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WO2001076600A2 - Inhibition de la production de cytokines - Google Patents

Inhibition de la production de cytokines Download PDF

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Publication number
WO2001076600A2
WO2001076600A2 PCT/US2001/011474 US0111474W WO0176600A2 WO 2001076600 A2 WO2001076600 A2 WO 2001076600A2 US 0111474 W US0111474 W US 0111474W WO 0176600 A2 WO0176600 A2 WO 0176600A2
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WO
WIPO (PCT)
Prior art keywords
desloratadine
allergic
basophils
inhibition
generation
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PCT/US2001/011474
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WO2001076600A3 (fr
Inventor
Robert P. Schleimer
John Schroeder
William Kreutner
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Merck Sharp and Dohme LLC
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Schering Corp
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Priority to EP01928407A priority Critical patent/EP1267870A2/fr
Priority to MXPA02009821A priority patent/MXPA02009821A/es
Priority to JP2001574117A priority patent/JP2003530351A/ja
Priority to CA002405413A priority patent/CA2405413A1/fr
Priority to AU2001255268A priority patent/AU2001255268A1/en
Publication of WO2001076600A2 publication Critical patent/WO2001076600A2/fr
Publication of WO2001076600A3 publication Critical patent/WO2001076600A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to pharmaceutical compositions and the use of desloratadine for the preparation of a medicament for the inhibition of the generation of pro-inflammatory cytokines, e.g., IL-4 and IL-13.
  • cytokines e.g., IL-4 and IL-13.
  • Cytokines are polypeptides secreted by cells that affect the function of other cells. Cytokines, including interleukins, differ widely in the types of cells affected and in biological activities exhibited. Desloratadine, a non-sedating antihistamine, is disclosed by Lippert, U., et a ⁇ , Experimental Dermatology, 1995, Vol.
  • Human basophils are a major source of the cytokines, IL-4 and IL-13 that are produced in vitro in mixed leukocyte cultures. Production of IL-4 and IL-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, IL-4 and IL-13 each induce secretion of IgE- and lgG-4 by human B-cells. (See Schroeder, J.T.,"The role of basophil-cytokine networks in asthma.” In Asthma and Allergic Diseases: Physiology, Immunopharmacology, and Treatment, Editors: G. Marone, K.F. Austen, ST Holgate, A.B. Kay, L.M.
  • the present invention provides a method of inhibiting generation of IL-4 and IL-13 in a human patient in need of such inhibiting which comprises administering to such a patient an effective amount of desloratadine.
  • IL-4 and IL-13 is generated from human basophils as well as other cells, e.g., human B-cells.
  • the present invention also provides a method of inhibiting generation of IL- 4 and IL-13 from human basophils in a patient exhibiting 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 IL-4 and IL-13 and to concurrently treat the symptoms of such an allergic and/or inflammatory conditions.
  • the present invention also provides a method of blocking generation of pro-inflammatory cytokines in a patient in need of such blocking which comprises administering to such a patient an effective amount of desloratadine.
  • the present invention also provides a method of inhibiting secretion of pro- inflammatory cytokines from human basophils in a patient in need of such inhibiting which comprises administering to such a patient an effective amount of desloratadine.
  • the preferred pro-inflammatory cytokines are IL-4 and IL-13.
  • the patients in need of such inhibiting are those having symptoms of allergic/inflammatory conditions of the airway passages, skin, eyes and intestinal tract.
  • the present invention also provides a method of treating a patient exhibiting the symptoms 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 patients 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 airway 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 IL-4 and IL-13 in a patient in need of such treating which comprises administering to such a patient an effective amount of desloratadine.
  • the present invention also provides a method of treating and/or preventing allergic rhinitis and inhibiting secretion of IL-4 and IL-13 in a patient in need of such treating which comprises administering to such a patient an effective amount of desloratadine.
  • the present invention also provides a method of treating and/or preventing atopic dermatitis and inhibiting secretion of IL-4 and IL-13 in a patient in need of such treating which comprises administering to such a patient an effective amount of desloratadine.
  • the present invention also provides a method of treating and/or preventing urticaria and inhibiting secretion of IL-4 and IL-13 in a patient in need of such treating which comprises administering to such a patient an effective amount of desloratadine.
