WO2004110424A1

WO2004110424A1 - Method and pharmaceutical formulation for reducing stress-induced accelerated colonic transit in a mammal

Info

Publication number: WO2004110424A1
Application number: PCT/US2004/016565
Authority: WO
Inventors: Frank C. Barone; Jeffrey J. Legos; Raymond F. White
Original assignee: Glaxo Group Limited
Priority date: 2003-05-28
Filing date: 2004-05-27
Publication date: 2004-12-23

Abstract

The present invention relates to a method of reducing stress-induced accelerated colonic transit, a representative model of irritable bowel syndrome and thus IBS, in a mammal using an inhibitor of phosphodiesterase 4 (PDE4).

Description

METHOD AND PHARMACEUTICAL FORMULATION FOR REDUCING STRESS-INDUCED ACCELERATED COLONIC TRANSIT IN A MAMMAL

Field of Invention

The present invention relates to a method of reducing stress-induced accelerated colonic transit and evaluating compounds effect on stress-induced accelerated colonic transit.

BACKGROUND OF THE INVENTION

Irritable Bowel Syndrome (IBS) is a commonly occurring gastrointestinal (GI) disorder and accounts for the greater majority of visits to gastroenterologists. The precise causes for this disease are unknown but relate to altered bowel function and discomfort with both upper and lower GI symptoms. Primary symptoms of the syndrome may include altered bowel habit, diarrhea and/or constipation, abdominal distension and pain. Stress is well documented to initiate or exacerbate the symptoms of IBS. Emotional factors, diet, drugs, or hormones may also precipitate or aggravate a heightened sensitivity to GI motility and exacerbate the symptoms of IBS. In non-infectious diarrhea like that which may be predominant in IBS, accelerated colonic transit occurs. This accelerated colonic transit and diarrhea may be induced by various types of psychological stress and may be modeled in small rodents.

Phosphodiesterases (PDEs) comprise a large, divergent family of en:zymes that catalyze the catabolism of cAMP and cGMP to AMP and GMP, respectively. Eleven members of this family have been identified to date based on substrate specificity, kinetic properties, sensitivity to specific inhibitors, tissue distribution, and primary sequences (Manganiello, VC et al., Arch Biochem Biophys, 322 (1): 1-13, (1995); Fawcett, L et al., Proc Natl Acad Sci USA₁ 97(7): 3702-3707 (2000)). Recognition of these differences has greatly stimulated interest in PDEs as drug targets. In particular, the cAMP specific PDE, termed PDE4, that is found in nearly all immune and inflammatory cells has been an attractive target for novel anti-asthmatic and anti-inflammatory therapies.

The present inventors have now discovered that PDE4 inhibitors have a property of reducing colonic transit. SUMMARY OF THE INVENTION

In a first aspect of the present invention, a method is provided for reducing stress- induced accelerated colonic transit in a mammal, comprising administering an effective amount of a phosphodiesterase 4 (PDE4) inhibitor to reduce said colonic transit. In a another aspect of the present invention, there is provided a pharmaceutical formulation comprising a PDE4 inhibitor in an amount effective to reduce colonic transit in a mammal.

DETAILED DESCRIPTION OF THE INVENTION In view of an unmet medical need of providing new treatments for disorders associated with accelerated colonic transit, a study was commenced that investigated inhibitors having a property of reducing colonic transit. The present invention was based, in part, on these studies.

The instant invention provides a method for reducing stress-induced accelerated colonic transit in a mammal, comprising administering an effective amount of a phosphodiesterase 4 (PDE4) inhibitor to reduce said colonic transit.

As used herein, "colonic transit" refers to movement of bowel contents including, but not limited to, excretion of feces.

As used herein, "normal colonic transit" refers to the number of fecal pellets or rate or extent of colonic transit in an animal in the absence of induced external stress or GI disorder.

