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WO1999049865A1 - Utilisation d'inhibiteurs du facteur d'activation des plaquettes sanguines (paf) pour entraver une activation ou une degranulation eosinophilique declenchee par l'interleukine 5 - Google Patents

Utilisation d'inhibiteurs du facteur d'activation des plaquettes sanguines (paf) pour entraver une activation ou une degranulation eosinophilique declenchee par l'interleukine 5 Download PDF

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Publication number
WO1999049865A1
WO1999049865A1 PCT/US1999/006859 US9906859W WO9949865A1 WO 1999049865 A1 WO1999049865 A1 WO 1999049865A1 US 9906859 W US9906859 W US 9906859W WO 9949865 A1 WO9949865 A1 WO 9949865A1
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Prior art keywords
eosinophils
paf
eosinophil
degranulation
induced
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PCT/US1999/006859
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English (en)
Inventor
Gerald J. Gleich
Hirohito Kita
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Mayo Foundation For Medical Education And Research
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Priority to AU33700/99A priority Critical patent/AU3370099A/en
Publication of WO1999049865A1 publication Critical patent/WO1999049865A1/fr

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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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • 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
    • A61K31/4425—Pyridinium derivatives, e.g. pralidoxime, pyridostigmine

Definitions

  • Eosinophils play important roles in the pathophysiology of asthma and other inflammatory diseases. Activated eosinophils adhere to endothelial cells and subsequently release inflammatory mediators (Lee et al., 1984; Gleich et al., 1994), superoxide anion (Sedgwick et al., 1988) and cationic granule proteins (Abu-Ghazaleh et al., 1989).
  • the toxic granule proteins include
  • MBP major basic protein
  • ECP eosinophil cationic protein
  • EDN eosinophil- derived neuro toxin
  • EPO eosinophil peroxidase
  • the eosinophil also possesses a series of enzymes, collagenase, Charcot-Leyden crystal protein, and the 92 kDa gelatinase
  • GM-CSF tumor necrosis factor-
  • TGF- tumor necrosis factor-
  • TGF- transforming growth factor-
  • PDGF platelet-derived growth factor
  • IL-5 is known to play an important role in the regulation of the immune system and is one of several cytokines regulating organ-specific eosinophil infiltration and degranulation during normal host immune function (Kita et al, 1992). Moreover, IL-5, as well as the cytokines IL-3, IFN- ⁇ and GM-CSF, prolong the survival of eosinophils in vitro (Valenus et al, 1990) and augment eosinophil -function (Rothenberg et al., 1988; Fujisana et al., 1990); Silberstein et al., 1986).
  • IL-5 also primes eosinophils for enhanced locomotor responses to chemotactic agents, such as PAF, leukotriene B4, and IL-8 (Sehmi et al., 1992).
  • chemotactic agents such as PAF, leukotriene B4, and IL-8.
  • Recent information indicates that IL-5 is present in the lung following allergen- induced pulmonary late allergic reactions (Sedgwick et al., 1991), and mRNA for IL-5 is expressed in the bronchial epithelium of patients with asthma (Hamid et al., 1991).
  • Eosinophil infiltration and deposition of granule proteins have been observed in bronchoalveolar lavage (BAL) fluids and in bronchial tissues obtained from patients with bronchial asthma (Broide et al., 1991; Filley et al, 1982). Both in vitro and in vivo studies have supported the conclusion that eosinophil granule proteins damage respiratory epithelium (Motojima et al, 1989; Frigas et al., 1986). Eosinophil degranulation can be stimulated with Ig (Abu-Ghazaleh et al., 1989), lipid mediators (Kroegel et al., 1989) and cytokines (Horie et al., 1994).
  • PAF activates platelets, eosinophils and neutrophils, and has been implicated in the pathophysiology of asthma, inducing bronchoconstriction, airway microvascular leakage, edema, and bronchial hyperresponsiveness (Chung et al., 1991).
  • PAF stimulates eosinophil adhesion (Kimani et al, 1988; Lamas et al., 1988) and causes eosinophils to release superoxide anion and eosinophil granule proteins such as eosinophil-derived neurotoxin (EDN) (Kroegel et al., 1988).
  • EDN eosinophil-derived neurotoxin
  • PAF also may act as a secondary messenger for activation of leukocytes (Stewart et al., 1990).
  • Eosinophil adhesion and PLA 2 activation are prerequisites for activated eosinophil degranulation. Recently, ⁇ 2 -integrin-dependent adhesion was shown to be necessary for degranulation of eosinophils activated with GM- CSF, IgG, PAF and PMA (Horie et al, 1997; Kaneko et al, 1995; Horie et al., 1994).
  • PLA 2 is an enzyme that initiates the synthesis of PAF via the remodeling pathway by catalyzing the hydrolysis of a phospholipid precursor (Prescott et al., 1990). Mepacrine inhibits this pathway (Vigo, 1980; Jain et al., 1984).
  • PLA 2 likely plays a role in these functions (White et al., 1993).
  • Glucocorticoids are the most useful class of drugs for treating many eosinophil-related disorders, including bronchial asthma (Schleimer et al., 1990).
  • Glucocorticoids e.g., dexamethasone, methylprednisolone and hydrocortisone, produce eosinopenia in normal persons, decrease circulating eosinophils in patients with eosinophilia, and reduce eosinophil influx at inflammatory sites (Butterfield et al., 1989). The mechanism of these effects is still uncertain.
  • glucocorticoids are the principal therapy and these patients may require glucocorticoid therapy for long periods of time, e.g., months to years.
  • the disease can be characterized as one of chronic glucocorticoid toxicity, in that the toxicity of these steroids can cause severe morbidity and even mortality in the patients.
  • cessation of glucocorticoid therapy leads to withdrawal symptoms, such as malaise and 4 muscle pain.
  • glucocorticoids are the only effective therapy for severe asthma, and are prescribed long-term despite their toxicity.