  • the present invention also provides a method of treating IL-4 and IL-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 generation of IL-4 and IL-13.
  • the effective amount of desloratadine is an amount sufficient to inhibit and preferably block generation of IL-4 and IL-13 from human cells.
  • the present invention also provides a method of inhibiting generation of IL-
  • FIGS 1a and 1b illustrate the effect of desloratadine on histamine, leukotriene C ("LTC 4 ”) and IL-4 secretion by human basophils.
  • Figure 2 graphically illustrates the effect of desloratadine on histamine release and IL-4 secretion in response to activation by ionomycin.
  • Figure 3 graphically illustrates the effect of desloratadine on IL-13 secretion by human basophils activated by anti-lgE and ionomycin.
  • Figure 4 graphically illustrates that desloratadine inhibits IL-13 secretion from basophils activated with IL-3 and PMA.
  • Figures 5a, 5b, 5c and 5d graphically illustrate the inhibition of IL-4 mRNA expression in basophils pretreated with desloratadine.
  • cytokines IL-4 and IL-13
  • desloratadine were equally inhibited by desloratadine following activation with ionomycin despite the lack of an effect on the histamine release induced with ionomycin.
  • Desloratadine had a lesser effect at inhibiting the IL-13 secreted in response to IL-3 and PMA, suggesting that the antihistamine differentially targets individual pathways of cytokine generation.
  • desloratadine was cytotoxic, i.e., that it mediated its inhibitory effects by causing decreased cell viability.
  • IL-4 mRNA accumulation was also remarkably inhibited, by as much as 80%, following pretreatment with desloratadine, suggesting that desloratadine targets signals regulating cytokine gene transcription in addition to those controlling mediator release.
  • an allergic and/or inflammatory condition means those allergic and inflammatory conditions and symptoms found on the skin, eyes, intestinal tract 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 rhinitis, non-allergic rhinitis, asthma including allergic and non-allergic asthma, sinusitis, colds, dermatitis, especially allergic and atopic dermatitis, and urticaria and symptomatic dermographism.
  • Typical allergic and/or inflammatory 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.
  • human as used herein includes a male or female pediatric 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.
  • pro-inflammatory cytokines as used herein means those cytokines associated with allergic and inflammatory reactions of the skin, eyes, intestinal tract, and airway passages of humans exposed to allergens.
  • suitable pro-inflammatory cytokines include IL-3, IL-4, IL-5, IL-6, IL-8, IL-9 and IL- 13.
  • 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.
  • the amount of desloratadine effective for treating or preventing such allergic and inflammatory conditions in an adult human of age older than 12 is in the range of about 2.5 mg/day 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 mg/day to about 10 mg/day, more preferably about 5.0 mg/day to about 7.5 mg/day, and most preferably about 5.0 mg/day in single or divided doses, e.g., 2 x 2.5 mg/day, or a single dose of 5.0 mg/day.
  • Desloratadine is a non-sedating long acting histamine antagonist with potent and selective peripheral H1 -receptor antagonist activity.
  • In vitro and in vivo animal pharmacology studies have been conducted to assess various pharmacodynamic effects of desloratadine and loratadine.
  • CNS central nervous system
  • desloratadine was relatively free of producing alterations in behavior, neurologic or autonomic function.
  • the potential for desloratadine to occupy brain H1 -receptors was assessed in guinea pigs following IP administration and results suggest poor access to central histamine receptors for desloratadine.
  • Desloratadine (5 g once daily) significantly reduced the total symptom scores (the sum of individual scores for rhinorrhea, sneezing, congestion/stuffiness, nasal itching, itchy/burning eyes, tearing, ocular redness, and itchy ears/palate). Desloratadine (5 mg) was significantly (p ⁇ 0.01) more effective than placebo in reducing nasal symptoms.
  • An important efficacy endpoint analyzed in the desloratadine studies is the AM NOW total symptom score. This parameter measures the total symptom relief by the patient after 24 hours before taking the next day dose. Statistically significant (p ⁇ 0.05) reductions were maintained for the full 24 hour dosing interval over the entire 5 mg to 20 mg dosage range
  • Desloratadine is particularly useful for the treatment and prevention of the nasal (stuffiness/congestion, rhinorrhea, nasal itching, sneezing) and non-nasal (itchy/burning eyes, tearing/watery eyes, redness of the eyes, itching of the ears/palate) symptoms of seasonal allergic rhinitis, including nasal congestion, in patients in need of such treating and/ or preventing.
  • U.S. Patent No. 4,659,716 discloses methods of making desloratadine, pharmaceutical compositions containing it and methods of using desloratadine and pharmaceutical compositions containing it to treat allergic reaction in mammals.