As used herein, "reduce colonic transit" or "reducing colonic transit" refers to a decrease in the number of fecal pellets excreted by an animal over the course of a defined increment of time compared with another interval of time of the same increment. Reduced colonic transit may be caused by application or administration of any of the methods or compounds of the current invention.

As used herein, "accelerated colonic transit" refers to an increase in the number of fecal pellets excreted by an animal over the course of a defined increment of time compared with another interval of time of the same increment. Accelerated colonic transit may be induced by external stress and/or a GI disorder.

As used herein, "inhibitor(s)" or "inhibiting," as it relates to PDE4, means a compound that causes or is related to a change in an amount, and/or quality, and/or effect of a particular response and/or activity of PDE4. As used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon having from one to twelve carbon atoms, optionally substituted with substituents selected from the group that includes, but is not limited to, Ci-C₆ alkyl, C₁-Ce alkoxy, Q-C₆ alkylsulfanyl, Ci-C₆ alkylsulfenyl, Ci-C₆ alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by a substituent selected from the group including alkyl, nitro, cyano, halogen and lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of "alkyl" as used herein may include, but are not limited to, methyl, ethyl, n-butyl, n-pentyl, isobutyl, isopropyl and the like. As used herein, the term "alkoxy" refers to the group R₃O-, where R₃ is alkyl.

As used herein, the term "aryl" refers to a 6 to 10 carbon aromatic moiety. Examples of "aryl" as used herein may include, but are not limited to, phenyl, pyridyl, pyrimidyl, pyridazyl, pyrazyl, and triazyl.

As used herein, the term "aryloxy" refers to the group R₃O-, where R₃ is aryl. As used herein the term "halogen" refers to the group fluorine, chlorine, bromine, and iodine.

As used herein the term "agent" is understood to mean a substance that produces a desired effect in a tissue, system, animal, mammal (in particular human), or other subject.

As recited above, the method of the present invention includes administering a specific PDE4 inhibitor agent. Typical PDE4 inhibitor agents may include, but are not limited to, rolipram, rolipram derivatives and rolipram mimetic compounds; and fused ring compounds such as benzopyrazoles, benzimidazoles, benzofurans, diazepinio-indoles, quinolines, quinolones, nitraquazone derivatives, purines and xanthines, and losartan analogues. Such PDE4 inhibitors are described, for instance, in "PDE4 inhibitors 1998", Norman, Peter; Exp. Opin. Ther. Patents (1998) 8(7); and "Chronic Pulmonary

Inflammation and Other Therapeutic Applications of PDEIV Inhibitors", Stafford, Jeffrey A. and Feldman, Paul L., Ch. 8 Annual Reports In Medicinal Chemistry Ch. 8, 71-80 (1996) which references are herein incorporated by reference to the extent of their disclosure of PDE4 inhibitor compounds. One class of PDE4 inhibitor compounds that may be usefully employed in the present invention includes compounds of the Formula I :

wherein: R₁ is -(CR₄ R₅)_n C(O)O(CR₄ R₅)_m Re, -(CR₄ Rs)_n C(O)NR₄ (CR₄ R₅)_ra R₆, -(CR₄ Rs)_n 0(CR₄ R₅)_m R₅, or -(CR₄ R₅)_r R5 wherein the alkyl moieties may be optionally substituted with one or more halogens; m is O to 2; n is 0 to 4; r is 0 to 6;

