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`PATENT APPLICATTON SERTAL NO. dt- vT rso 6
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`U.S. DEPAR'III\{BNT OF COMMERCE
`PATENT AND TRADEMARK OIIF'ICE
`FEE RECORD SHEET
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`04tet/200t smDIEr 000000{6 190365 09011506
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`FOñ
`UN'lEt SfArEs PA;Êr NÞ -rn^c€uaRK OFFtcE
`lMlrìÈorÞì O C, z9?Jr
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`CONFIRMATION NO.5627
`
`CLASS
`514
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`GROUP ART UNIT
`1 646
`
`ATTORNEY
`DOCKET NO.
`AL01 1 43K
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`y' ouß
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`STATE OR
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`SHEEÍS
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`rrffiililtr|[ililffiffiffi[l!ilffil[ilil
`Biþ Dãla Sheel
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`SERIAL NUMBER
`09/841,506
`
`FILING DATE
`04t24t2001
`
`RULE
`
`Robert P. Schleimer, Baltimore, MD;
`John Schroeder, Baltlmore, lVlD;
`William Kreutner, West Paterson, NJ;
`coNTlNulNG DATA å¡¡..irÈrr*¡iir*ftr.riil¡
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`FOREIGN AppLlCATlON g rÑüft rilr*iil*rrrr
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`ADDRESS
`24265
`
`rITLE
`
`nhibition of cytokine generation
`
`FILING FEE
`RECEIVED
`950
`
`FEES: Authority has been given in paper
`No. --
`to charge/credit DEPOSIT ACCOUNT
`No.
`fo¡ followino:
`
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`

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`Erprls! Mrll LrDef Nà,
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`r.'.t 403237585 US
`
`Drtø
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`April 24. 200 I
`
`FEE TRANSMITTAL
`for FY 2001
`
`Palanl løes üo aub¡øcl lo annuâl ftvlslon,
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`! TOTALAMOUNI OF PAYMENT
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`D. HOFFMAN
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`(908) 298-5037
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`I horeby codlry l}lâl thl! corerpondonoo ls balng daposltcd wlth lhe Unllod SlÊt€s Po8tsl
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`
`

`

`Attorney Docket Number 4L01143K
`
`PATENT APPLICAT]ON COVER SHEËT
`
`INHIBITION OF CYTOKINE GENERATION
`
`ROBERT P. SCHLEIMER, a citizen of the United States, residing at
`704 Stags Head Road, Baltimore, Maryland 21286, U.S.A.
`
`JOHN SCHROEDER, a citizen of the United States, residing at 47
`Dunmore Road, Baltimore, Maryland 21228, U,S,A.
`
`WILLIAM KREUTNER, a citizen of the United States, residing at 18
`Woodland Dríve, West Paterson, New Jersey 07424, U.S.A,
`
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`
`ASSIGNEE: SCHERING CORPORATION
`
`"Express Mail" Label No. EL403237585US
`Date of Deposit: April24,2001
`
`Thomas D. Hoffman
`Schering-Plough Corporation
`Patent Department, K-6-1, 1990
`2000 Galloping Hill Road
`Kenilworth, New Jersey 07033-0530
`Telephone: (908)298-5037
`Fax: (908) 298:5388
`
`6
`
`