  • the present invention provides a therapeutic method which counteracts or prevents eosinophil-associated pathologies mediated by cytokines, such as IL-5, growth factors, such as GM-CSF, or by binding of immunoglobulms.
  • the method comprises the administration of one or more agents that are inhibitors of platelet activating factor (PAF), e.g., a PAF receptor antagonist, to an afflicted mammal, e.g., a human, in an amount effective to inhibit or reduce eosinophil activation and/or degranulation.
  • PAF platelet activating factor
  • the agents of the invention inhibit or reduce (antagonize) the activity of cytokines, growth factors and/or immunoglobulms on eosinophils which, in turn, limits or blocks the pathogenic effects of the proteins secreted and/or released by eosinophils on the tissue of the mammal in need of said treatment.
  • Inhibitors of PAF include, but are not limited to, agents that reduce or block the synthesis of PAF, the secretion of PAF, the binding of PAF to a receptor on eosinophils, or the intracellular signal generated by the binding of PAF to a receptor on eosinophils, although the invention is not limited to an agent which has a particular mechanism of action.
  • Preferred agents are those which are permeant to eosinophils, e.g., they are intracellular inhibitors of the PAF synthetic or signaling pathway.
  • autocrine PAF is an essential mediator in eosinophil effector functions, e.g., degranulation and superoxide production, induced by either IL-5 or immobilized Ig, as shown by the effect of PAF, an agonist of eosinophils, released by activated eosinophils on the magnitude of the same eosinophils' response to the original stimuli.
  • human eosinophils produced superoxide and released granule proteins when stimulated with immobilized IgG or soluble IL-5.
  • the invention also provides a method to inhibit or treat an eosinophil-associated pathology, e.g., bronchial asthma.
  • the method comprises administering to a mammal in need of said therapy an amount of an inhibitor of platelet activating factor effective to counteract at least one symptom of the pathology.
  • the inhibitor is an inhibitor of the platelet activating factor receptor.
  • the inhibitor is administered to the respiratory tract of the mammal.
  • such a method may be useful for studying the mechanism of action of other therapeutic agents or potential therapeutic agents that effect eosinophil effector function(s) so as to further elucidate the beneficial or pathological effects of eosinophils in biological systems.
  • PAF receptor antagonists include, but are not limited to, CV6209, WEB 2086, kadsurenome, kadsurin B, UK74505, SM-10661, BN52063, SR27417A, CV3988, ONO-6240, ginkolide B, etizolam, Y24180, C166985, U66985, methyl- WEB 2086, Y24180, BN52111, desmethyl-Y24180, SRI63- 072, R019-3704, SDZ64-412, L-652,731, 48740RD, BN52021 and analogs of PAF such as those described in Grigoriadis et al. (1991) and Tokumura et al. (1985).
  • PAF 6 antagonists such as Y24180 and SR24717A are not orally administered.
  • the PAF receptor antagonist is not CV6209.
  • inhibitors of PAF include, but are not limited to, inhibitors of PLA 2 such as mepacrine, local anesthetics, phenothiazines, peptides related to lipocortin, 7J-dimethyleicosadienoic acid, quinacrine and aristolochic acid.
  • Figure 1 depicts a graph of superoxide production by eosinophils stimulated with PMA, immobilized IgG or IL-5 as a function of mepacrine concentration.
  • Figure 2 depicts a graph of the amount of PAF released per 10 6 eosinophils, which had been stimulated with IL-5, immobilized IgG or PMA, at 15 minutes and 45 minutes post-stimulation.
  • Figure 3 shows a graph of the amount of superoxide released by eosinophils stimulated with various amounts of PAF in the presence of increasing amounts of CN6209.
  • Figure 4 shows the amount of superoxide released by eosinophils over time after stimulation with immobilized IgG (A); IL-5 (B); PMA (C); or PAF (D), in the presence of varying amounts of CV6209.
  • Figure 5 depicts the superoxide release by eosinophils after stimulation with immobilized IgG (A); IL-5 (B); PMA (C); or PAF (D), as a function of CN6209 concentration.
  • Figure 6 depicts the release of ED ⁇ by eosinophils stimulated with immobilized IgG or IL-5 in the presence or absence of CN6209.
  • Figure 7 shows the amount of superoxide released by eosinophils stimulated with various agents in the presence or absence of pertussis toxin (PTX).
  • PTX pertussis toxin
  • Figure 8 shows a comparison of the effect of hexanolamine PAF versus Y24180 on superoxide production by eosinophils.
  • Figure 9 shows a graph of LTC4 release by eosinophils stimulated with IL-5, immobilized IgG or PAF in the presence of CN6209. 7
  • Conditions, indications or diseases amenable to treatment by the therapeutic agents of the invention include upper and lower respiratory tract diseases, eosinophilic myocitis, Chrohn's disease, renal or hepatic graft rejection, eosinophilic endomyocardial disease, acute necretizing myocarditis, primary biliary cirrhosis, sclerosing cholangitis, retroperitoneal fibrosis, medistinum fibrosis, Ridell's invasive fibrosis, thyroiditis, cirrhosis, urticaria, skin diseases, nasal polyps, and inflammatory diseases, e.g., allergic disorders, that are associated with eosinophiha.
  • Eosinophiha is the infiltration of eosinophils into tissues such as blood or lung, and the activation of those eosinophils, which results in the production of eosinophil-derived proteins that in turn mediate pathogenic effects.
  • the infiltration of eosinophils into tissues, and the subsequent degranulation of the eosinophils, is associated with hypersensitivity diseases such as bronchial asthma, chronic eosinophilic pneumonia, vernal conjunctivitis, allergic conjunctivitis, giant papillary conjunctivitis, allergic rhinitis, allergic sinusitis, and allergic gastroenteropathy.
  • eosinophil-associated diseases include eosinophilic gastroenteritis, atopic dermatitis, bullous pemphigoid, episodic angioedema associated with eosinophiha, ulcerative colitis, and inflammatory bowel disease.