R₄ and R₅ are independently selected from hydrogen or a Ci_-2 alkyl; Rs is hydrogen, methyl, hydroxyl, aryl, halo substituted aryl, aryloxy C₁.₃ alkyl, halo substituted aryloxy C₁.₃ alkyl, indanyl, indenyl, C_7-π polycycloalkyl, tetrahydrofuranyl, furanyl, tetrahydropyranyl, pyranyl, tetrahydrothienyl, thienyl, tetrahydrothiopyranyl, thiopyranyl, C₃.₆ cycloalkyl, or a C_4-6 cycloalkyl containing one or two unsaturated bonds, wherein the cycloalkyl and heterocyclic moieties may be optionally substituted by 1 to 3 methyl groups or one ethyl group; provided that: when R₆ is hydroxyl, then m is 2; or when R₆ is hydroxyl, then r is 2 to 6; or when R₆ is 2-tetrahydropyranyl, 2-tetrahydrothiopyranyl, 2- tetrahydrofuranyl, or 2-tetrahydrothienyl, then m is 1 or 2; or when R₅ is 2-tetrahydropyranyl, 2-tetrahydrothiopyranyl, 2- tetrahydrofuranyl, or 2-tetrahydrothienyl, then r is 1 to 6; when n is 1 and m is 0, then R₆ is other than H in -(CR₄ Rs)_nO(CR₄ R₅)_m R₆

X is YR₂, halogen, nitro, NR₄ R5, or formyl amine; Y is O or S(O)_1n ¹; m' is 0, 1, or 2; X₂ is O or NR₈ ; X3 is hydrogen or X;

X₄ ΪS

C(Z')NHR₁₅, CR₄(NOC(O)R₁₆), or CR₂ ⁵R₃ ³CR₄ ⁵R₅ ⁵R₆';

X₅ is H, R₉, OR₈, CN, C(O)R₈, C(O)OR₈₅ C(O)NR₈ R₈, or NR₈ R₈ ;

R₂ is independently selected from the group consisting Of-CH₃ and -CH₂ CH₃ optionally substituted by 1 or more halogens;

R₂ ⁵ is a hydrogen, halogen, or ORi₇; s is O to 4;

R₃ ⁵ and R₄ ⁵ are each independently -(CH2)_tX₆; t is O, 1, 2, or 3; v is 0, 1, 2, or 3;

X₆ is a mono- or bicyclic aryl group optionally containing one or more heteroatoms selected from sulfur, oxygen, or nitrogen;

R₅ ⁵ and R₆ ⁵ are each independently hydrogen or an optionally substituted alkyl;

R₃ is hydrogen, halogen, C₁₄ alkyl, CH₂NHC(O)C(O)NH₂, halo-substituted C_1-4 alkyl, - CH=CR₈ ⁵R₈ ⁵, cyclopropyl optionally substituted by R₈ ', CN, OR₈, CH₂OR₈, NR₈Ri₀,

CH₂NR₈Ri₀, C(Z')H, C(O)OR₈, C(O)OR₈, C(O)NR₈Ri₀, or C≡CR₈;

Z' is O, S, NR₈, NOR₈, NCN, C(-CN)₂, CR₈CN, CR₈NO₂, CR₈C(O)OR₈, CR₈C(O)ONR₈R₈,

C(-CN)N0₂, C(-CN)C(O)OR₉, 0(-CN)C(O)NR₈R₈;

Z is COORi₄, C(O)ORu, COf)NRi₀ Ri₄, C(NRi₀)NR₁₀ R_M, CN, C(NOR₈)Ri₄, C(O)NR₈ NR₈ C(O)R₈, C(O)NR₈ NRi₀ R_M, C(NORi₄)R₈, C(NR₈)NRi₀ RM, C(NR_I4)NR₈ R₈

C(NCN)NRi₀ Ri₄, C(NCN)SR₉, (2-, 4- or 5-imidazolyl), (3-, 4- or 5-pyrazolyl), (4- or 5- triazolyl[l,2,3]), (3- or 5-triazolyl[l,2,4]), (5-tetrazolyl), (2-, 4- or 5-oxazolyl), (3-, 4- or 5- isoxazolyl), (3- or 5-oxadiazolyl[l,2,4]), (2oxadiazolyl[l,3,4]), (2-thiadiazolyl[l,3,4]), (2-,