  • U.S. Patent No. 5,595,997 discloses pharmaceutical compositions containing desloratadine and methods of using desloratadine for treating and preventing various disease states, e.g., allergic rhinitis.
  • U.S. Patent No. 6,100,274 discloses stable pharmaceutical compositions containing desloratadine suitable for oral administration to treat allergic reactions, e.g., allergic rhinitis.
  • compositions of desloratadine can be adapted for any mode of administration e.g., for oral, parenteral, e.g., subcutaneous (“SC"), intramuscular (“IM”), and intraperitoneal (“IP”), topical or vaginal administration or by inhalation (orally or intranasally).
  • SC subcutaneous
  • IM intramuscular
  • IP intraperitoneal
  • desloratadine is administered orally.
  • compositions may be formulated by combining desloratadine or an equivalent amount of a pharmaceutically acceptable salt 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.
  • suitable pharmaceutically acceptable acids include the mineral acids, e.g., HNO 3) H 2 SO 4 , H 3 PO , HCI, HBr, organic acids, including, but not limited to, acetic, trifluoroacetic, propionic, lactic, maleic, succinic, tartaric, glucuronic and citric acids as well as alkyl or arylsulfonic acids, such as p-toluenesulfonic acid, 2-naphthalenesulfonic acid, or methanesulfonic acid.
  • the preferred pharmaceutically acceptable salts are trifluoroacetate, tosylate, mesylate, and chloride.
  • Desloratadine is more stable as the free base than 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.
  • 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 from 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 ingredient.
  • Suitable solid carriers 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 and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pennsylvania.
  • Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for 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 buffers. Opacifiers may be included in 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 may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g., nitrogen.
  • a pharmaceutically acceptable carrier such as an inert compressed gas, e.g., nitrogen.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
  • liquid forms include solutions, suspensions and emulsions.
  • the compounds of the invention may also be deliverable transdermally.
  • 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 conventional in the art for this purpose.
  • the pharmaceutical preparation is in a unit dosage 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.
  • desloratadine may be administered 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 an upper airway passage decongestant 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 HCI is preferred.
  • the desloratadine used in these experiments was supplied by The Schering-Plough Research Institute. 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 NaCI, and 50 mM KCL, pH 7.3 and stored at 4°C). Isotonic Percoll, (referred to as 100% Percoll) was made by mixing 9 parts Percoll with 1 part 10X PIPES. PAG contained one-tenth 10X PIPES. 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 100% Percoll with IX PIPES.
  • the basophils were additionally purified to >99.9% using a negative-selection protocol (Miltenyi Corp., Auburn, CA).
  • a negative-selection protocol Miltenyi Corp., Auburn, CA.
  • the cells were cultured in 96-well 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 ⁇ g/ml gentamicin (C-IMDM).
  • IL-4 mRNA expression cells in C-IMDM were cultured in autoclaved (RNase-free) 1.5 ml microcentrifuge 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.
  • leukocyte suspensions containing approximately 100,000-500,000 basophils in 100 ⁇ l of C-IMDM were prewarmed to 37°C before adding 100 ⁇ l of desloratadine concentrations in C-IMDM also prewarmed to 37°C. After 15 minutes preincubation, the cells were then activated by adding 50 ⁇ l of 5x (5 times the final concentration) of stimulus.
  • IL-4 was also measured at this time point, particularly when ionomycin was used, since the kinetics for the secretion of this cytokine extend beyond 4 hours with this stimulus (Schroeder, et al., J. Leuk. Biol.. 1998, Vol. 63:692-698).
  • IL-13 protein measurements were also made using a commercial ELISA (Immunotech). RNA Isolation and Semi-Quantitative Analysis of IL-4 mRNA Expression
  • RNA was resuspended in 25 ⁇ l of diethlpyrocarbonate (DEPC)-treated water and stored at -80°C.
  • RT Reverse Transcription
  • PCR polymerase chain reaction
  • serial dilutions of RNA as previously detailed (MacGlashan, et al., J. Immunol. 1994; Vol. 152:3006-3016; Schroeder, at al.,J_. Immunol.. 1997, Vol. 158: 5448-5454) using the GeneAmp RNA PCR kit (Perkin- Elmer Cetus, Norwalk, CT). PCR products were visualized in 3% agrose gels using ethidium bromide staining.
  • Figures 1a and 1b graphically illustrate the effect of desloratadine on histamine, LTC , and IL-4 secretion by human basophils.