  • lung diseases associated with pulmonary eosinophiha include helminth infection, e.g., tropical pulmonary eosinophiha syndrome (TPE), pulmonary infiltrates with eosinophiha (PIE), and pulmonary strongyloidiasis, Churg-Strauss syndrome, allergic bronchopulmonary aspergiUosis, chronic obstructive pulmonary disease (COPP), bronchocentric granulomatosis, drug-induced lung disease, acute eosinophilic pneumonia, pulmonary fibrosis, Spanish toxic oil syndrome (TOS), eosinophilia-myalgia syndrome (EMS), eosinophilic granuloma and the hypereosinophilic syndrome (HES).
  • helminth infection e.g., tropical pulmonary eosinophiha syndrome (TPE), pulmonary infiltrates with eosinophiha (PIE), and pulmonary strongyloidia
  • pulmonary eosinophilic infiltrations include Wegener's granulomatosis, lymphoidmatoid granulomatosis, eosinophilic granuloma of the lung, adult respiratory distress 8 syndrome, and post-trauma pleural effusions which contain eosinophils or eosinophil containing pleural effusions associated with infections, such as tuberculosis (see Spry, In: Eosinophils, Oxford University Press, pp. 205-212 (1988), the disclosure of which is specifically incorporated by reference herein).
  • Preferred Therapeutic Agents for Use in the Methods of the Invention include agents that inhibit or block eosinophil activation and/or degranulation that is mediated by a cytokine, a growth factor or immunoglobulms.
  • a preferred agent is a compound of formula I:
  • R is (C 14 -C 22 )alkoxy, (C 14 -C 22 )alkanoyloxy, N-[ (C ]4 - C 22 )alkyl]aminocarbonyloxy, (C ]4 -C 22 )alkoxycarbonyloxy, (C 14 -C 22 )alkylthio, (C 14 -C 22 )alkanoylthio, N-[ (C 14 -C 22 )alkyl]aminocarbonylthio, or (C ]4 - C 22 )alkoxycarbonylthio;
  • R 2 is (C r C 6 )alkoxy, (C,-C 6 )alkylthio, (C r C 6 )alkanoyloxy, or (C,- C 6 )alkanoylthio;
  • R a is hydrogen or ( -C ⁇ alkyl;
  • R b , R c , and R d are each independently -(C,-C 8 )alkyl-R f or -(C 2 -
  • R e is hydrogen, (C r C 6 )alkyl, or (C r C 6 )alkanoyl;
  • R f is 2-pyridyl, 3-pyridyl, or 4-pyridyl
  • R g and R h are each independently (C 1 -C 3 )alkyl; or R g and R h together with the nitrogen to which they are attached are pyrrolidino, piperidino, morpholino, imidazolidin-1-yl, or piperazin-1-yl; or a pharmaceutically acceptable salt thereof.
  • R is not octadecylaminocarbonyloxy, when R 2 is methoxy and R 3 is:
  • Another preferred agent for use in the methods of the invention is a compound of formula (II):
  • R 7 II wherein R 4 and R 5 are each independently hydrogen, (C,-C 6 )alkyl, (C
  • R 6 and R 7 are each independently aryl or aryl(C,-C 6 )alkyl; wherein any aryl or aryl(C r C 6 )alkyl may optionally be substituted by 1, 2, or 3 halo, hydroxy, (C r C 6 )alkyl, (C,-C 6 )alkoxy, (C r C 6 )alkanoyl, (C [ -C 6 )alkanoyloxy, trifluoromethyl, trifluoromethoxy, nitro, cyano, or amino; 10 or a pharmaceutically acceptable salt thereof.
  • Preferred agents for use in the practice of the methods of the invention include CV6209, Y24180, hexanolamine PAF, and analogs thereof.
  • halo is fluoro, chloro, bromo, or iodo.
  • Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl” embraces only the straight chain radical, a branched chain isomer such as "isopropyl” being specifically referred to.
  • Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic.
  • (C ] -C 6 )alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;
  • (C 14 -C 22 )alkyl can be tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosanyl, heneicosanyl, or docosanyl;
  • (C r C 6 )alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;
  • (C C 6 )alkylthio can be methylthio, ethoxy, propylthioy, is
  • a specific value R is (C ]4 -C 22 )alkoxy or N-[ (C ]4 - C 22 )alkyl]aminocarbonyloxy.
  • Another specific value for R ⁇ is N-[ (C 16 - C 20 )alkyl]aminocarbonyloxy.
  • a preferred value for R is (C 14 -C 20 )alkoxy.
  • Another preferred value for R is N-[ (C 16 -C 20 )alkyl]aminocarbonyloxy.
  • R 2 is (C r C 6 )alkoxy, or (C r C 6 )alkanoyloxy.
  • R b is -(C 5 -C 8 )alkyl-N(R g )(R h ).
  • Another specific value for 1 ⁇ is -(CH 2 ) 6 -N(R g )(R h ).
  • R d is -(C j -C ⁇ alkyl-R f .
  • Another specific value for R d is -CH ⁇ R f .
  • a specific value for R f is 2-pyridyl.
  • a sprcific value for R 4 is hydrogen. Another specific value for R 4 is methyl.
  • a specific value for R 5 is methyl. 12
  • a specific value for A " is halo (e.g. chloro or bromo).
  • R d is -(C,-C 8 )alkyl-R f ;
  • R e is (C r C 6 )alkanoyl;
  • R f is 2-pyridyl, 3-pyridyl, or 4-pyridyl, substituted at the 1-position with (C,- C 6 )alkyl to form an ammonium salt; or a pharmaceutically acceptable salt thereof.
  • a preferred group of compounds are compounds of formula I which are quaternary ammonium salts of the nitrogen of R b , R c , R d , or R f .
  • a preferred salt of a compound of formula I is a salt wherein R b , R c , or R d is -(C 2 -C 8 )alkyl- ⁇ (R g )(R h )(-R J ) A " ; wherein R j is (C r C 3 )alkyl; and A " is a pharmaceutically acceptable anion.