4-, or 5-thiazolyl), (2-, A-, or 5-oxazolidinyl), (2-, 4-, or 5-thiazolidinyl), or (2-, A-, or 5- imidazolidinyl); wherein all of the heterocylic ring systems may be optionally substituted one or more times by R_M ; the dotted line in the first formula of X₄ represents a single or double bond; Y' is O or S; R₇ is -(CR₄ R₅)_q Rn or Cμ.₆ alkyl wherein the Rj₂ or Ci_-6 alkyl group is optionally substituted one or more times by Ci_-2 alkyl optionally substituted by one to three fluorines, - -F, -Br, -Cl, -NO₂, --NRi₀ R_n, -C(O)R₈, -C(O)OR₈, -OR₈, -CN, -C(O)NRi₀ Rn, ~ OC(O)NR₁₀ R_n, -OC(O)R₈, -NRi₀ C(O)NR₁₀ R_n, -NRi₀ C(O)Rn, -NRj₀ C (O)OR₉, - NRio C(O)Ri₃, -C(NRio)NRio Rn, -C(NCN)NRi₀ Rn, -C(NCN)SR₉, -NR₁₀ C (NCN)SR₉, -NR₁₀ C(NCN)NRi₀ Rn, -NR₁₀ S(O)₂ R₉, -S(O)_n, ¹R₉, ~NR_]0 C(O)C(O)NR₁₀ R_u, -NRi₀ C(O)C(O)Ri₀, thiazolyl, imidazolyl, oxazolyl, pyrazolyl, triazolyl, or tetrazolyl; q is O, 1, or 2;

Ri₂ is C₃ -C₇ -cycloalkyl, (2-, 3- or 4-pyridyl), pyrimidyl, pyrazolyl, (1- or 2-imidazolyl), thiazolyl, triazolyl, pyrrolyl, piperazinyl, piperidinyl, morpholinyl, furanyl, (2- or 3-thienyl), (4- or 5-thiazolyl), quinolinyl, naphthyl, or phenyl; R₈ is independently selected from hydrogen or R₉ ; R₈ ' is R₈ or fluorine;

R₉ is Ci_-4 alkyl optionally substituted by one to three fluorines;

; Rn is hydrogen, or C_M alkyl optionally substituted by one to three fluorines; or when Ri₀ and Rn are as NR]₀ Rn they may together with the nitrogen form a 5 to 7 membered ring optionally containing at least one additional heteroatom selected from O/N/or S; Ri₃ is oxazolidinyl, oxazolyl, thiazolyl, pyrazolyl, triazolyl, tetrazolyl, imidazolyl, imidazolidinyl, thiazolidinyl, isoxazolyl, oxadiazolyl, or thiadiazolyl, and each of these heterocyclic rings is connected through a carbon atom and each may be unsubstituted or substituted by one or two Ci_-2 alkyl groups;

Ri₄ is hydrogen or R₇ ; or when Ri₀ and R₁₄ are as NR₁₀ R₁₄ they may together with the nitrogen form a 5 to 7 membered ring optionally containing one or more additional heteroatoms selected from O, N, or S; R₁S is optionally substituted pyridyl, pyrazinyl, pyrimidinyl, isoxazolyl; or an N-oxide thereof; or a optionally substituted phenyl, wherein s optional substituents are one or more selected from the group consisting of halogen, haloalkyl, aryl, arylalkyl, alkoxy, aryloxy, alkylthio, arylthio, alkoxycarbonyl, alkanoyl, aroyl, alkylsulphonyl, arylsulphonyl, alkylsulphinyl, arylsulphinyl, hydroxy, hydroxyalkyl, formyl, alkanoylamino, aroylamino, cyano, and nitro;

Ri₆ is amino, Ci-C₆ alkylamino, arylamino, C₁-C₆ alkoxy, or aryloxy;

R₁₇ is hydrogen, or optionally substituted alkyl, alkenyl, alkqxyalkyl, or alkanoyl, or a formyl, carboxamido, or thiocarboxamido;

R₁₈ is (=0), hydrogen, -C(O)R₂, -CH₃, -CH₂CH₃, -C(O)OH; provided that: when R₁₂ is N-pyrazolyl, N-imidazolyl, N-triazolyl, N-pyrrolyl, N-piperazinyl, N- piperidinyl, or N-morpholinyl, then q is not 1; or the pharmaceutically acceptable salts thereof.

