Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals

Definitions

• the present invention relates to the use of a CSBP/p38 inhibitor in the treatment of a CSBP/p38 mediated disease.
• HRV Human rhinovirus
• Symptoms are believed to arise more from the host's response to infection, than an acute cytotoxic effect, since only a small fraction of upper respiratory epithelial cells are demonstrably infected, and there is minimal epithelial cell damage (Winther et al, JAMA 256: 1763-1767 (1986). Increased intranasal levels of kinins, IL-1, IL-8, LL- 6, LL-11, and neutrophils are found in normal individuals infected with rhinoviruses. A correlation between L-8 concentration in nasal secretions with local myeloperoxidase levels and with symptom severity has been demonstrated in several recent studies (Grieff, et al. Eur Respir J 13 : 41-47 (1999); Teren, et al.
• LL-1, IL-6, and LL-8 are also produced in response to infection with other respiratory viruses (influenza, respiratory syncytial virus) which can cause the common cold and associated sequelae.
• the present invention relates to the use of a CSBP/p38 kinase inhibitor for the treatment, including prophylaxis, of the common cold, or respiratory viral infection caused by human rhinovirus infection (HRV), other enteroviruses, coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, or adenovirus infection in a human in need thereof which method comprises administering to said human an effective amount of a CBSP/p38 inhibitor.
• HRV human rhinovirus infection
• Another aspect of the present invention is a method of treating, including prophylaxis of influenza induced pneumonia in a human in need thereof which method comprises administering to said human an effective amount of a CBSP/p38 inhibitor
• the present invention also relates to the use of the CSBP/p38 kinase inhibitor for the treatment, including prophylaxis, of inflammation associated with a viral infection of a human rhinovirus (HRV), other enteroviruses, coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, or adenovirus.
• HRV human rhinovirus
• Figure 1 demonstrates Cytokine Production by Rhinovirus infected BEAS- 2B cells. Culture supernatants were collected 72 hours post-infection of BEAS-2B cells with rhinovirus-39 (MOI 1). Uninfected cells served as controls. Protein concentrations in supernatants were determined by ELISA (R&D Systems). Results represent the mean concentration values obtained from 6 experiments.
• Figure 2 demonstrates Inhibition of cytokines by CSATDs: BEAS-2B cultures infected with rhinovirus-39 were cultured in the presence of various concentrations of drug. Cytokine levels in supernatant were determined 72 hours post-infection using commercially available ELISA kits. Results are expressed as % inhibition from infected untreated cultures. Cytokine concentrations in infected control cultures were 4902 pg/ml LL-6, 4520 pg/ml LL-8, and 28 pg/ml GM-CSF.
• Figure 3 demonstrates Tyrosine phosphorylation of p38 kinase by rhinovirus infection.
• BEAS-2B cells were incubated with rhinovirus-39 for various times as indicated.
• Cell lysates were separated by 10% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with specific antibody to phosphorylated p38 kinase (A) or total p38 kinase (B).
• A phosphorylated p38 kinase
• B total p38 kinase
• Figure 4 demonstrates Tyrosine phosphorylation of p38 kinase by rhinovirus infection.
• BEAS-2B cells were incubated with various doses (MOI) of rhinovirus- 39 for 30 minutes.
• Cell lysates were separated by 10% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with specific antibody to phosphorylated p38 kinase or total p38 kinase.
• Amounts of p38 kinase were quantitated by image analyzer and are presented as relative amounts of total or phosphorylated p38 kinase compared to control cells incubated with media alone (fold increase).
• Figure 5 demonstrates the effect of Compound VI, l-(l,3-Dihydroxyprop-2- yl)-4-(4-fluorophenyl)-5-[2-phenoxypyrimidin-4-yl]imidazole on improvement of pulmonary function with increasing doses.
• BALB/c mice were treated from days 3-8 post-infection with a sub-lethal dose of Influenza A. Pulmonary resistance was determined using whole body plethysmography.
• Figure 6 demonstrates the effect of Compounds V, 1 -(4-Piperidinyl)-4-(4- fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)imidazole and Compound VI, on prevention of weight loss in animals in an in vivo influenza model.
• Figure 7 demonstrates the efficacy of of Compounds V and VI at improving arterial blood oxygen levels (%SpO2) upon treatment. SpO2 was determined using daily pulse oximetry.