`

`ALO1143KUS
`
`!NHIBITION OF CYTOKINE GENERATION
`
`BACKGROUND OF THE INVENTION
`
`The present invention relates to methods of using desloratadine to inhibit
`generation of pro-inflammatory cytokines, e.9., lL-4 and lL-13.
`
`Mast cells and basophils play a role in allergic and inflammatory diseases,
`5 These cells produce a wide variety of mediators such as prostaglandins, e.fJ.,
`orostaglandin D2, leukotrienes, e.9., leukotriene C4cytokines and histamine,
`Cytokines are poiypeptides secreted by cells that affect the function of other cells.
`Cytokinos, including interleukins, differ widely in the types of cells affected and ln
`
`biologicalactivities exhibited. Desloratadine, a non-sedating antihistamine, is
`'10 disclosed by Lippert, U., et al, Experimental Dermatology, 1995, Vol. 4, 272-2761o
`be active in vitro in inhibiting the release of the cytokines lL-6 by up to 40% and
`lL-8 by up to 50% from human mast and basophilic cell lines. Human Fce Rln cells
`play a considerable pro-inflammatory role through the release of histamine,
`tryptase and chymase, However, since human mast and basophilic cell lines are
`'15 defective in signaling through the high affinity immunoglogulin-E(lgE) receptor,
`Fco Rl, it is difficult to predict what effect, if any, desloratadine has on the lgE-
`mediated release of lL-6 and lL-8. . S. Molet, el al., Clinical and Expenmental
`Allergy,1997, Vol, 27, pages 1167-1174 disclose that desloratadine reduces
`histamine-induced release of lL-6 and lL-8 in an ln r/fro study in endothelial cells.
`20 Kleine-Tebbe, J., et al, J. Allergy Clin. Immunol. 1994, Vol. 93, No, 2, pages 494-
`500 disclose that desloratadlne lnhibits lgE- and non-lgE-mediated histamine
`release in an in vlfro study in human basophilic leukocyte cells.
`
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`Human basophils are a major source of the cytokines, IL4 and lL-13 that
`25 are produced in vitro in mixed leukocyte cultures. Production of lL4 and lL-13 by
`basophils may play a role in modulating a variety of activities that are involved in
`the pathogenesis of allergic inflammatory conditions. For example, lL-4 and lL-13
`each induce secretion of lgE- and lgG4 by human B-cells. (See Schroeder,
`J.T.,''The role of basophil-cytokine networks in asthma." ln Asthma and Allergic
`30 Diseases: Physiology, lmmunopharmacology, and Treatment, Editors: G. Marone,
`
`12741 1
`
`7
`
`

`

`2
`
`K.F. Austen, ST Holgate, A.B, Kay, L,M. Lichtenstein, Academic Press, San '
`Diego, 75-84,1998a). However, there is a no information on the'effect of
`desloratadine on the generation of cytokines such as lL-4 and lL-13 in basophils
`or any other cell types, One study by Gibbs, et al. Naunyn Schmiedebegs Arch,
`5 Pharmacoi ,1998, Vol. 357: 573-578 reports moderate (500/o) inhibition of ll-4 and
`lL-13 secretion in vitro in basophilic cells using relativelv high concentrations of
`terfenadine, a non-sedating antihistamine. This stLrdy did not test whether the
`terfenadine concentrations used were toxic. The FDA has withdrawn the two
`terfenadine NDAs because of the finding that terfenadine was no longer safe for
`10 cardiac reasons for use in treating seasonal allergic rhinitis.(Federal Register,
`October 5,1998, Vol, 63, page 53444).
`
`t,u
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`
`There is a need for a safe, effective therapy io inhibit secretion of prc-
`infiammatory cytokines such as lL-4 and lL-13 to treat dÍsease statos, for
`example, allergic and/or infl ammatory conditionsr/
`
`IL 15
`
`SUMMARY OF THE INVENTION
`
`The present invention provides a method of inhibiting generation of lL-4
`20 and lL-13 in a human patient in need of such inhibiting which comprises
`administering to such a patient an effective amount of desloratadine.
`
`Typically. lL-4 and lL-13 is generated from human basophils as well as
`othercells, e.9., human B-cells,
`
`25
`
`Ïhe present invention also provides a method of inhibiting generation of lL.-
`4 and lL-13 from human basophils in a patient exhíbiting the symptoms of an
`allergic and/or inflammatory condition which comprises administering to such a
`patient an amount of desloratadine effective to inhibit the generation of lL-4 and
`30 lL-13 and to concurrently treat the symptoms of such an allergic and/or
`inflammatory conditions.
`
`12741 1127t1 1
`
`8
`
`