In one embodiment, X₂ is O; R₁ is (CR(RsXRe, where r=0 and R₅ is C₃-C₆ cycloalkyl, or cyclopentyl; X=YR₂; R₂ is -CH₃; Y=O; X₄ is

R3 is -CN; X₅ is hydrogen; s=0; Z is C(O)OR_M; and R14 is hydrogen.

Of particular interest within the PDE4 inhibitors of Formula I are czs-4-cyano-4-[3- (cyclopentyloxy)-4-methoxyphenyl]cyclohexane-l-carboxylic acid and 4-cyano-4-(3- cyclopentyloxy-4-methoxyphenyl)cyclohexan-l-one (SB 200473) and salt thereof. The first compound has the trademark of ARIFLO, represented by Figure 9(a) of WO 01/93909. The second compound is described in Example 1 of US 5,643,946. Other compounds within the scope of Formula I are also described in WO 01/93909 and/or US5,643,946.

SB 200473 Another PDE4 inhibitor that may be usefully employed in the present invention includes filaminast, l-[3-cyclopentyloxy)-4-methoxyphenyl] ethanone (E)-O- {aminocarbonyl}oxime, represented by Figure 10(a) of WO 01/93909, and related compounds. Filaminast and related compounds are described, as well as the synthesis thereof, in European Patent Application EP 470805A1.

Still another group of Formula I PDE4 inhibitor compounds that may be usefully employed in the process of the present invention includes (+/-)-4-[2-(3-cyclopentyloxy-4- methoxyphenyl)-2-phenylethyl]pyridine (CDP-840), represented by Figure 10(b) of WO 01/93909, and related compounds. These compounds are described, as well as the synthesis thereof, in International Patent Applications WO 94/14742; 97/23460; 97/23461; 97/38976; 97/42172; and 98/12178.

Additional PDE4 inhibitors that may be utilized in the present invention may include, but are not limited to:

(i) nitroquazone, depicted in Figure 1 l(a) of WO 01/93909, and nitraquazone derivatives, such compounds being described in WO 93/07146 including the synthesis thereof;

(ii) denbufylline, i.e., 7-acetonyl,l,3dibutylxanthine, made by SmithKline Beecham and depicted in Figure 1 l(b) of WO 01/93909;

(iii) rolipram, depicted in Figure 1 l(c) of WO 01/93909; (iv) RS-25344, depicted in Figure 12(a) WO 01/93909 and related compounds, such compounds being described in WO 93/07146 and 93/19068 including the synthesis thereof;

(v) CP-77059, depicted in Figure 12(b) WO 01/93909 and related compounds, such compounds being described in WO 96/40636 and 97/05105 including the synthesis thereof;

(v) GI 193600X, available from Glaxo Wellcome, Inc., depicted in Figure

12(c) WO 01/93909; and (vi) Roflumilast described in The Journal of Pharmacology and Experimental

Therapeutics, VoI 297, No. 1, pp267-279 (2001) In one embodiment, a PDE4 inhibitor agent is a compound of Formula I. In another embodiment, a PDE4 inhibitor is czs-4-cyano-4-[3-(cyclopentyloxy)-4- methoxyphenyl]cyclohexane-l-carboxylic acid or salt thereof; N-(3,5-dichloropyrid-4-yl)-3- cyclopentyloxy-4-methoxybenzamide; 1 -[3 -cyclopentyloxy)-4-methoxyphenyl] ethanone (E)-O- {aminocarbonyl}oxime; (+/-)-4-[2-(3-cyclopentyloxy-4-methoxyphenyl)-2- phenylethyl]pyridine; SB200473; or roflumilast.