• LL-1, TNF, and other cytokines affect a wide variety of cells and tissues and these cytokines as well as other leukocyte derived cytokines are important and critical inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these cytokines is of benefit in controlling, reducing and alleviating many of these disease states.
• the present invention is directed to the treatment of a viral infection in a human, which is caused by the human rhinovirus (HRV), other enterovirus, coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, or an adenovirus.
• HRV human rhinovirus
• the invention is directed to respiratory viral infections that exacerbate asthma (induced by such infections), chronic bronchitis, chronic obstructive pulmonary disease, otitis media, and sinusitis. While inhibiting LL-8 or other cytokines may be beneficial in treating a rhinovirus may be known, the use of an inhibitor of the p38 kinase for treating HRV or other respiratory viral infections causing the common cold is believed novel.
• the respiratory viral infection treated herein may also be associated with a secondary bacterial infection, such as otitis media, sinusitis, or pneumonia.
• treatment may include prophylaxis for use in a treatment group susceptible to such infections. It may also include reducing the symptoms of, ameliorating the symptoms of, reducing the severity of, reducing the incidence of, or any other change in the condition of the patient, which improves the therapeutic outcome.
• the mechanism of action for inhibition of a cytokine by a cytokine suppressive anti-inflammatory drug (CSAID) versus inhibition of virus - induced IL-8 production in airway epithelial cells is believed to be different.
• CSAID inhibition of IL-8 synthesis is independent of IL-1 and TNF production, whereas the published studies have focused on IL-1 and TNF - induced IL-8 production.
• the treatment herein is not directed to the elimination or treatment of the viral organism itself but is directed to treatment of the respiratory viral infection that exacerbates other diseases or symptoms of disease, such as asthma (induced by such infections), chronic bronchitis, chronic obstructive pulmonary disease, otitis media, and sinusitis.
• CSAID inhibitors are useful in the treatment of symptoms associated with HRV, including exacerbations of underlying conditions such as asthma, COPD, sinusitis and otitis media amongst others.
• a preferred virus for treatment herein is the human rhinovirus infection
• Another aspect of the present invention is a method of treating, including prophylaxis of influenza induced pneumonia in a human in need thereof which method comprises administering to said human an effective amount of a CBSP/p38 inhibitor. Therefore, for this usage, a preferred virus for treatment is the influenza virus.
• a CSBP/p38 kinase inhibitor for the treatment, including prophylaxis, of inflammation associated with a viral infection of a human rhinovirus (HRV), other enteroviruses, coronavirus, influenza virus, parainfluenza virus, respiratory syncytial virus, or adenovirus.
• HRV human rhinovirus
• the viral infection is HRV or RSV, or the influenza or parainfluenza virus.
• Suitable CSALD compounds are well known in the art, and an assay for determining CBSP/p38 inhibition is also readily available using assays disclosed in the below noted patents or applications. For instance, see US Patents 5,716,972, US 5,686,455, US 5,656,644, US 5,593,992, US 5,593,991, US
• Preferred compounds of this invention include those contained in WO 99/01131, and a representative genus is described below. Also preferred for use herein are the compounds disclosed in WO 99/61426 Scios, Inc.; and those compounds disclosed in WO 98/27098 containing the compound known as VX-745; (also known as 5-(2,6-Dichloro-phenyl)-2-(2,4-difluoro-phenylsulfanyl)-l,7,8a- triaza-naphthalen-6-one), the Johnson & Johnson compound RWJ-68354 disclosed in WO 98/47899, RPR compound RPR-200765A, the Zeneca compound ZM 336372 disclosed in WO 99/15164; the Sugen compound SU 4984 disclosed in WO 98/50356.