`

`3
`
`The present invention also provides a method of blocking þoneration of
`pro-inflammatory cytokines in a patient in need of such blocking which compríses
`administering to such a patient an effective amount of desloratadine.
`
`5
`
`The present invention also provides a method of inhibiting socretion of pro-
`inflammatory cytokines from human basophils in a patient in need of such
`inhíbiting which comprisos administering to such a patient an effective amount of
`desloratadine.
`
`10
`
`ïhe preferred pro-inflarnmatory cytokines are lL-4 and lL-13.
`
`1:
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`
`25
`
`30
`
`The patients in need of such inhibiting are those having symptoms of
`allorgic/inflammatory conditions of the a¡rway passages, skin, eyes and intestinal
`tract.
`
`The present invention also provides a method of treating a patient
`exhibiting the syrnptoms of allergic/inflammatory conditions of the skin, eyes,
`intestinal tract,and/or upper and lower airway passages which comprises
`administering to such a patient an effective amount of desloratadine.
`
`The palients in need of such inhibiting are those having symptoms of an
`allergic and/or inflammatory condition of the skin, eyes, intestinal tract, and/or the
`upper and lower airuvay passages.
`
`The present invention also provides a method of treating a disease state
`that has an allergic component and an inflammatory component which comprises
`administering to a patient in need of such treatment an amount of desloratadine
`effective to produce an anti-inflammatory effect,
`
`The present invention also provides a method of treating and/or preventing
`allergic asthma and inhibiting secretion of lL-4 and lL-13 in a patient in need of
`such treating which comprises administering to such a patient an effective ämount
`of desloratadine.
`
`12741 _112741 _1
`
`9
`
`

`

`4
`
`The present invention also provides a method of treating andior preventing
`allergic rhinitis and inhibiting secretion of lL-4 and lL-13 in a pâtient in need of
`such treating which comprises administering to such a patient an effective amount
`5 of desloratadine.
`
`The present invention also provides a method of treating and/or preventing
`atopic dermatitis and inhibiting secretion of lL-4 and lL-13 in a patient in need of
`such treating whích comprises administering to such a patient an effective amount
`10 of desloratadine.
`
`The present invention also provides a method of treating and/or preventing
`urticaria and inhibiting seoretion of lL-4 and lL-13 in a patient in need of such
`treating which comprises administering io such a patient an effective amount oi
`15 desloratadine.
`
`The presenl invention also provides a method of treating lL-4 and lL-13
`mediated disease states in a patient in need of such treating which comprises
`administering to such patients an effective amount of desloratadine to inhibit
`20 generation of lL4 and lL-13.
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`The efiective amount of desloratadine is an amount sufficient to inhibit and
`preferably block generation of lL4 and iL-13 from human cells.
`
`25
`
`The present invention also provides a method of inhibiting generation oÍ lL-
`4 and lL-13 from human basophils in a patient exhibiting the symptoms of an
`allergic and/or inflammatory condition of the skin, eyes, intestinal tract, andior the
`upper and lower airway passages which comprises administering to such a patient
`an amount of desloratadine effective to inhibit the generation of lL-4 and lL-13 and
`30 to concurrently treat the symptoms of such an allergic andior inflammatory
`condition.
`
`1274i_i12741_-1
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`5
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`BRIEF DESCRIPT]ON OF THE FIGURES
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`Figures 1a and 1b illustrate the effect of desloratadine on histamine,
`leukotriene çn 1"LTC+") and lL-4 secretion by human basophils'
`
`Figure 2 graphically illustrates the effect of desloratadine on histamine
`release and lL-4 secretion in response to activation by ionomycin.
`
`10
`
`Figure 3 graphically illustrates the effect of desloratadine on lL-13 secretion
`by human basophils activated by anti-lgE and ionomycin'
`
`Figure 4 graphicatly illustrates that desloratadine inhibits lL-13 secretion
`15 from basophils activated with lL-3 and PMA'
`
`Figures 5a, 5b, 5c and 5d graphically illustrate the inhibition of lL-4 mRNA
`expression in basophils pretreated with desloratadine'
`
`20
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`ln accordance with the methods of the present invention, we demonstrated
`the ability of desloratadine to inhibit the generation of lL-4 and lL-13 from human
`basophils while concurrently comparing its efficacy in preventing mediator release
`25 from these cells using a variety of stimuli. Desloratadine was found to be
`remarkably (nearly 6-7) times more potent in reducing the secretion of lL4 and lL-
`13 from human basophils induced by anti-lgE than it was at inhibiting the
`mediators, histamine and LTC¿, released in the same culture supernatanls' The
`cytokines, lL4 and lL-13, were equally inhibited by desloratadine following
`g0 activation with ionomycin despite the lack of an effect on the histamine release
`induced with ionomycin. Desloratadine had a lesser effect at inhibiting the lL-13
`secreted in response to lL-3 and PMA, suggesting that the antihistamine
`differentially targets individual pathways of cytokine generation. Finally, there was
`
`127+1 112741 1
`
`11
`
`

