse width); VTt is the minimum pulse current (in μA) to evoke a sustained episode of VT (0.2msec pulse width and 50 Hz) (Howard, P.G. and Walker, M.J.A, Proc. West. Pharmacol. Soc. 33:123-127 (1990)). Blood pressure (BP) and electrocardiographic (ECG) parameters are recorded and analyzed using LabView (National Instruments) with a customized autoanalysis software (Nortran Pharmaceuticals Inc.) to calculate mean BP (mmHg, 2/3 diastolic + 1/3 systolic blood pressure), HR (bpm, 60/R-R interval ); PR (msec, the interval from the beginning of the P-wave to the peak of the R-wave), QRS (msec, the interval from the beginning of the R-wave due to lack of Q wave in rat ECG, to the peak of the S- wave), QT (msec, the interval from the beginning of the R-wave to the peak of the T- wave). The initial infusion dose is chosen based on a previous toxicology study of the test compound in conscious rats. This is an infusion dose that did not produce a 10% change from pre-drug levels in haemodynamic or ECG parameters. The animal is left to stabilize prior to the infusion treatment according to a predetermined random and blind table. The initial infusion treatment is started at a rate of 0.5 mL/hr/300g (i.e., 0.5μmol/kg/min). Each infusion dose is doubled (in rate) every 5 minutes. All experiments are terminated at 32 mL/hr/300g (i.e., 32 μmol/kg/min).
Electrical stimulation protocols are initiated during the last two minutes of each infusion level. Responses to test compounds are calculated as percent changes from pre- infusion values; this normalization is used to reduce individual variation. The mean values of BP and ECG parameters at immediately before the electrical stimulation period (i.e., 3 min post-infusion) are used to construct cumulative dose-response curves. Data points are fit using lines of best fit with minimum residual sum of squares (least squares; SlideWrite program; Advanced Graphics Software, Inc.). D25's (infused dose that produced 25% change from pre-infusion value) are interpolated from individual cumulative dose-response curves and used as indicators for determining the potency of compounds of the present invention. * * * * * All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
Claims
WHAT IS CLAIMED IS 1. A serum protein conjugate comprising an ion channel modulating compound and a serum protein.
2. The serum protein conjugate of claim 1 , wherein the ion channel modulating compound is attached to the serum protein via a linker.
3. The serum protein conjugate of claim 2, wherein the linker comprises a first linkage group, a spacer group, and a second linkage group.
4. The serum protein conjugate of claim 3, wherein the first linkage group is selected from a functional group selected from the group consisting of ester, amide, carbamate, urea, boronate and maleimido groups.
5. The serum protein conjugate of claim 3, wherein the spacer group is selected from the group consisting of a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, alkaryl, alkoxy, and polyethylene glycol.
6. The serum protein conjugate of claim 5, wherein the spacer group is a substituted or unsubstituted alkaryl group, wherein one of the hydrogen atoms bonded to the terminal carbon of a straight-chain (C C20)alkyl, (C2-C20)alkenyl or (C2-C20)alkynyl group is replaced with an (C5-C20)aryl moiety.
7. The serum protein conjugate of claim 6, wherein the spacer group is 3- phenyl-prop-1-yl, and wherein the second linkage group is attached to the spacer group by the substitution of the hydrogen at the para position of the phenyl ring with a bond to the second linkage group.
8. The serum protein conjugate of claim 7, wherein the second linkage group is a pyrrolidin-yl-2,5-dione-3-yl group, and wherein the bond from the spacer group to the second linkage group is a bond from the phenyl ring of the spacer group to the nitrogen of the pyrrolidin-yl-2,5-dione-3-yl group.
9. The serum protein conjugate of claim 5, wherein the spacer group is a substituted or unsubstituted aryl group.
10. The serum protein conjugate of claim 9, wherein the aryl group is a C5-C20aryl ring that is further substituted with an amino group.
11. The serum protein conjugate of claim 10, wherein the second linkage group is attached to the spacer group by a bond from the nitrogen of the amino substituent on the aryl ring to the second linkage group.
12. The serum protein conjugate of claim 11 , wherein the second linkage group comprises an acetyi moiety, and wherein the second linkage group is attached to the spacer group via an amide bond between the carbonyl functionality of the acetyl moiety and the amino group on the spacer group.
13. The serum protein conjugate of claim 5, wherein the spacer group is a substituted or unsubstituted d-C6alkyl group.
14. The serum protein conjugate of claim 13, wherein the spacer group is an ethyl group.
15. The serum protein conjugate of claim 3, wherein the second linkage group is a thiol-reactive group or an amino-reactive group.
16. The serum protein conjugate of claim 15, wherein the second linkage group is a thiol-reactive group selected from the group consisting of pyrrolidin-yl-2,5- dione-3-yl, acetyl-2-yl, and methyl-thi-yl.
17. The serum protein conjugate of claim 1 , wherein the serum protein is a serum mobile protein.
18. The serum protein conjugate of claim 1 , wherein the serum protein is a serum protein selected from the group consisting of ceuroplasmin, immunogiobulin, serum albumin, ferritin, transferrin, thyroxin binding protein, steroid binding protein, and a/pt7a-2-macroglobulin.
19. The serum protein conjugate of claim 1 , wherein the serum protein comprises an unpaired thiol residue.
20. The serum protein conjugate of claim 17, wherein the serum mobile protein is human serum albumin.
21. The serum protein conjugate of claim 1 , wherein the molar ratio of serum protein to ion channel modulating compound is 1 :1.