• VX-745 also known as 5-(2,6-Dichloro-phenyl)-2-(2,4-difluoro-phenylsulfanyl)-l,7,8a- triaza
• Rl is 4-pyridyl, pyrimidinyl, 4-pyridazinyl, l,2,4-triazin-5-yl, quinolyl, isoquinolinyl, or quinazolin-4-yl ring, which ring is substituted with Y-Ra and optionally with an additional independent substituent selected from C ⁇ _4 alkyl, halogen, hydroxyl, Ci-4 alkoxy, C ⁇ _4 alkylthio, Ci-4 alkylsulfinyl, CH2OR12, amino, mono and di- Ci-6 alkyl substituted amino, an N-heterocyclyl ring which ring has from 5 to 7 members and optionally contains an additional heteroatom selected from oxygen, sulfur or NR15, N(R ⁇ o)C(O)Rb or NHR a ;
• Y is oxygen or sulfur
• R4 is phenyl, naphth-1-yl or naphth-2-yl, or a heteroaryl, which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-l-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro, C(Z)NR ⁇ Ri7, C(Z)ORi6, (CRioR20)vCORi2, SR5, SOR5, OR12, halo-substituted-Ci-4 alkyl, Ci-4 alkyl, ZC(Z)Ri2, NR ⁇ oC(Z)Ri6, or (CRifjR2 ⁇ )vNRl ⁇ R20 and which, for other positions of substitution, is halogen, cyano, C(Z)NRi3R]4, C(Z)OR3,
• A is an optionally substituted aryl, heterocyclyl, or heteroaryl ring, or A is a substituted Ci -io alkyl; R22 is an optionally substituted CJ.IQ . alkyl;
• R a is aryl, arylC ⁇ _6alkyl, heterocyclic, heterocyclylCi -6 alkyl, heteroaryl, heteroarylCi- ⁇ alkyl, wherein each of these moieties may be optionally substituted;
• Rb is hydrogen, Ci -6 alkyl, C3.7 cycloalkyl, aryl, arylCi-4 alkyl, heteroaryl, heteroarylCi-4alkyl, heterocyclyl, or heterocyclylC ⁇ _4 alkyl, wherein each of these moieties may be optionally substituted;
• R3 is heterocyclyl, heterocyclylCi-io alkyl or Rs;
• R5 is hydrogen, Ci-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the moieties SR5 being SNR7R17 and SOR5 being SOH;
• R6 is hydrogen, a pharmaceutically acceptable cation, Ci-io alkyl
• Ci-io alkanoyl is each independently selected from hydrogen or Ci-4 alkyl or R7 and
• Rl7 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
• R8 is Ci-io alkyl, halo-substituted Ci-io alkyl, C2-10 alkenyl, C2-10 alkynyl, C3.7 cycloalkyl, C5.7 cycloalkenyl, aryl, arylCi-l ⁇ alkyl, heteroaryl, heteroarylCi-io alkyl, (CRioR20)nORl l, (CRl ⁇ R2 ⁇ )nS(O) m Rl8, (CR ⁇ oR2 ⁇ )nNHS(O)2Rl8, (CRloR2 ⁇ )nNRi3Ri4; wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl may be optionally substituted;
• R Q is hydrogen, C(Z)Rn or optional
• Rl2 is hydrogen or Ri ⁇ ;
• Rl3 and R14 is each independently selected from hydrogen or optionally substituted C1-.4 alkyl, optionally substituted aryl or optionally substituted aryl-Ci-4 alkyl, or together with the nitrogen which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9 ;
• Rl5 is Rio or C(Z)-Ci -4 alkyl;
• Rl6 is Ci -4 alkyl, halo-substituted-Ci-4 alkyl, or C3-.7 cycloalkyl
• Ri8 is Ci-io alkyl, C3-.7 cycloalkyl, heterocyclyl, aryl, aryli -ioalkyl, heterocyclyl, heterocyclyl-Ci-ioalkyl, heteroaryl or heteroaryl i-ioalkyl; or a pharmaceutically acceptable salt thereof.
• R2 is a substituted alkyl derivative. It is recognized that the first methylene carbon in this chain is a tertiary carbon, and it will contain one hydrogen moiety. This methylene group will have has two additional substituents, an R22 moiety and an A moiety, -C(H)(A)(R22)- Both A and R22 may not be unsubstituted Ci _ ⁇ Q alkyl moieties.
• R2 is a -C(AA])(A) moiety, wherein AAi is the R22 moiety, but is specifically the side chain residue (R) of an amino acid, as is further described herein.
• A is an optionally substituted C3_7cycloalkyl, aryl, heteroaryl, or heterocyclic ring, or A is a substituted C ⁇ .JQ alkyl moiety.