`

`6
`
`no evidence that desloratadine was cytotoxic, i.e., that ii mediated its inhibitory
`effects by causing decreased cell viability. ln accordance with the present
`invention, lL-4 mRNA accumulation was also remarkably inhibited, by as much as
`80%, following pretreatment with desloratadine, suggesling that desloratadine
`5 targets signals regulating cytokine gene transcríption in addition to those
`controlling mediator release.
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`The phrase "an allergic and/or inflammatory condition" means those allergic
`and inflamrnatory conditions and symptoms found on the skin,eyes, intestinal tract
`10 and/ or in the upper and lower airway passages from the nose to the lungs
`Typical allergic and/or inflammatory conditions of the skin or upper and lower
`airway passages include seasonal and perennial allergic rhinilis, non-allergic
`rhinitis, asthma including allergic and non-allergic asthma, sinusitis, colds,
`dermatitis, especially allergic and atopic dermatitis, and urticaria and symptomatic
`15 dermographism. Typical allergic and/or infìammatory conditions of the eyes
`include, but are not limited to, allergic conjunctivitis. Typical allergic and/or
`inflammatory conditions of the intestinal tract, but are not limited to, food allergies.
`
`The term "human" as used herein includes a male or female pediatric
`20 subject of less than 12 years of age to less than '18 years of age, a male or female
`pediatric subject of greater than 12 years of age to less than 18 years, and male
`and female adults of 18 years of age and older.
`
`The term "pro-inflammatory cytokines" as used herein means those
`25 cytokines associated with allergic and inflammatory reactions of the skin, eyes,
`intestinal tract, and airway passages of humans exposed to allergens. Typically
`suitable pro-inflammatory cytokines include lL-3, lL-4, lL-5, lL-6, lL-8, lL-9 and lL-
`
`13,
`
`30
`
`The amount of desloratadine effective for treating or preventing allergic and
`inflammatory conditions of the skin or airway passages will vary with the age, sex,
`body weight and severity of the allergic and inflammatory condition of the patient.
`Typically, lhe amount of desloratadine effective for treating or preventing such
`
`12741 1'12741 1
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`12
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`allergic and inflammatory conditions in an adult human of age older than 12 i5 in
`the range of about 2.5 mglday to about 45 mg/day, preferably about 2.5 mg/day to
`about 20 mg/day, or about 5.0 mg/day to about 15 mg/day, or about 5.0 mgiday to
`about 10 mg/day, more preferably about 5.0 mg/day to about 7.5 mg/day, and
`5 most preferably about 5.0 mg/day in single or divided doses, e,9., 2 x 2.5 mg/day,
`or a single dose of 5.0 mg/day.
`
`Desloraladine is a non-sedaling long acting histamine antagonist with
`potent and selective peripheral H1-receptor antagonist activity. ln vitro and in vivo
`10 animal pharmacology studies have been conducted to assess various
`pharmacodynamic effects of desloratadine and loratadine. ln assessing central
`nervous system ('CNS") activity in mice, desloratadine was relatively free of
`producing alterations in behavior, neurologic or autonomicfunction. The potentiai
`for desloratadine to occupy brain H1-receptors was assessed in guinea pigs
`15 following lP administration and results suggest poor access to central histamine
`receptors for desloratadine.
`
`The clinical efficacy and safety of desloratadine has been documented in
`20 over 3,200 seasonal allergic rhinitis patients in 4 double-bfinded, randomized
`clinical trials. The results of these clinical studies demonstrated the efficacy of
`desloratadine in the treatment of adult and adolescent patients rvith seasonal
`
`rhinitis.
`
`25
`
`Efficacy endpoints in all the studies were Total Symptom Score, Total
`
`Nasal Symptom Score, Total Non-nasalSymptom Score, and Health Quality of
`Life (HAOL) analysis in efficacy trials. Desloratadine (5 mg once daily)
`significantly reduced the total symptom scores (the sum of individual scores for
`rhinorrhea, sneezing, congestionlstuffiness, nasal itching, ítchy/buming eyes,
`30 tearing, ocular redness, and itchy ears/palate). Desloratadine (5 mg) was
`significantly (p<0,01) more effective than placebo in reducing nasalsymptoms.
`An important effìcacy endpoint analyzed in the desloratadine studies is the AM
`NOW total symptom score. This parameter measures the total symptom relief by
`
`12741 112741 1
`
`13
`
`