  • Another preferred salt of a compound of formula I is a salt wherein R f is 2-pyridyl, 3-pyridyl, or 4-pyridyl, substituted at the 1 -position with (C r C 6 )alkyl.
  • a specific group of compounds are compounds of formula II wherein R 6 is benzyl or phenethyl, optionally substituted by 1, 2, or 3 halo, hydroxy, (C,-C 6 )alkyl, (C,-C 6 )alkoxy, (C r C 6 )alkanoyl, (C r C 6 )alkanoyloxy, trifluoromethyl, trifluoromethoxy, nitro, cyano, or amino.
  • R 6 is phenethyl, optionally substituted with (C r C 6 )alkyl. 13
  • a compound of formula I can conveniently be prepared from glycerol using techniques which are known in the art.
  • a compound of formula II can conveniently be prepared using procedures similar to those used to prepare Y-24180 or WEB 2086.
  • compounds of formula I wherein R f is 2-pyridyl, 3-pyridyl, or 4-pyridyl can be alkylated to give a compound of formula I wherein R f is 2-pyridyl, 3-pyridyl, or 4-pyridyl, substituted at the 1 -position with (C r C 6 )alkyl.
  • administration of the compounds as salts may be appropriate.
  • Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, -ketoglutarate, and ⁇ -glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
  • Compounds of formula I or II may comprise more than one acidic or basic site capable of salt formation. Accordingly, the pharmaceutically acceptable salts of the invention include mono, di, tri, etc. salts of compounds of formula I or II. 14
  • Therapeutic agents for use in the methods of the invention are those that inhibit the cytokine, growth factor and/or immunoglobulin mediated pathogenic activity of eosinophils.
  • Preferred therapeutic agents for use in the methods of the invention are those that inhibit cytokine, e.g., IL-5, induced degranulation of eosinophils, e.g., PAF receptor antagonists, as well as derivatives or analogs thereof.
  • In vitro methods for determining whether a particular agent is within the scope of the invention include, but are not limited to, those provided below in Example 1.
  • in vitro methods may also be employed, such as methods to detect or determine the effect of the agent on MBP, ECP or EPO release from eosinophils, or LTC4 or cytokine production (Kita et al., 1995) from eosinophils.
  • the efficacy of the agent on eosinophils can be measured in the presence of a moiety that inhibits eosinophil adherence, e.g., anti-CD18, to determine whether the agent inhibits adherence- independent degranulation of eosinophils.
  • mice for a particular disorder or disease may also be employed to screen for therapeutic agents useful in the practice of the methods of the invention.
  • a murine model of the allergen-induced IgE- mediated eosinophil-rich late phase reaction induced by immunization of BALB/c mice with ragweed extract is available (Kaneko et al., Int. Arch. Allergy Appl. Immunol., 26, 41 (1991)).
  • mice are sensitized by a series of five injections of a ragweed pollen extract and on day 20 are challenged by an intraperitoneal injection of 0.2 ml of a 1:1,000 dilution of ragweed pollen extract (Greer Laboratories, Inc., Lenoir, NC).
  • Agents that inhibit platelet activating factor can be administered prior to, concurrently, and/or subsequent to ragweed pollen extract administration. Forty-eight hours after challenge, peritoneal lavage fluids are collected, and the numbers and kinds of cells determined by total and differential leukocyte counts (after staining of the peritoneal cells with May-Grunwald-Giemsa stain). With this model, in the absence of agent administration, marked eosinophiha occurs at 48 hours and is associated with eosinophil degranulation. RT-PCR analyses have shown that peritoneal cells express IL-5.
  • eosinophil degranulation can be estimated by measuring the amount of cationic granule proteins, e.g., EPO 15 activity using o-phenylendiamine as substrate, in peritoneal fluids.
  • CN6209 is administered by intraperitoneal injection 6 hours prior to allergen challenge.
  • a suitable dose of CN6209 can include about 0.01 to about 10 mg/kg, preferably about 0J to about 1 mg/kg, although other dosages may be efficacious.
  • Pulmonary late phase reactions may also be used, because of the ability to administer CN6209 to the lung by local instillation in such reactions. For the experiments described above, it may be desirable to give large doses of agents of the invention. If the desired concentrations of drugs with the peritoneal model of the late phase reaction cannot be achieved, the same reagents can be employed to sensitize mice by pulmonary challenge and elicitation of BAL eosinophiha and eosinophil degranulation.
  • bronchial hyperactivity is manifested in patients as a marked irritability of the respiratory tract to nonspecific stimuli including cold air, dust, and, in the laboratory, to inhaled methacholine. Indeed, this hyperactivity is a diagnostic criterion for asthma ( ⁇ J. Gross et al., in Allergy, Principles and
  • Guinea pigs can be sensitized with ovalbumin by nebulization, and bronchial hyperreactivity assessed by administration of aerosolized acetylcholine. Following sensitization and challenge of guinea pigs with ovalbumin, a significant increase in the number of eosinophils are present in the BAL fluids at 24 hours, and at 72 hours after ovalbumin inhalation, bronchial activity to aerosolized acetylcholine is increased, as shown by a significant decrease in the concentrations needed to cause changes in lung resistance and dynamic compliance.
  • Guinea pigs administered an agent of the invention show suppressed bronchial hyperreactivity to aerosolized acetylcholine, and BAL eosinophiha.
  • therapeutic interventions designed specifically to inhibit PAF may inhibit alterations in bronchopulmonary function.
  • agent efficacy can include the measurement of forced expiratory volume (FEV 1 ⁇ or peak expiratory flow rate (PFR), a decrease in steroid amount or use, inhibition of an exudate of eosinophils, degenerating bronchial epithelial cells, Creola bodies and Charcot-Leyden crystals in the bronchial lumen.