• the ring may be substituted independently one or more times, preferably, 1 to 3 times by Ci-io alkyl; halogen; halo substituted Ci -io alkyl, such as CF3; (CR ⁇ oR2 ⁇ )tORl i; (CR ⁇ oR2 ⁇ )tNRl3Rl4, especially amino or mono- or di-C ⁇ _4 alkylamino; (CRioR2 ⁇ )tS(O)mRl8, wherein m is 0, 1 or 2; SH; NRioC(Z)R3 (such NHCO(Ci -K) alkyl)); or NRioS(O) m R8 (such as NHSO2(Ci-io alkyl)).
• t is 0, or an integer of 1 to 4.
• A is an optionally substituted cycloalkyl it is as defined below with the R22 substitution.
• the ring is preferably a morpholino, pyrrolidinyl, piperazinyl or a piperidinyl ring.
• A is an optionally substituted aryl moiety, it is preferably a phenyl ring.
• A is an optionally substituted heteroaryl ring, it is as defined below in the definition section.
• the alkyl chain may be straight or branched.
• the chain is substituted independently 1 or more times, preferably 1 to 3 times by halogen, such as fluorine, chlorine, bromine or iodine; halosubstituted Ci-io alkyl, such as CF3; C3-7cycloalkyl, Ci-io alkoxy, such as methoxy or ethoxy; hydroxy substituted Ci-io alkoxy; halosubstituted Ci-io alkoxy, such as OCF2CF2H; OK u ; S(O)mR 18 (wherein m is 0, 1 or 2); NR13R14; C(Z)NRI3RH;
• A is a 03.7 cycloalkyl, or a Ci _g alkyl, more preferably a Ci _2 alkyl, i.e. a methylene or ethylene moiety, more preferably a methylene moiety which is substituted by one of the above noted groups.
• A when A is a C io alkyl, it is substituted by OR1 1 where R] ⁇ is preferably hydrogen, aryl or arylalkyl; NRi 3R14; OC(Z)R ⁇ 1 ; or C(Z)OR ⁇ 1. More preferably, A is substituted by ORi 1 where Rj 1 is hydrogen.
• R22 is a Ci .I Q alkyl chain, which chain may be straight or branched and which may be optionally substituted independently, one or more times, preferably
• halogen such as fluorine, chlorine, bromine or iodine
• halo substituted Ci-io alkyl such as methoxy or ethoxy
• Ci -io alkoxy such as methoxy or ethoxy
• hydroxy substituted C ⁇ _ ⁇ o alkoxy halosubstituted Ci-io alkoxy, such as OCF2CF2H; ORn; S(O) m Ri8;
• the optional substituents on these cycloalkyl, aryl, heteroaryl, and heterocyclic moieties are as defined herein below.
• R22 substituent groups which contain carbon as the first connecting group i.e. C(Z)OR ⁇ 1; C(Z)NR] 1OR9, C(Z)R ⁇ 1, C(Z)NRi3Rl4, and
• R22 is a Cj _6 unsubstituted or substituted alkyl group, such as a Ci .3 alkylene, such as methyl, ethyl or isopropyl, or a methylene or ethylene moiety substituted by one of the above noted moieties, or as noted above those substituent groups which contain a carbon may substitutent for the first methylene unit of the alkyl chain, such as carboxy, C(O)OR ⁇ 1 , C(O)NRi3Ri4, or R22 is an optionally substituted aryl group, such as a benzyl or phenethyl.
• R22 can be an optionally substituted alkyl group, or R 2 can be C(Z)ORi 1 , C(Z)NRi 1 OR9, C(Z)R ⁇ 1 ,
• R22 is a Ci. ⁇ unsubstituted or substituted alkyl group, more preferably a C ⁇ _2 alkylene chain, such as a methylene or ethylene moiety, more preferably methylene.
• the alkyl chain is substituted by ORi 1 , where Ri 1 is preferably hydrogen, aryl or arylalkyl; S(O)mR ⁇ g, where m is 0 and R1 g is a Ci .g alkyl; or an optionally substituted aryl, i.e. a benzyl or phenethyl moiety.
• R22 i phenyl, benzyl, CH2OH, or CH2-O-aryl.