`

`I
`
`the patient after 24 hours before taking the next day dose. Statistically signiflcant
`(p<0.05) reductions were maintained for the full 24 hour dosing interual over the
`entire 5 mg to 20 mg dosage range
`
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`There were no significant differences in the effectiveness of desloratadine
`(over the entire 5 mg to 20 mg dosage range) across subgroups of patients
`defìned by gender, age, or race. Desloratadine is particularly useful for the
`trealment and prevention of the nasal (stuffiness/congestion, rhinorrhea, ìlasal
`itching, sneezing) and non-nasal (itch/burning eyes, tearing/watery eyes, redness
`10 of the eyes, itching of the ears/palate) symptoms of seasonal allergic rhinitis,
`including nasal congestion, in patients in need of such treating andi or preventing,
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`U,S. Patent No, 4,659,716 discloses methods of making desloratadine,
`pharmaceutical compositions containing it and methods of using desioratacline
`15 and pharmaceutical compositions containing it to ireat allergic reaction in
`mammals.
`
`U.S. Patent No. 5,595,997 discloses pharmaceutical compositions
`containing desloratadine and methods of using desloraladine for treating and
`20 preventing various disease states. e.9., allergic rhinitis.
`
`U.S. Patent No. 6,100,274 discloses stable pharmaceutical compositions
`containing desloratadíne suitable for oral administration to treat allergic reactions,
`e.9., allergic rhinitis.
`
`25
`
`The pharmaceutical compositions of desloratadine can be adapted for any
`mode of administration e.9., for oral, parenteral, e.g., subcutaneous (',SC,').
`intramuscular ("1M"), and intraperitoneal ("1P"), topical or vaginal administration or
`by inhalation (orally or intranasally). Preferably desloratadine is administered
`30 orally,
`
`Such pharmaceutical compositions may be formulaied by combining
`desloratadine or an equivalent amount of a pharmaceutically acceptable salt
`
`12741 \12741 t
`
`14
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`