  • Control release for each product Histamine: 40 ⁇ 6% of total, IL-4: 403+207 pg/10 6 basophils, and LTC 4 : 860+127 pg/10 6 basophils.
  • Figure 1b shows the effect of desloratadine on histamine, LTC 4 , and IL-4 secretion from human basophils, 99% purity.
  • Control release for histamine, LTC 4 , and IL-4 were 60% of total, 903 pg/10 6 basophils, and 854 pg/10 6 basophils, respectively.
  • Figure 2 illustrates the effect of desloratadine on histamine release (HR) and IL-4 secretion in response to ionomycin.
  • Basophil suspensions of 42, 99, and 98% purity were pretreated 15 min. with 0.1 , 1.0, and 10 ⁇ M desloratadine.
  • Cells were then activated with ionomycin (500 ng/ml) for 4 hours.
  • Control levels of IL-4 were 3398, 253, and 348 pg/10 6 basophils. Percent histamine release in the same cultures was 91 , 25, and 10%, respectively.
  • FIG. 3 illustrates the effect of desloratadine on IL-13 secretion by human basophils activated by anti-lgE or ionomycin.
  • Basophil suspensions ranging in purity between 6-99% were pretreated 15 min. with the indicated concentrations of desloratadine.
  • Cells were activated with either anti-lgE (10-20 ng/ml) or ionomycin (500 ng/ml).
  • IL-13 protein in control cultures averaged 169+35 and 334+144 pg/10 6 basophils for anti-lgE and ionomycin, respectively. Values indicated by the open circles are the mean for two experiments (control levels of IL-13 were 260 and 230 pg/10 6 basophils).
  • FIG. 4 graphically illustrates that desloratadine inhibits IL-13 secretion from basophils activated with IL-3 and PMA.
  • Basophils up to 94% purity
  • Cells were pretreated with the indicated concentrations of desloratadine before adding IL-3 (100 ng/ml) or PMA (2 ng/ml).
  • the amount of IL-13 secretion in the absence of desloratadine averaged 570 ⁇ 142 pg/10 6 basophils for IL-3 activation and 216+120 pg/10 6 basophils for PMA induction.
  • Figures 5a-d graphically illustrate the inhibition of IL-4 mRNA expression in basophils pretreated with desloratadine.
  • Cells were incubated 15 min. with desloratadine (10 ⁇ M) or DMSO (1 :10,000) before receiving anti-lgE (10 ng/ml) or medium alone.
  • Total RNA was isolated after 2h and used for dilutional RT-PCR analysis to compare message expression for IL-4 and the housekeeping gene, hypoxanthine phosphoribosyl transferase (HPRT) (panel a).
  • Densitometric analysis is shown for undiluted RNA expression (panel b).
  • Desloratadine also inhibited histamine from pure basophils much as it did for impure suspensions.
  • Figure 2 shows that the secretion of histamine induced by the calcium ionophore, ionomycin, was unaffected by desloratadine at concentrations of 0.1 , 1.0, and 10 ⁇ M.
  • the high levels of IL-4 that were secreted in response to ionomycin stimulation were remarkably inhibited by the same concentrations of desloratadine (i.e. 0.1 , 1.0 and 10 ⁇ M).
  • the histamine and LTC 4 made in response to FMLP stimulation was also not inhibited by desloratadine (data not shown).
  • Figure 3 shows that IL-13 generated in response to either anti-lgE or ionomycin.
  • concentration-response curve of desloratadine for inhibition of IL-13 induced by either stimulus.
  • These curves in Figure 3 were nearly identical to that seen for the inhibition of IL-4 by desloratadine as shown in Figures 1 and 2.
  • an initial plateau was seen at 1 ⁇ M at the level of 50-70% inhibition.
  • Dose-dependent inhibition of IL-13 continued as desloratadine concentrations were increased up to 10 ⁇ M.
  • the drug inhibited IL- 3-mediated IL-13 production with an IC 50 of approximately 2 ⁇ M, which was nearly 5-fold that necessary to inhibit the IL-13 secreted with anti-lgE or ionomycin activation.
  • Desloratadine inhibited the IL-13 induced by PMA in similar fashion, with inhibition seen between 1 ⁇ M and 10 ⁇ M concentrations of drug.
  • IL-4 protein is first detected after 1-2 hours and IL- 13 some 48 hours after activation. Therefore, it seemed possible that the kinetics of mediator release vs. cytokine production might account for the differences in their sensitivity to desloratadine, particularly if cell lysis was a factor. However, we saw no evidence that 10 ⁇ M desloratadine, even when combined with stimulus for 18 hours, was cytotoxic, i.e., caused cell death as assessed by trypan blue exclusion in pure basophil cultures (data not shown). Cells did stain with trypan blue when cultured with 100 ⁇ M desloratadine alone. Furthermore, histamine was detected in these cultures, indicating that this concentration of desloratadine caused cell lysis.
  • Panel b of Figure 5 shows the average densitometric analysis of three experiments, indicating that desloratadine pretreatment caused approximately an 80% reduction in the IL-4 message accumulated with anti-lgE activation.
  • desloratadine (10 ⁇ M) also inhibited the histamine and IL-4 protein secreted into the supernatants of these cultures, as shown in panels c and d, respectively, of Figure 5.
  • desloratadine targets at least two signals along with common pathway triggered by both stimuli.