• one or both of A and R22 contain hydroxy moieties, such as in Ci _g alkyl ORj ⁇ , wherein R 1 is hydrogen, i.e.CH2CH2OH.
• R 1 is hydrogen, i.e.CH2CH2OH.
• AA] is the (R) side chain residue of an amino acid, it is a Ci .g alkyl group, which may be straight or branched. This means the R group off the core amino acid of the structure R-C(H)(COOH)(NH2).
• the R residue term is for example,
• CH 3 for alanine (CH 3 ) 2 CH- for valine, (CH 3 ) 2 CH-CH2-for leucine, phenyl-CH 2 - for phenylalanine, CH3-S-CH2-CH2- for methionine, etc.
• All generally recognized primary amino acids are included in this groups, such as but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine, hydroxylysine, methylhistidine, and other naturally occurring amino acids not found in proteins, such as ⁇ -alanine, ⁇ -aminobutyric acid, homocysteine, homoserine, citrulline, ornithine, canavanine, djenkolic acid, and ⁇ - cyanoalanine, or other naturally occurring non-mammalian amino acids.
• AAi is the residue of phenylalanine, or alanine.
• A is a hydroxy substituted Ci .1 Q alkyl, and R22 i a Ci _ ⁇ Q alkyl or a hydroxy substituted C ⁇ _ ⁇ Q alkyl.
• R22 i a Ci _ ⁇ Q alkyl or a hydroxy substituted C ⁇ _ ⁇ Q alkyl.
• Suitable compounds for use herein include but are not limited to, tr ⁇ «5- l-(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)pyrimidin-4- yl]imidazole; l-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4- pyrimidinyl)imidazole; or (4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4- pyridy ⁇ )-imidazole. Methods of using and dosage amounts are the same as those disclosed in the references cited above.
• the daily oral dosage regimen will be from about 0.1 to about 80 mg/kg of total body weight, preferably from about 0.2 to 30 mg/kg, more preferably from about 0.5 mg to 15mg.
• the daily parenteral dosage regimen about 0.1 to about 80 mg/kg of total body weight, preferably from about 0.2 to about 30 mg/kg, and more preferably from about 0.5 mg to 15mg/kg.
• the daily topical dosage regimen will preferably be from 0.1 mg to 150 mg, administered one to four, preferably two or three times daily.
• the daily inhalation dosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kg per day.
• CSALD compounds herein may also be used in association with the veterinary treatment of mammals, other than humans, in need of inhibition of CSBP/p38 or cytokine inhibition or production for treatment of influenza pneumonia, and other sequelae associated with viral infection.
• the CSBP/p38 inhibitor may also be administered with a second therapeutic agent.
• the second therapeutic agent may be an antiviral agent such as ribavirin, amantidine, rimantidine, Pleconaril,AG 7088 or BTA-188; it may also be an antiviral agent such as an influenza neuraminidase inhibitor, such as zamanivar (Relenza), oseltamivir (Tamiflu) or RWJ-270201; it may be an antihistamine, such as Benadryl®, chlorpheneramine and salts thereof, brompheneramine or salts thereof, and the generally accepted non-sedating antihistamines, such as loratadine (Claritin®), descarboethoxyloratadine (DCL), fexofenadine (Allegra®), and cetirizine hydrochloride (Zyrtec®) etc., a decongestant, such as phenylpropanolamine and salts thereof
• the above noted agents may be administerd as immediate release, or as extended release dosage forms, either together with a suitable CSALD compound, or seperately.
• the compositions may be administered sequentially, in combination with, or contemporaneously with a CSAID agent.
• the administration route of the second agent may also differ from that of the CSALD agent, and hence the dosing schedule may vary accordingly.
• Cetirizine HCl manufacture and dosing is described in US Patent 4,525,358; fexofenadine manufacture and dosing is described in US Patents 4,524,129; US 5,375,693; US 5,578,610; US 5,855,912; US 5,932,247; and US 6,037,353.
• Loratadine and DCL manufacture and dosing are described in US patent 4,282,233; US 4,371,516; US 4,659,716; US 4,863,931; US 5,314,697; and US 5,595,997.
• CSPB/p38 inhibitor may be administered systemically or non- systemically, such as orally, bucally, topically (intranasal) or via inhalation (aerosol), or both topically and via inhalation.