In another embodiment, PDE4 inhibitors of the present invention are PDE4 specific inhibitors. By "PDE4 specific inhibitors" they are meant that activities of inhibitors against PDE IV en2yme is at least 400 times or more potent in terms of IC₅₀ as compared to activities against other PDE isoenzymes, e.g. PDE I, II, HI, or V. For example the activities of flumilast, rolipram and cilomilast (AREFLO) against PDE IV are more than 400 times potent against PDE I, II, IH and V. {J Pharmacol Exp Titer 2001 Apr;297(l):267-79). The present use of PDE4 inhibitors to reduce colonic transit are not limited to human use, but to a veterinary use as well. However, it is well understood that the effectiveness of a particular PDE4 inhibitor in a particular species is limited, such as, by its bioavailability in that species. Which PDE4 inhibitor is more effective in a particular species may be readily ascertained by conventional pharmacological methods.

Thus, one aspect of the present invention is a method of reducing colonic transit in a mammal, comprising administering an amount effective for reducing said colonic transit with a phosphodiesterase 4 (PDE4) inhibitor. In another aspect, the mammal is suffering from accelerated colonic transit. In another aspect, the mammal is suffering from irritable bowel syndrome. In another aspect, the PDE4 inhibitor is PDE4 specific inhibitor. A PDE4 specific inhibitor may be rolipram, roflumilast, or SB200473, among others. PDE4 specific inhibitors may be administered separately or in combination with one another or with other pharmaceutical agents. In another aspect, accelerated colonic transit may be aggravated by stress.

In another embodiment, this invention provides a pharmaceutical formulation comprising a PDE4 inhibitor in an amount effective to reduce colonic transit in a mammal suffering from accelerated colonic transit. In another aspect, the mammal is suffering from accelerated colonic transit. In another aspect, the mammal is suffering from irritable bowel syndrome. In some instances, accelerated colonic transit may be induced by stress. In another aspect, a PDE4 inhibitor is PDE4 specific inhibitor. A PDE4 specific inhibitor may be rolipram, roflumilast, or SB200473, among others. These PDE4 inhibitors may be administered separately or in combination with one another or with other pharmaceutical agents.

PDE4 inhibitors of the present invention may be administered by any appropriate route. Suitable routes may include, but are not limited to, oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intraveneous, intradermal, intrathecal, and epidural). It will be appreciated that a route may vary with, for example, the condition of the recipient.

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; chewable gum; or oil-in-water liquid emulsions or water-in-oil liquid emulsions, among others.

For instance, for oral administration in the form of a tablet or capsule, an active drug component may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Powders are prepared by comminuting a compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing and coloring agent may also be present.

Capsules are made, for example, by preparing a powder mixture as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol may be added to a powder mixture before a filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate may also be added to improve the availability of a medicament when a capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated into a mixture. Suitable binders may include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms may include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators may include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing a compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate, among others. A powder mixture may be granulated by wetting with, for example, a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, a powder mixture may be run through a tablet machine forming imperfectly formed slugs broken into granules. Granules may be lubricated to prevent sticking to tablet-forming dies by means of addition of stearic acid, a stearate salt, talc or mineral oil, among others. A lubricated mixture is then compressed into tablets. Compounds of the present invention may also be combined, for example, with free flowing inert carrier and compressed into tablets directly without going through granulating or slugging steps. A clear or opaque protective coating consisting of, for example, a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating of wax may be provided. Dyestuffs or other compounds may be added to these coatings to distinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs may be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups may be prepared by dissolving a compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions may be formulated by dispersing a compound in a non-toxic vehicle. Solubilizers and emulsifϊers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like may also be added.

Where appropriate, dosage unit formulations for oral administration may be microencapsulated. A formulation may also be prepared to prolong or sustain release as for example by coating or embedding particulate material in polymers, wax or the like. In addition, oral formulation may be in the form of a chewable gum.