22. The serum protein conjugate of claim 21 , wherein the serum protein is human serum albumin.
23. The serum protein conjugate of claim 22, wherein the ion channel modulating compound is conjugated to human serum albumin at the Cys34 thiol functionality of the protein.
24. The serum protein conjugate of clam 1 , wherein the ion channel modulating compound comprises an aminocyclohexyl ether moiety.
25. The serum protein serum protein conjugate of Claim 1 , wherein the ion channel modulating compound is a compound of formula (I):
26. The serum protein conjugate of Claim 1 , wherein the ion channel modulating compound is a compound of formula (IA), or solvates, pharmaceutically acceptable salts, esters, amides, complexes, chelates, stereoisomers, stereoisomeric mixtures, geometric isomers, crystalline or amorphous forms, metabolites, or metabolic precursors thereof:
27. The serum protein conjugate of claim 26, wherein the ion channel modulating compound is attached to the protein by the substitution of the valency occupied by hydrogen in the 'ww QH moiety with a bond to the protein.
28. The serum protein conjugate of Claim 1 , wherein the ion channel modulating compound is the Compound A of the following formula:
29. The serum protein conjugate of claim 28, wherein the ion channel modulating compound is attached to the protein by the substitution of the valency occupied by hydrogen in the """''''OH moiety with a bond to the protein.
30. A thiol-reactive ion channel modulating compound of the formula (IV-R), or a solvate or pharmaceutically acceptable salt thereof:
31. The thiol-reactive ion channel modulating compound of claim 30, wherein Z' is a first linkage group selected from the group consisting of ester, amide, carbamate, urea, boronate and maleimido; Z" is a spacer group selected from the group consisting of substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, alkaryl, alkoxy, and polyethylene glycol; Z'" is selected from the group consisting of maleimidyl, haloacetyl, alkenyl sulfone, alkylsulfonethi-yl and pyridyl disulfide.
32. The thiol-reactive ion channel modulating compound of claim 30, wherein Z'" is selected from the group consisting of iodoacetyl, 2-pyridylthiol, pyridin-2- yl disulfane, vinyl sulfonyl, methylsulfonethi-yl, thiosulfonate and maleimidyl.
33. The thiol-reactive ion channel modulating compound of claim 30, whererin Z" is a substituted or unsubstituted alkaryl group, wherein one of the hydrogen atoms bonded to the terminal carbon of a straight-chain (CrC20) alkyl, (C2-C20) alkenyl or (C2-C20) alkynyl group is replaced with an (C5 -C20) aryl moiety.
34. A method of conjugating an ion channel modulating compound to a serum protein comprising contacting a thiol-reactive ion channel modulating compound with a serum protein comprising an unbound thiol residue.
35. The method of claim 34, wherein the serum protein is human serum albumin.
36. The method of claim 35, wherein the human serum albumin is conjugated to the ion channel modulating compound in vivo, ex vivo or in vitro.
37. The thiol-reactive ion channel modulating compound of claim 30, wherein the compound is selected from the group consisting of :
38. The serum protein conjugate of claim 1 , wherein the serum protein conjugate comprises human serum albumin and wherein the serum protein conjugate is selected from the group consisting of:
39. A thiol-reactive ion channel modulating compound of formula (Al), or a solvate or pharmaceutically acceptable salt thereof:
3-azabicyclo[3.1.0]hexan-3-yl and 3-azabicyclo[3.2.0]heptan-3-yl; wherein the bicyclic ring may be substituted with a Z'-Z"-Z'" moiety; R3 and R4 are independently attached to the cyclohexane ring shown in formula (Al) at the 3-, 4-, 5- or 6- positions and are independently selected from hydrogen, hydroxy, C C6alkyl and d-C6alkoxy, and, when both R3 and R4 are attached to the same cyclohexane ring atom, may together form a spiro five- or six-membered heterocyclic ring containing one or two heteroatoms selected from oxygen and sulfur; R5l R6 and R14 are independently selected from hydrogen, d-Cealkyl, aryl and benzyl, or R6 and R 4, when taken together with the carbon to which they are attached, may form a spiro C3-C5cycloalkyl; A is selected from formula (Alll):
40. A thiol-reactive ion channel modulating compound of formula (IV-C), or a solvate or pharmaceutically acceptable salt thereof: 
wherein, independently at each occurrence, X is selected from a direct bond, -C(R6,R14)-Y- and -C(R13)=CH-, Y is selected from a direct bond, O, S and d-C4alkylene; R13 is selected from hydrogen, d-C6alkyl, C3-C8cycloalkyl, aryl and benzyl; RT and R2 are independently selected from hydrogen, d-C8alkyl, C3-C8alkoxyalkyl, d-dhydroxyalkyl, and C7-C12aralkyl; or R-i and R2, when taken together with the nitrogen atom to which they are directly attached in formula (IV-C), form a ring denoted by formula (All):
41. A pharmaceutical composition comprising a serum protein conjugate of any one of Claims 1-40 and a pharmaceutically acceptable excipient.
42. A method of treating or preventing arrhythmia in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a serum protein conjuate of any one of Claims 1-40 or a pharmaceutical composition of Claim 41.
43. A method for modulating ion channel activity in a subject, wherein the method comprises administering to the subject an effective amount of a serum protein conjugate of any one of Claims 1-40 or a pharmaceutical composition of Claim 41.
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