  • FEV 1 ⁇ or peak expiratory flow rate (PFR) a decrease in steroid amount or use
  • FEV 1 ⁇ or peak expiratory flow rate (PFR) a decrease in steroid amount or use
  • FEV 1 ⁇ or peak expiratory flow rate (PFR) a decrease in steroid amount or use
  • Therapeutic agents of the invention may be targeted to a specific therapeutic site by linking the therapeutic agent to a moiety that specifically binds to a cellular component, e.g., antibodies or fragments thereof, and small molecule drugs, so as to form a therapeutic conjugate.
  • a cellular component e.g., antibodies or fragments thereof, and small molecule drugs
  • binding peptides of the present invention may be identified and constructed or isolated in accordance with known techniques.
  • Preferred binding peptides of these embodiments of the present invention bind to a target epitope with an association constant of at least about 10 "6 M.
  • Targeting of the therapeutic agents of the invention can result in increased concentration of the therapeutic agent at a specific anatomic location or to a specific cell type, i.e., eosinophils.
  • the linking of a therapeutic agent of the invention to a binding moiety to form a conjugate may increase the stability of the therapeutic agent in vivo.
  • eosinophil-binding proteins e.g., polypeptides or carbohydrates, proteoglycans and the like, that are associated with the cell membranes of eosinophils can be employed to prepare therapeutic conjugates.
  • antibodies to the bronchial epithelial or to eosinophils may be useful to prepare immunoconjugates for use in the methods of the invention.
  • Such targeting agents include, but are not limited to, the IL-5 receptor, or the or ⁇ chain of the IL-5 receptor, or a fragment thereof which 17 specifically binds to eosinophils.
  • the ⁇ chain of the IL-5 receptor, or a fragment thereof, is a preferred targeting agent of the invention.
  • an antibody or fragment thereof, or other targeting moiety, having a specificity for a surface antigen on eosinophils is attached to a therapeutic agent of the invention.
  • the attachment is via a linker that is covalently attached to both the antibody and the therapeutic agent.
  • the linker is a peptide which has a proteolytic cleavage site. This site, interposed between the targeting moiety, e.g., immunoglobulin, and the agent, can be designed to provide for proteolytic release of the agent at, or more preferably, within the eosinophil.
  • plasmin and trypsin cleave after lysine and arginine residues at sites that are accessible to the proteases.
  • Many other site-specific endoproteases and the amino acid sequences they attack are well known.
  • the antigen binding activity of the purified immunoconjugates can then be measured by methods well known to the art, such as described in Gillies et al. (J. Immunol. Methol., 125, 191 (1989)).
  • immunoconjugate activity can be determined using antigen-coated plates in either a direct binding or competition assay format.
  • humanized antibodies are prepared and then assayed for their ability to bind antigen. Methods to determine the ability of the humanized antibodies to bind antigen may be accomplished by any of numerous known methods for assaying antigen-antibody affinity.
  • Humanized antibodies are useful tools in methods for therapeutic purposes. When determining the criteria for employing humanized antibodies or antibody conjugates for in vivo administration for therapeutic purposes, it is desirable that the general attainable targeting ratio is high and that the absolute dose of therapeutic agent delivered to the target tissue or cell is sufficient to elicit a significant therapeutic response.
  • Methods for utilizing the humanized antibodies can be found, for example, in U.S. Patent Nos. 4,877,868, 5,175,343, 5,213,787, 5,120,526, and 5,202,169. It will be recognized that the inventors also contemplate the utility of human monoclonal antibodies or "humanized" murine antibody as a eosinophil binding protein in the therapeutic conjugates of their invention.
  • murine monoclonal 18 antibody may be "chimerized” by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., containing the antigen binding sites) with the nucleotide sequence encoding a human constant domain region and an Fc region, e.g., in a manner similar to that disclosed in European Patent Application No. 0,411,893 A2.
  • Humanized eosinophil binding partners will be recognized to have the advantage of decreasing the immunoreactivity of the antibody or polypeptide in the host recipient, which may thereby be useful for increasing the in vivo half-life and reducing the possibility of adverse immune reactions. See also, N. Lonberg et al. (U.S. Patent Nos. 5,625,126; 5,545,806; and 5,569,825); and Surani et al. (U.S. Patent No. 5,545,807).
  • Coupled methods for linking the therapeutic agent through covalent or non-covalent bonds to the targeting moiety include chemical cross-linkers and heterobifunctional cross-linking compounds (i.e., "linkers") that react to form a bond between reactive groups (such as hydroxyl, amino, amido, or sulfhydryl groups) in a therapeutic agent and other reactive groups (of a similar nature) in the targeting moiety.
  • reactive groups such as hydroxyl, amino, amido, or sulfhydryl groups
  • This bond may be, for example, a peptide bond, disulfide bond, thioester bond, amide bond, thioether bond, and the like.
  • conjugates of monoclonal antibodies with drugs have been summarized by Morgan and Foon (Monoclonal Antibody Therapy to Cancer: Preclinical Models and Investigations, Basic and Clinical Tumor Immunology, Vol. 2, Kluwer Academic Publishers, Hingham, MA) and by Uhr, J. of Immunol. 13 :i-vii, 1984).
  • U.S. Patent No. 4,897,255, Fritzberg et al is instructive of coupling methods that may be useful.
  • an effective therapeutic effective dosage and treatment protocol This will depend upon factors such as the particular selected therapeutic agent, route of delivery, the type of target site(s), affinity of the targeting moiety for target site of interest, any cross- 19 reactivity of the targeting moiety with normal tissue, condition of the patient, whether the treatment is effected alone or in combination with other treatments, among other factors.
  • a suitable dosage ranges from about 0.001 to about 10 mg, and more preferably from about 0J to 2 mg, may be employed.
  • the conjugates may be administered by known methods of administration.
  • Known methods of administration include, by way of example, intraperitoneal injection, intravenous injection, intramuscular injection, intranasal administration, among others. Intravenous administration is generally preferred.
  • the agents of the invention may be administered locally or systemically. While it is possible that for use in therapy the PAF receptor antagonist (the "active agent”) or their salts may be administered as the pure dry chemicals, as by inhalation of a fine powder via an insufflator, it is preferable to present the active ingredient as a pharmaceutical formulation.