  • the inhibition may result from disruption of a signal involved only in cytokine generation, while at concentrations above 1 ⁇ M, a second signal may also be affected, resulting in inhibition of preformed mediator release for most stimuli.
  • desloratadine was less effective at preventing mediator release induced by IgE-independent stimuli, such as ionomycin, fmlp, and phorbol ester. In fact, we found no evidence that the histamine and/or LTC 4 released with these stimuli were inhibited by any of the desloratadine concentrations tested (data not shown). In contrast, desloratadine was quite effective in blocking the production of IL-4 induced by ionomycin and the IL-13 generated in response to either ionomycin, PMA, or IL-3.
  • desloratadine does not block the IL-13 made in response to IL-3 or PMA, both of which are thought to activate separate pathways.
  • desloratadine may inhibit IgE-dependent cytokine secretion and mediator release by simply preventing changes in cytosolic calcium. This effect, however, does not fully explain why desloratadine inhibited cytokine induced by ionomycin while having no effect on mediator release. Since IgE-crosslinking and Ca 2+ ionophores appear to activate calcineurin in basophils, it is possible that desloratadine targets a factor within pathway initiated by this phosphatase.
  • desloratadine inhibits the accumulation of IL-4 mRNA, supporting the concept that desloratadine can negatively regulate factors important for cytokine gene transcription.
  • desloratadine pretreatment resulted in nearly 80% inhibition of the IgE-mediated increase of IL-4 mRNA.
  • the secretion of protein for this cytokine (IL-4) in the same cultures was below detection, and was thus inhibited >95%.
  • IL-4 and IL-13 help regulate the synthesis of IgE, activate endothelium for VCAM-1 -mediated eosinophil transmigration, and that IL-4 promotes the development of the T-helper 2 phenotype, we expect that desloratadine will mediate anti-allergic activity, not only by inhibiting mediator release, but also by blocking the generation of pro-inflammatory cytokines IL-3, IL- 4, IL-5 , IL-6, IL-8, IL-9 and IL-13.
  • Basophils and lymphocytes will be isolated using standard Percoll gradient techniques and their RNA will be extracted and tested using a real time PCR assay with primers for IL-3, IL-4, IL-5, IL-6, IL-9 IL-13, IL-5 and RANTES.
  • mRNA for these cellular products will be quantitated by comparison to the PCR product of a housekeeping gene (GAPDH). Leukocyte differential, and total eosinophil and basophil counts will be used to assess the potential effect of changes in cell numbers on allergen-induced changes in cytokine/chemokine mRNA.
  • GPDH housekeeping gene
  • Dose-titrated allergen-induced skin wheal reaction testing will be done at the time of initial blood drawing above (i.e., 3 days prior to the first nasal allergen challenge) and one week after the last nasal allergen challenge.
  • Nasal symptom severity scores will be monitored during each nasal allergen provocation day. It is expected that induction of cytokine transcription will correlate to worsening nasal symptomatology.
  • the expected primary outcome should be allergen- induced increases in inflammatory cells and up-regulation of IL-4 and IL-13 transcription in the skin biopsy tissue.
  • Secondary Endpoints Changes from baseline in peripheral leukocytes, including eosinophils, basophils and lymphocytes.
  • nasal allergen provocation of atopic individuals induces systemic effects, e.g., peripheral blood eosinophil counts are increased between 6 to 24 hours following this procedure and consistent increases in lower airway responsiveness, in patients with allergic rhinitis and asthma, have followed nasal allergen provocation. It is possible that the systemic effects of allergic rhinitis are mediated through increased proliferation of peripheral leukocytes and increased cytokine production by these cells. The underlying rationale for these hypotheses is that these phenomena are dependent on the state of activation in immune cells, including basophils and lymphocytes.
  • Study No. 2 The clinical efficacy will involve the same essential nasal allergen challenge design and blood specimen collections and allergen-induced skin wheal testing as in Study No. (See below.) Study No. 2, however, will be a double-blind, placebo- controlled, cross-over design during which desloratadine (5 mg once daily) and placebo will be taken daily for 5-7 days prior to the first blood drawing and throughout the remainder of the testing period: the repeat allergen-induced skin wheal test will occur one week after the last nasal allergen challenge. The number of subjects for this study will be determined by power analysis dependent on the results of the preliminary study. A two-week washout period will intervene between the two treatment periods. Flow Chart (Study Days)
  • Desloratadine is expected to provide combined anti-histamine and antiallergic inflammatory effects to control both early and late phase allergic reactions and clinical symptoms in humans.