• the second therapeutic agent may be administered by any suitable means, including parenteral, suppository, etc. which means of administration is not necessarily by the same route, nor concurrent therewith.
• topically shall include non-systemic administration. This includes the application of a compound externally to the epidermis or the buccal cavity and/or the instillation of such a compound into the ear, eye and nose.
• systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration, subcutaneous intranasal, intrarectal, or intravaginal.
• the optimal quantity and spacing of individual dosages of a CSBP/p38 inhibitor will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a CSBP/p38 inhibitor given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
• BEAS-2B cells were cultured according to instructions provided by ATCC using BEGM (bronchial epithelial growth media) purchased from Clonetics Corp.
• BEGM bronchial epithelial growth media
• HELA cell cultures used for detection and titration of virus, were maintained in Eagle's minimum essential media containing 10% fetal calf serum, 2mM 1-glutamine, and 10 mM HEPES buffer (MEM).
• Virus yield was also determined from culture supernatants using a microtitration assay in HELA cell cultures (Subauste et al, supra 1995). In cultures treated with p38 kinase inhibitors, drug was added 30 minutes prior to infection. Stocks of compounds were prepared in DMSO (10 mM drug) and stored at -20°C.
• Influenza A/PR/8/34 sub type H1N1 VR-95 American Type Culture Collection, Rockville, MD
• Allantoic fluid was harvested, pooled, and centrifuged (1,000 rcf; 15 min; 4°C) to remove cells.
• Supernatent was aliquoted and stored at -70°C.
• Tissue Culture Infective Dose/ml (TCID50) Inoculation procedure: Four-six week old female Balb/cAnNcrlBr mice were obtained from Charles River, Raleigh, NC. Animals were infected intranasally. Mice were anesthetized by intraperitioneal injection of Ketamine (40mg/kg; Fort Dodge Labs, Fort Dodge, la) and Xylazine (5 mg/kg; Miles, Shawnee Mission, Ks) and then inoculated with 100 TCLD50 of PR8 diluted in PBS in 20 ul. Animals were observed daily for signs of infection. All animal studies were approved by SmithKline Beecham Pharmaceuticals Institutional Animal Care and Use Committee.
• Virus titration At various times post infection, animals were sacrificed and lungs were aseptically harvested. Tissues were homogenized, in vials containing 1 micron glass beads (Biospec Products, Bartlesville, OK) and 1 ml. of Eagles minimal essential medium. Cell debris was cleared by centrifugation at 1,000 rcf for 15 minutes at 4°C, and supernatants were serially diluted on Madin-Darby canine kidney (MDCK) cells. After 5 days of incubation at 37°C (5% CO 2 ), 50 ⁇ l of 0.5% chick red blood cells were added per well, and agglutination was read after 1 hour at room temperature. The virus titer is expressed as 50% tissue culture infective dose (TCLD50) calculated by logistic regression.
• TCLD50 tissue culture infective dose
• ELISA Cytokine levels were measured by quantitative ELISA using commercially available kits. Ear samples were homogenized using a tissue minser in PBS. Cell debris was cleared by centrifugation at 14,000 rpm for 5 minutes. The cytokine concentrations and thresholds were determined as described by the manufacturer; IL-6, EFN- ⁇ , and KC (R&D Systems, Minneapolis, MN). Myeloperoxidase Assay: Myeloperoxidase (MPO) activity was determined kinetically as described by Bradley et al. (1982). Briefly, rabbit cornea were homogenized in Hexadecyl Trimethyl-Ammonium Bromide (HTAB) (Sigma Chemical Co. St.
• HTAB Hexadecyl Trimethyl-Ammonium Bromide
• Measurements were performed by using a Beckman Du 640 Spectrophotometer (Fullerton, Ca.) fitted with a temperature control device. 50 ul of material to be assayed was added to 950 ul of ODI and change in absorbance was measured at a wave length of 460 nm for 2 minutes at 25°C.
• Penh [(expiratory time / relaxation time)-l] x (peak expiratory flow / peak inspiratory flow) where relaxation time is the amount of time required for 70% of the tidal volume to be expired. Determination of arterial oxygen saturation.