  • the invention provides a pharmaceutical formulation comprising one or more active agent, or pharmaceutically acceptable salts thereof, together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients.
  • the carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions include those suitable for oral or parenteral (including intramuscular, subcutaneous and intravenous) administration.
  • forms suitable for parenteral administration also include forms suitable for administration by inhalation or insufflation or for nasal, or topical (including buccal, rectal, vaginal and sublingual) administration.
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with liquid carriers, solid matrices, semi-solid carriers, finely 20 divided solid carriers or combinations thereof, and then, if necessary, shaping the product into the desired delivery system.
  • compositions suitable for oral administration may be presented as discrete unit dosage forms such as hard or soft gelatin capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or as granules; as a solution, a suspension or as an emulsion; or in a chewable base such as a synthetic resin or chicle.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents.
  • the tablets may be coated according to methods well known in the art, i.e., with enteric coatings.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
  • the compounds according to the invention may also be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • the compounds may be formulated as ointments, creams or lotions, or as the active ingredient of a transdermal patch.
  • Suitable transdermal delivery systems are disclosed, for example, in A. Fisher et al. (U.S. Patent No. 4,788,603), or R. Bawa et al. (U.S. Patent Nos. 4,931,279; 4,668,506 and 4,713,224).
  • Ointments and creams may, 21 for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • the active ingredient can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051,842.
  • the above-described formulations can be adapted to give sustained release of the active ingredient employed, e.g., by combination with certain hydrophilic polymer matrices, e.g., comprising natural gels, synthetic polymer gels or mixtures thereof.
  • compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories.
  • Suitable carriers include cocoa butter and other materials commonly used in the art, and the suppositories may be conveniently formed by admixture of the active compound with the softened or melted carrier(s) followed by chilling and shaping in molds.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • compositions include those suitable for administration by inhalation or insufflation or for nasal, intraocular or other topical (including buccal and sub-lingual) admimstration.
  • the formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active compound with liquid carriers or finely divided solid carriers or both.
  • the compounds according to the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, 22 dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the composition may take the form of a dry powder, for example, a powder mix of the active agent and a suitable powder base such as lactose or starch.
  • a powder mix of the active agent and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatine or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.
  • the active agent may be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler.
  • atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).
  • Drops such as eye drops or nose drops, may be formulated with an aqueous or nonaqueous based also comprising one or more dispersing agents, solubilizing agents or suspending agents.
  • Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
  • Formulations suitable for topical administration in the mouth or throat include lozenges comprising active agent in a flavored base, usually sucrose and acacia or tragacanth; pastille comprising the active agent in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
  • compositions according to the invention may also contain other active ingredients such as antimicrobial agents, or preservatives.
  • the active agent may also be used in combination with other therapeutic agents, for example, bronchodilators or anti-inflammatory agents.
  • bronchodilators or anti-inflammatory agents.
  • the amount of the active agent required for use in treatment will vary not only with the particular compound selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at 23 the discretion of the attendant physician or veterinarian.
  • a suitable unit dose for counteracting respiratory tract symptomatology will deliver per day from about 0.001 to about 100 mg/kg, preferably about 0.01 to about 10 mg/kg, of body weight.
  • Useful dosages of the agents of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two-, three-, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye or nose.
  • the invention will be further described by reference to the following detailed examples.
  • Example 1 Attenuation of Eosinophil Activation by Inhibiting Autocrine Production of PAF Materials and Methods Reagents.
  • Platelet activating factor (PAF) and CN6209 were purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA) and were dissolved in absolute ethanol at a concentration of 40 mM and stored at -20°C.
  • Phorbol myristate acetate (PMA) was purchased from Calbiochem (La Jolla, CA), dissolved with dimethyl sulfoxide at a concentration of 5 mg/ml and stored at -20°C.
  • Human IL-5 was a generous gift from Schering-Plough
  • IgG was diluted to 50 mg/ml with PBS and 50 24 ⁇ l per well added to plates for overnight incubation at 4°C.
  • Catalase, superoxide dismutase, human serum albumin (HSA), taurine and cytochrome c were purchased from Sigma Chemical Co.
  • Pertussis toxin (PTX) was purchased from Calbiochem.
  • Eosinophil isolation Eosinophil isolation was performed with minor modifications of the method described by Ide et al. by a magnetic cell separation system (MACS; Becton Dickinson, San Jose, CA).
  • venous blood anticoagulated with 50 U/ml heparin was obtained from normal volunteers and diluted with PBS at a 1 :1 ratio.
  • Diluted blood was overlaid on isotonic Percoll solution (density, 1.085 g/ml; Sigma Chemical Co.) and centrifuged at 1000 g for 30 minutes at 4°C.
  • the supernatant and mononuclear cells at the interface were carefully removed, and erythrocytes in sediment were lysed by two cycles of hypotonic water lysis.
  • Isolated granulocytes were washed twice with piperazine-N,N'bis[2-ethanesulfonic acid] (Pipes) buffer (25 mM Pipes, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose, pH 7.4) with 1% fetal calf serum (FCS) (HyClone Laboratories; Logan, UT), and an approximately equal volume of anti-CD 16-conjugated magnetic beads (Miltenyi Biotech Inc.; Auburn, CA) was added to the cell pellet. After 60 minutes of incubation on ice, cells were loaded onto the separation column positioned in the strong magnetic filed of the MACS.