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`I
`
`thereof with a suitable, inert, pharmaceutically acceptable carrier or diluent that
`may be either solid or liquid, Desloratadine may be converted into the
`pharmaceutically acceptable acid addition salts by admixing it with an equivalent
`amount of a pharmaceutically acceptable acid. Typically suitable pharmaceutically
`5 acceptable acids include the mineral acids, e.9., HNO3 H2SO+, H¡PO+, HCl, HBr,
`organic acids, including, but not llmited to, acetic, trifluoroacetic, propionic, lactic,
`maleic, succinic, tartaric, glucuronic and citric acids as well as alkyl or arylsulfonic
`acids, such as p{oluenesulfonic acid, 2-naphthalenesulfonic acid, or
`methanesulfonic acid. The preferred pharmaceutically acceptable salts are
`10 trifluoroacetate, tosylate, mesylate, and chloride, Desloratadine is more stable as
`the free base lhan as an acid addition salt and the use of the desloratadine free
`base in pharmaceutical compositions of the present invention is more preferred.
`
`See U.S.Patent No. 6,100,274.
`
`15
`
`Solid form preparations include powders, tablets, dispersible granules,
`capsules, cachets and suppositories. The powders and tablets may be comprised
`of from about 2.5 to about 95 percent active ingredient preferably frorn about 2.5
`to about 20 percent, more preferably from about 2^5 to about 10 percent, or from
`about 2,5 to about 5 percent and most preferably 5 percent of the active
`20 ingredient. Suitable solid caniers are known in the art, e,g. magnesium
`carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets
`and capsules can be used as solid dosage forms suitable for oral administration.
`Examples of pharmaceutically acceptable carriers ancl methods of manufacture
`for various compositions may be found in A. Gennaro (ed.), Remington's
`25 Pharmaceutical Sciences, 18th Editíon, (1990), Mack Publishing Co,, Easton,
`Pennsylvania.
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`Liquid form preparations include solutions, suspensions and emuisions. As
`an example may be mentioned water or water-propylene glycol solutions for
`30 parenteral injection. Solid form preparations may be converted into liquid
`preparations shortly before use for either oral or administration. Parenteral forms
`to be injected intravenously , intramuscularly or subcutaneously are usually in the
`form of sterile solutions and may contain tonicity agents (salts or glucose), and
`
`12741 112741 1
`
`15
`
`

`

`10
`
`buffers. opacifìers may be included ín oral solutions, suspensions and emulsions,
`Liquid form preparations may also include solutions for intranasal.administration.
`The liquid form preparations may comprise the same ranges of active ingredient is
`as used in solid form preparations.
`
`Aerosol preparations suitable for inhalation rnay inciude solutions anci
`solids in powder form, which may be in combination with a pharmaceutically
`acceptable carrier, such as an inert compressed gas, e.g., nítrogen.
`
`Also included are solid form preparations which are intended to be
`converted, shortly before use, to liquid form preparations for either oral or
`parenteral administration. such liquid forms include solutions, suspensions and
`emulsions,
`
`The compounds of the invention may also be deliverable transdermally
`The transdermal compositions can take the form of creams, lotions, aerosols
`and/or emulsions and can be included in a transdermal patch of the matrix or
`reservoir type as are convenlional in the art for this purpose.
`
`Preferably, the pharmaceutical preparation is in a unit dosage form. ln
`such form, the preparation is subdivided into suitably sized unit doses containing
`appropriate quantities of the active component, e.g,, an effective amount to
`achieve the desired purpose.
`
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`Further, desloratadine may be adminlstered in association with
`therapeutically effective amounts of steroids, e.g, mometasone furoate,
`beclomethasone dipropinate or fluticasone propinate, leukotriene inhibitors, e,g.,
`montelukast sodium or zafirlukast, and/or en upper airway passage decongestanl
`including, but not limited to phenylephedrine, pseudoephedrine and
`phenylpropanolamine or pharmaceutically acceptable salts thereof, in accordance
`with the dosing levels known to those skilled in the art and as described in the
`Physicians' Desk Reference. The use of the upper airway passage decongestant,
`pseudoephedrine HCl, is preferred.
`
`12741 112741 1
`
`16
`
`