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, an active ingredient may be delivered from a patch by iontophoresis as generally described in Phaπnaceutical Research, 3(6), 318 (1986).

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas, among others. Pharmaceutical formulations adapted for parenteral administration may include, but are not limited to, aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes that render a formulation isotonic with the blood of an intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. Formulations may be presented in unit- dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only addition of a sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. It should be understood that in addition to the ingredients particularly mentioned above, formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

Salts encompassed within the term "pharmaceutically acceptable salts" refer to non- toxic salts of the compounds of this invention that are prepared, for example, by reacting a free base with a suitable organic or inorganic acid or by reacting an acid with a suitable organic or inorganic base.

Typically, a therapeutically effective amount of a PDE4 inhibitor of the present invention will depend upon a number of factors including, for example, age and weight of a mammal, at least one precise condition requiring treatment, severity of a condition, nature of a formulation, and route of administration. Ultimately, a therapeutically effective amount will be at the discretion of an attendant physician or veterinarian.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.

Example 1

Preparation ofPDE4 Inhibitor Suspension.

PDE4 inhibitors, rolipram (TOCRIS, USA), roflumilast, and SB200473, were suspended in 1% carboxymethylcellulose (CMC) solution (vehicle) with a sonicator and utilized in the following examples.

Example 2

Loperamide as target validation for the reduction of stress-induced accelerated colonic transit Male C57BL/6 mice (n=10 per dose group), 18-24 g in weight, were used for the experiment. Loperamide, a known opiate agonist that reduces colonic transit, was orally administered to the mice at doses between 0.3 mg/kg and 10.0 mg/kg. The control group was administered with vehicle. Sixty minutes after dosing, the mice were held stationary in a wire mesh at room temperature (~25°C) for 30 minutes to induce stress. Fecal pellets were counted at 20 minutes after the start of stress induction.

A dose-related effect was observed among mice treated with increasing levels of loperamide. A statistically significant decrease in fecal pellet output was observed in mice receiving all doses of loperamide compared with vehicle. Mice treated with vehicle

(control group) excreted about nine fecal pellets within 20 minutes of the start of induced stress, while mice treated with 10 mg/kg of loperamide excreted about one fecal pellet within 20 minutes of the start of induced stress as shown in Table 1. Table 1 presents mean±SE fecal pellets excreted within 20 minutes of start of induced stress of mice treated with loperamide.

Oral treatment of mice with loperamide dose-dependently reduced colonic transit (Table 1).

Table 1. Effects of lo eramide on fecal ellet count from stressed mice

Example 3

Effect ofPDE4 inhibitors on stress-induced accelerated colonic transit.

Male C57BL/6 mice, 18-24 g in weight, were used for the experiment. Ten mice were used in each dose group as shown in Table 2. PDE4 inhibitors suspended in 1% carboxymethylcellulose (CMC) (vehicle) were orally administered to mice 60 minutes prior to restraint at doses between 1 mg/kg to 30 mg/kg (rolipram), and at doses between 3 mg/kg and 30 mg/kg (roflumilast and SB200473). The control group was administered with the vehicle. The mice were then held stationary in a wire mesh at room temperature (25⁰C) for 30 minutes to induce stress. Fecal pellets were counted at 30 minutes after the start of stress induction. A dose-related effect was observed among mice treated with increasing levels of rolipram, roflumilast and SB200473. Mice treated with vehicle excreted mean±SE fecal pellet counts of 8.8+0.7, 11.7+1.2, and 8.1+0.9 for the rolipram, roflumilast and SB200473 controls, respectively. Mean±SE fecal pellet counts for mice treated with the highest dose of rolipram, roflumilast and SB200473 were 4.1+1.0, 4.1±0.9 and 4.9±1.7, respectively. A statistically significant reduction in mean fecal pellet counts at 30 minutes after the start of stress induction was observed in mice treated with >10 mg/kg of rolipram, >3.0 mg/kg of roflumilast, and 30 mg/kg of SB200473 compared with vehicle.