  • Pipes piperazine-N,N'bis[2-ethanesulfonic acid]
  • Eosinophil superoxide production was induced by various stimuli in polystyrene 96-well flat-bottom tissue culture plates (Falcon #3072, Becton Dickinson). To immobilize IgG onto the wells of the plate, selected wells were coated overnight at 4°C with 50 ⁇ l of IgG dissolved in PBS at 50 ⁇ g/ml. The solution was aspirated; all wells were coated with 50 ⁇ l of 2.5% HSA (Sigma, A3782) dissolved in PBS and incubated at 37°C for 2 hours. After incubation, wells were washed twice with 0.9% NaCl and used immediately for the experiments. Generation of superoxide by eosinophils was measured by reduction of cytochrome c, as described previously (Sedgwick et al., 1988), with slight modifications. 25
  • eosinophils were washed with Hanks' balanced salt solution (HBSS) with 10 mM HEPES and resuspended to 5 10 5 cells/ml in cytochrome c (Sigma) solution (2.4 mg/ml cytochrome c in HBSS medium).
  • HBSS Hanks' balanced salt solution
  • cytochrome c Sigma
  • Fifty ⁇ l of antagonist (CV6209 or mepacrine) at 4X concentrated stock (the final concentration) or medium was added to each well.
  • One hundred microhters of cell suspension was dispensed onto plates and the reactions were initiated by adding 50 ⁇ l of soluble stimulus diluted in HBSS with 10 mM HEPES.
  • Wells coated with immobilized Ig received 50 ⁇ l of HBSS with 10 mM HEPES.
  • the reaction wells were measured for absorbance at 550 nm in a microplate autoreader (Thermomax, Molecular
  • Eosinophils were stimulated to produce PAF using a modification of the method described by van der Bruggen et al. (1994). Generation of PAF by eosinophils was performed in 96-well flat-bottom tissue culture plates, prepared with immobilized Ig and 2.5% HSA as described previously. All incubations were performed in enriched
  • HEPES medium HEPES medium (HEPES supplemented with 1 mM CaCl 2 , 5 mM glucose, and 0.5% (w/v) HSA) containing O 2 -radical scavengers (2.5 mM taurine, 5000 U/ml catalase, and 380 U/ml superoxide dismutase) to prevent lipid degradation.
  • O 2 -radical scavengers 2.5 mM taurine, 5000 U/ml catalase, and 380 U/ml superoxide dismutase
  • Freshly isolated eosinophils were suspended in enriched HBSS with scavengers at 5 X 10 3 cells/ml; and 100 ⁇ l aliquots were added to prepared tissue culture plates. Enriched HBSS with scavengers or IL-5 diluted in enriched HBSS with scavengers was added.
  • Eosinophil degranulation Eosinophil degranulation was performed in 96-well flat-bottom tissue culture plates, prepared with 26 immobilized IgG and 2.5% HSA as described previously. Eosinophils were washed with RPMI-1640 medium (Celox Inc., Hopkins, MN) with 10 mM HEPES and 0.02% HSA, and resuspended in the same medium at 5 x 10 5 cells/ml. The soluble stimuli (IL-5, PMA and PAF) and the agonist CV6209 were diluted in the same medium. Fifty microhters of 4-fold concentrated solutions of CV6209 or medium were added to wells, followed by 100 ⁇ l of cell suspension.
  • EDN eosinophil-derived neurotoxin
  • EDN radioimmunoassay To quantitate eosinophil degranulation, concentrations of EDN in the sample supernatants and lysates were measured by radioimmunoassay.
  • the radioimmunoassay is a double-antibody competition assay in which radioiodinated EDN, rabbit anti-EDN and burro anti-rabbit IgG are used as reported elsewhere (Abu-Ghazaleh et al., 1989). Total cellular EDN contents were measured simultaneously using supernatants from cells lysed with 0.5% Nonidet P-40 detergent. All assays were carried out in duplicate. Statistical analysis. Data are presented as mean (SEM from the numbers of experiments indicated).
  • mepacrine inhibited IgG-induced superoxide production and IL-5 -induced superoxide production in a concentration-dependent manner.
  • PMA-induced superoxide production was not affected by the addition of up to 100 ⁇ M mepacrine.
  • PLA 2 is likely involved in superoxide production by eosinophils stimulated with immobilized IgG or IL-5.
  • Agonist-stimulated eosinophils release PAF in a time-dependent manner. Because PLA 2 is involved in the metabolism of arachidonic acid, it was determined whether PAF is produced when eosinophils are stimulated with soluble IL-5, immobilized-IgG or PMA. As a result, human eosinophils released PAF in response to IL-5 and immobilized IgG in a time-dependent manner ( Figure 2). PAF was detectable 15 minutes after stimulation with immobilized IgG or soluble PMA. After 45 minutes, PAF was also detectable in supernatants from eosinophils stimulated with IL-5.
  • Eosinophils responded more vigorously to immobilized IgG (2.9 ⁇ 0.4 ng PAF/10 6 cells) and PMA (3.6 ⁇ 1.0 ng PAF/10 6 cells), but less vigorously to IL-5 (0.5 ⁇ 0.1 ng PAF/10 6 cells). At 45 minutes, all three stimuli induced significant release of PAF, p ⁇ 0.05 compared to medium alone (MWU). Eosinophils incubated in medium alone did not release detectable levels of PAF at any time.
  • CV6209 is a competitive antagonist for the PAF receptor.
  • CV6209 is known as a potent PAF receptor antagonist (Terashita et al., 1987).
  • PAF PAF induced superoxide production in a concentration-dependent manner ( Figure 3).
  • Figure 3 PAF induced superoxide production in a concentration-dependent manner
  • CV6209 was increased, superoxide release dropped.
  • CV6209 at 0J ⁇ M blocked 60% of the superoxide released from eosinophils stimulated with 0J but had little or no effect on superoxide release from eosinophils incubated with 0.3, 1 or 28
  • IL-5- and PAF- induced eosinophils appeared equally sensitive to CV6209, with an ED 50 of approximately OJ ⁇ M.
  • Immobilized-IgG-induced eosinophils appeared to be less sensitive, with an ED 50 of approximately 1 ⁇ M.
  • IL-5-induced superoxide production is PTX sensitive.
  • the receptor for IL-5 belongs to the hemopoietin receptor family, which has not been shown to be coupled to G-proteins.