`

`11
`
`EXAMPLES
`
`MATERIALS AND METHODS
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`SpecÍal Reagents
`All reagents were purchased unless otherwise noted. Piperazine-N.N'-bis-
`2-ethanesulfonic acid (PIPES), ionomycin, FMLP, PMA. and fetal bovine serum
`(FBS)from Sigma Chemical Co. (St. Louis, MO); RPMI-1640 and lscove's
`modified Dulbecco's medium (IMDM) both with L-glutamine and containing 25 mM
`N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), gentamicin,
`nonessential amino acids (100x)from Life Technologies, lnc,, Grand lsland, NY);
`and Percoll from Pharmacia (Piscataway, NJ), The desloratadine used in these
`experiments was supplied by The Scheríng-Plough Research lnstitute. A 0,1 M
`stock solution was made in DMSO, aliquoted, and frozen at -20'C. All pipes-
`containing buffers were made from stock 10X PIPES (250 mM PIPES, 1 .20 M
`NaCl, and 50 mM KCL, pH 7 ,3 and stored at 4"C). lsotonic Percoll, (referred to as
`100% Percoll)was made by mixing 9 parts Percollwith 1 part 10X PIPES. PAG
`contained one-tenth 10X PÍPES. 0.003% HAS, and 0.1% D-glucose. PAG-EDTA
`additionally contained 4 mM EDTA. Percoll solutions used for cell isolation were
`all made by mixing the appropriate amounts of 1000/o Percollwith lX PIPES,
`
`Cell Preparatlon and Culture
`Mixed leukocyte suspensions containing basophils were prepared either
`25 using double-Percoll density centrifugation as described (Schroeder, et al., J.
`lmmunol.,.1994, Vol. '153: 1808, or by a combination of countercurrent elutriation
`and Percoll density centrifugation protocols (MacGlashan, et al., J.
`lmmunol,,1994, Vol, 153:3006-3016). The percentages of basophils obtained
`using these protocols typically ranged between 5-50% and 10-30%, respectively,
`30 and were determined by counting Alcian blue positive and negative stained cells
`on a Spiers-Levy chamber (Gilbert and Ornstein, 1975). For some experiments,
`the basophils were additionally purified to >99.9% using a negative-selection
`protocol (Miltenyi Corp., Auburn, CA). For all experiments other than those
`
`^,2741 i12741 1
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`assessing lL-4 mRNA expression, the cells were cultured in g6-wäll flat-bottom
`microtiter plates (in duplicate) using IMDM supplemented with 5% heat-inactivated
`(56"C for 30 min.) FBS, 1 x non-essential amino acids, and 5 ¡rg/ml gentamicin
`(C-IMDM) For the analysis of lL-4 mRNA expressíon, cells in C-IMDM were
`5 cultured in autoclaved (RNase-free) 1.5 ml mícrocentrifuge tubes (see below),
`since this allowed for a more precise way to quantitatively isolate mRNA without
`
`the need for transferring cells from culture wells before extraction. For each
`condition, leukocyte suspensions containing approximately 100,000-500,000
`basophils in 100 ¡rl of C-IMDM were prewarmed to 37"C before adding 100 ¡rl of
`10 desloratadine concentrations in C-IMDM also prewarmed to 37"C. After 15
`minutes preincubation, the cells were then activated by adding 50 pl of 5x (5 times
`
`the final concentration) of stimulus. lt is important to note here that the
`concentrations of stimuli used were optimal for lL-4 generation rather than
`mediator release, This is particularly true for anti-lgE antibody, which has been
`15 shown to induce lL4 at concentrations 1O-fold less than those causing optimal
`histamine release (Schroeder, et al., ibid). For harvesting, cullures were
`centrifuged and cell-free supernatânts collected for mediator release and cytokine
`analysis, as described (Schroeder, et al., ibid), Histamine, LTC¿, and lL-4 were all
`measured in aliquots of culture supernatant taken at 4 hours. For histamine, this
`20 meant taking 20-50 ¡rl of supernatant and diluting it in 1 ml of PAG buffer
`containing 1,6% HCIO¿, After an overnight precipitation at 4"C, the samples were
`assayed by automated fluorimetry (Siraganian,R.P.. Anal. Biochem,. 1974,
`Vol.57:383-394). LTC4 was measured by an in-house RIA (MacGlashan, ot al,, J.
`lnnCI!¡qL 1986, Vol.136:2231-2239). lL4 protein was measured by a commercial
`25 ELISA (Biosource lnternational), Culture supernatants were harvested after 20
`hours incubation for all experfments investigating the ef