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Abstract

L'invention concerne l'utilisation de desloratadine pour préparer un médicament destiné à inhiber la production de cytokines pro-inflammatoires telles que IL-4 et IL-13 chez un patient humain nécessitant une telle inhibition.
PCT/US2001/011474 2000-04-07 2001-04-05 Inhibition de la production de cytokines Ceased WO2001076600A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP01928407A EP1267870A2 (fr) 2000-04-07 2001-04-05 Inhibition de la production de cytokines
MXPA02009821A MXPA02009821A (es) 2000-04-07 2001-04-05 Inhibicion de la generacion de citocina.
JP2001574117A JP2003530351A (ja) 2000-04-07 2001-04-05 サイトカイン生成の阻害
CA002405413A CA2405413A1 (fr) 2000-04-07 2001-04-05 Inhibition de la production de cytokines
AU2001255268A AU2001255268A1 (en) 2000-04-07 2001-04-05 Inhibition of cytokine generation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US19601600P 2000-04-07 2000-04-07
US60/196,016 2000-04-07

Publications (2)

Publication Number Publication Date
WO2001076600A2 true WO2001076600A2 (fr) 2001-10-18
WO2001076600A3 WO2001076600A3 (fr) 2002-06-27

Family

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Family Applications (1)

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PCT/US2001/011474 Ceased WO2001076600A2 (fr) 2000-04-07 2001-04-05 Inhibition de la production de cytokines

Country Status (6)

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EP (1) EP1267870A2 (fr)
JP (1) JP2003530351A (fr)
AU (1) AU2001255268A1 (fr)
CA (1) CA2405413A1 (fr)
MX (1) MXPA02009821A (fr)
WO (1) WO2001076600A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1670427A4 (fr) * 2003-09-15 2009-01-07 Combinatorx Inc Procedes et reactifs pour le traitement des troubles immuno-inflammatoires

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5595997A (en) * 1994-12-30 1997-01-21 Sepracor Inc. Methods and compositions for treating allergic rhinitis and other disorders using descarboethoxyloratadine

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1670427A4 (fr) * 2003-09-15 2009-01-07 Combinatorx Inc Procedes et reactifs pour le traitement des troubles immuno-inflammatoires

Also Published As

Publication number Publication date
MXPA02009821A (es) 2003-03-27
EP1267870A2 (fr) 2003-01-02
JP2003530351A (ja) 2003-10-14
WO2001076600A3 (fr) 2002-06-27
AU2001255268A1 (en) 2001-10-23
CA2405413A1 (fr) 2001-10-18

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