  • the PAF receptor is coupled to G-proteins (Houslay et al., 1986; Hwang et al., 1986; Uhing et al., 1989).
  • PTX catalyzes the ADP-ribosylation of certain G-protein ⁇ -subunits in intact cells, resulting in the uncoupling of these G-proteins from cell surface receptors (Moss, 1988).
  • PTX pertussis toxin
  • PTX-sensitive G-proteins are likely involved in eosinophil activation stimulated by IL-5 as well as by PAF.
  • Eosinophils were preincubated with various concentrations of CV6209, and then stimulated with IL-5, PAF, or immobilized human IgG for 60 minutes. Supernatants were collected and analyzed by a LTC4 ELISA kit. As shown in Figure 9, LTC4 production and release by eosinophils stimulated with IL-5 and immobilized IgG as well as that stimulated with PAF were inhibited by CV6209 in a concentration-dependent manner. These findings suggest that autocrine PAF is involved in LTC4 production by eosinophils activated by IL-5 or immobilized IgG. 32
  • IL-5 and IgG effectively activate eosinophils, causing cellular adhesion, degranulation, superoxide anion production and release of lipid mediators such as PAF (Gleich et al, 1994; Lee et al., 1984; Sedgwick et al., 1988; Trigianni et al., 1992; White et al, 1993; Cromwell et al, 1990).
  • PAF itself induces eosinophil activation, specifically adhesion, degranulation and superoxide production (Gleich et al, 1994).
  • Eosinophil growth factors such as IL-5, IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are released by activated eosinophils in vitro and are known to activate eosinophils themselves, suggesting an autocrine role for these cytokines in eosinophil- associated inflammation (Gleich et al., 1994). Whether PAF acts similarly, in an autocrine fashion, has not been studied. As described above, eosinophils stimulated with immobilized IgG or with soluble IL-5 produced biologically active levels of PAF. Moreover, PAF produced by activated eosinophils enhanced degranulation and superoxide anion production in those same eosinophils and that this auto-upregulation most likely occurs via a G-protein coupled receptor.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • eosinophils Many agonists induce eosinophils to release PAF.
  • calcium ionophores, fMLP, C5a and ECF-A stimulate rapid (1 to 3 minutes) release of PAF from eosinophils (White et al., 1993).
  • IgG coated sepharose beads and soluble GM-CSF also stimulate eosinophils to release PAF (Cromwell et al, 1990; Triggiani et al., 1992). Further, eosinophils stimulated with either immobilized IgG or soluble IL-5 released nM levels of PAF ( Figure 2).
  • PAF-activated cells are prevented from adhering, either by blocking ⁇ 2 -integrin adhesion using antibody to Mac-1 (CD1 lb/CD 18) or simply by constant stirring, EDN release is blocked (Horie et al., 1994).
  • PAF 34 production is dependent on adhesion, indicating that adhesion is necessary not only for eosinophil function (degranulation and superoxide production) but also for the production of PAF, which in turn upregulates degranulation and adhesion.
  • treatment of eosinophils with PTX has been shown to completely abolish EDN release from immobilized slgA-activated eosinophils and to temporarily inhibit EDN release from cells activated with immobilized IgG (Kita et al., 1991).
  • neutrophils primed with PAF and subsequently stimulated with TNF release superoxide in a PTX sensitive manner Braquet et al., 1991).
  • PAF receptors are thought to signal through a G protein, although whether that response is sensitive to PTX appears to depend on the cell involved (Houslay et al, 1986; Hwang et al, 1986; Uhing et al., 1989).
  • eosinophils pretreated with PTX PAF-induced increases in intracellular free calcium were significantly attenuated (Kernen et al., 1991).
  • Eosinophils pretreated with PTX and stimulated with IL-5 or PAF released significantly less superoxide than cells that were not pretreated (Figure 7), suggesting that PTX- sensitive G proteins are involved in eosinophil activation stimulated by IL-5 and PAF.
  • IL-5 receptor is coupled to a G protein, and, indeed, structurally such a configuration appears unlikely. For this reason, it appears that IL-5 activates eosinophils not just via the IL-5 receptor, but through another G protein coupled receptor as well, indicating that an IL-5 induced mediator, such as PAF, is necessary.
  • eosinophils Upon stimulation with immunoglobulms (IgG) or cytokines (IL- 5), eosinophils produce and release PAF.
  • This autocrine PAF stimulates cellular adhesion of eosinophils, which further upregulates PAF production.
  • autocrine PAF and cellular adhesion induce eosinophil effector functions, such as degranulation and superoxide production.
  • Inhibition of either autocrine PAF or adhesion markedly suppresses eosinophil function.
  • Bo-ting, A., William, T. J., and Collins, P. D. Zymosan-induced IL-8 release from human neutrophils involves activation via the CD 11 b/CD 18 receptor and endogenous platelet-activating factor as an autocrine modulator.
  • Platelet- activating factor may act as a second messenger in the release of icosanoids and superoxide anions from leukocytes and endothelial cells. Proc. Natl. Acad. Sci. USA, £2:3215-3219, 1990.
  • Trigianni M. Characterization of platelet- activating factor synthesized by normal and granulocyte-macrophage colony-stimulating factor-primed human eosinophils. Tmmunology. 22:500-504, 1992. Uhing, R.J., Perpic, V., Hollenbach, P.W., and Adams, D.O.

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Abstract

Cette invention porte sur une méthode thérapeutique visant à mettre fin à des états pathologiques associés à une activation, à une adhérence ou à une dégranulation éosinophiliques ou bien à les traiter. Cette méthode consiste à administrer au mammifère nécessitant cette thérapie une quantité efficace d'un inhibiteur du facteur d'activation des plaquettes sanguines (PAF).
PCT/US1999/006859 1998-03-31 1999-03-31 Utilisation d'inhibiteurs du facteur d'activation des plaquettes sanguines (paf) pour entraver une activation ou une degranulation eosinophilique declenchee par l'interleukine 5 WO1999049865A1 (fr)

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