• a Nonin veterinary hand held pulse oximeter 8500V with lingual sensor (Nonin Medical, Inc., Madison MN) was used to determine daily arterial oxygen saturation %SpO2 as described (Sidwell et al. 1992 Antimicrobial Agents and Chemotherapy 36:473-476).
• specific p38 kinase inhibitors SB203580, Compound II, Compound III, and an inactive analog, SKF106978 were tested for their ability to inhibit cytokine production in rhinovirus-infected BEAS-2B cell cultures.
• the compound (4-Fluorophenyl)-2-(4- methylsulfinylphenyl)-5-(4-pyridyl)-imidazole is alternatively referred to as SB 203580 and may be found in US patent 5,656,644.
• the compound trans- ⁇ -(4- Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)pyrimidin-4-yl]imidazole also known as Compound JJ may be found in WO 97/25048.
• the compound 4-(4- Fluorophenyl-5-[(2-phenylamino)pyrimidin-4-yl]-l-(piperdin-4-yl)imidazole, also known as Compound III may be found in US Patent 5,658,903.
• the compound 2- (4-Methylsulfinyl)-3-[4-(2-methylpyridyl]-6,7-dihydro[5H]pyrrolo[l,2-a]imidazole, is also known as SB 106978.
• Concentrations of LL-8, LL-6 and GM-CSF in culture supernatants from infected cells treated with inhibitors were all lower than those from untreated infected cultures (figure 2).
• LL-6 was the most sensitive to inhibition with significant inhibition (40%) being observed with SB 203580 concentrations as low as 30 nM .
• GM-CSF was the least sensitive to inhibition by SB 203580, with an IC50 of approximately 4 uM.
• Another inhibitor of p38 kinase was slightly more potent in inhibiting GM-CSF with an IC50 of approximately 1 uM.
• Compound II was comparable to SB 203580 in inhibiting LL-6 and LL-8 production.
• cytokine inhibition was greatest with Compound III, with an IC50 value ⁇ 10 nM for LL-6, while SKF 106978 was inactive at all concentrations tested.
• Maximum effect obtained by any of the p38 kinase inhibitors against any of the three cytokines was 50% -70% inhibition.
• the inhibition of cytokine production by CSALDs was not due to general cell cytotoxicity as determined by standard XTT assays (CC50 > 40 uM for all compounds tested)
• tyrosine phosphorylated p38 kinase was measured by immunoblot at various times after the addition of virus to BEAS-2B cultures. Rhinovirus infection of BEAS-2B cells resulted in an increase in phosphorylated p38 kinase that was both dose and time-dependent. Increases in phosphorylated p38 kinase were evident by 15 minutes post exposure to rhinovirus-39 (MOI 10), appeared to peak by 30 minutes after addition of virus and remained elevated 60 minutes post-infection (figure 3). In addition, rhinovirus-induced tyrosine phosphorylation of p38 kinase was dose- dependent (figure 4).
• the antiviral Tamiflu was used as control and demonstrated 47% improvement in pulmonary functions (p ⁇ 0.01 days 5-12), 64% improved %Sp0 2 (p ⁇ 0.01 days 5-18), and prevention of weight loss relative to placebo.
• the optimal dose of Compound VT was 10 mg/kg leading to 39% improvement in pulmonary functions (p ⁇ 0.01 days 5-12), 30% improvement in %Sp ⁇ 2 (p ⁇ 0.01 days 5-13, p ⁇ 0.05 days 14-15), and a similar effect on weight loss as Tamiflu treatment. Efficacy was observed in doses as low as 1 mg/kg: 27% improvement in pulmonary functions (p ⁇ 0.01 days 6-9), 11.6% (p ⁇ 0.01
• VI was equally effective to Compound V at 30 mg/kg.
• Samples were collected for evaluation of virus titers and cytokines in lung homogenates. A non-significant trend for inhibition of lung cytokines LL-6, KC, LFN-gamma, and RANTES was observed. There was no negative effect on lung virus titers.
• mice treated with Compound VI or Compound V during acute PR/8 (H1 ⁇ 1) influenza infection were protected from a lethal challenge with the same virus as demonstrated by 100% survival and normal pulmonary functions. All control primary infection animals died by Day 7. Thus, the CSAIDS have no effect on immunity to a homologous challenge.

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