Oral treatment of mice with PDE4 inhibitors dose-dependently reduced colonic transit (Table 2).

Table 2. Effects of rolipram, roflumilast and SB200473 on fecal pellet count from stressed mice.

Example 4

Effect ofPDE4 inhibitors on stress-induced accelerated colonic transit in rats.

Male Sprague Dawley rats, 260-300 g in weight, were used for the experiment. Loperamide was orally administered to rats (n=15-41 per dose group including control) at doses of 5 mg/kg, 10 mg/kg, or 20 mg/kg in an oral suspension 60 minutes prior to the start of stress induction. PDE4 inhibitors suspended in 1% CMC (vehicle) were orally administered to rats at doses of 3 mg/kg, 10 mg/kg, or 30 mg/kg (n=10-12 per dose group including control) one hour prior to the start of stress induction. Each control group was administered with the vehicle. Sixty minutes after dosing, the rats were held stationary in a wire mesh in a refrigerated room (~4°C) for 60 minutes to induce stress. Fecal pellets were counted at 60 minutes after the start of stress induction. Mean±SE fecal pellet counts were 9.17±1.08, 10.8±1.33, 8.9+0.6 for the rolipram, roflumilast, and loperamide control groups, respectively as shown in Table 3. Oral treatment of the rats with PDE4 inhibitors dose-dependently reduced stress-induced accelerated colonic transit as shown in Table 3. A statistically significant reduction in mean fecal pellet counts at 60 minutes was observed in rats treated with 30 mg/kg of rolipram and roflumilast compared with vehicle, also shown in Table 3. Total mean fecal output at 60 minutes after the start of stress induction was reduced by a statistically significant amount in rats treated with 10 mg/kg or 20 mg/kg of loperamide compared with vehicle, also shown in Table 3.

Oral treatment of rats with PDE4 inhibitors dose-dependently reduced colonic transit (Table 3).

Table 3 : Effects of rolipram, roflumilast and SB200473 on fecal pellet count from stressed rats.

The above results demonstrate that PDE4 inhibitors are effective in reducing colonic transit in mammals suffering from stress-induced accelerated colonic transit.

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

Claims

What is claim is:

1. A method of reducing colonic transit in a mammal, comprising administering an amount effective for reducing said colonic transit with a phosphodiesterase 4 (PDE4) inhibitor.

2. The method of claim 1 wherein the mammal is suffering from accelerated colonic transit.

3. The method of claim 1, wherein the mammal is suffering from irritable bowel syndrome.

4. The method of claim 1, wherein the PDE4 inhibitor is PDE4 specific inhibitor.

5. The method of claim 4, wherein the PDE4 specific inhibitor is rolipram.

6. The method of claim 4, wherein the PDE4 specific inhibitor is roflumilast.

7. The method of claim 4, wherein the PDE4 specific inhibitor is SB200473.

8. The method of claim 1, wherein the accelerated colonic transit may be aggravated by stress.

9. A pharmaceutical formulation comprising a PDE4 inhibitor in an amount effective to reduce colonic transit in a mammal.

10. The pharmaceutical formulation of claim 9, wherein said mammal is suffering from accelerated colonic transit.

11. The pharmaceutical formulation of claim 9, wherein the mammal is suffering from irritable bowel syndrome.

12. The pharmaceutical formulation of claim 9, wherein the PDE4 inhibitor is PDE4 specific inhibitor.

13. The pharmaceutical formulation of claim 12, wherein PDE4 specific inhibitor comprises rolipram, roflumilast, and SB200473.

14. The pharmaceutical formulation of claim 12, wherein PDE4 specific inhibitor is • selected from the group consisting of rolipram, roflumilast, or SB200473.