AU7073898A - Methods and use of compositions comprising tnf-rii(p75) agonists for treating asthma and other allergic conditions - Google Patents

Description

WO 99/59632 PCT/IB98/00761 METHODS AND USE OF COMPOSITIONS COMPRISING TNF-RII(P75) AGONISTS FOR TREATING ASTHMA AND OTHER ALLERGIC CONDITIONS BACKGROUND OF THE INVENTION Asthma is a complex inflammatory disease of the airways that is 5 characterized by recurrent exacerbations due to exposure to specific allergens, exercise, cold air or stress. The hallmarks of inflammation associated with asthmatic condition are the presence of activated eosinophils, an increased sensitivity of the airways to non-specific stimuli (airways hyperresponsiveness: AHR), edema, mucus hypersecretion and cough. This inflammatory process is 10 believed to be mediated, in part, by the generation and activation of Th2-type lymphocytes which secrete a variety of cytokines. In addition, allergic conditions other than asthma may be associated with similar generation and activation of Th2-type lymphocytes and cytokines secretion, such as rhinoconjuctivitis, conjuctivitis, rhinitis, dermatitis, urticaria, anaphylaxis, and others. 15 Multiple therapeutic agents are used for treating asthma, notably containing steroids and B-2 agonists. Despite these treatments, the morbidity and mortality of asthma remain high and is even increasing. There is certainly a need for a drug that can rapidly reverse the inflammatory response seen in asthma, as current treatments are only partially effective. The same could be 20 said from other allergic diseases. For more information on potential therapeutic approaches for asthma, see Bousquet J. et al. (Clinical and Experimental Allergy, 25(2), 39-42, 1995). Tumor Necrosis Factor TNFc exerts a key role in the cytokine network with regard to the pathogenesis of many infectious and inflammatory 25 diseases. TNFc was purified, sequenced and cloned in the mid 1980s. Since then, several biological properties of this cytokine have been demonstrated. Human TNF is synthesized as pro-protein comprising 233 amino acids with a molecular mass of 26 kDa. This protein is cleaved by specific metalloprotease (TNFa-converting enzyme TACE) to yield a monomeric form of 30 17 kDa comprising 157 non-glycosylated amino acids. Under physiological conditions TNFc forms a non-covalently bound cone-shaped homotrimer.

WO 99/59632 PCT/IB98/00761 2 TNF effects are transmitted via cross-linking of the membrane-bound receptor molecules TNF-Receptor I (TNF-RI, p55) and TNF Receptor 1I (TNF RII,p75). The extracellular portions of both TNF receptors can be shed, and these soluble receptors retain the ability to bind TNF. After binding to its 5 membrane-bound receptors, TNF mediates diverse effects in different organs and tissues. Although several cell types produce TNF, the main source for the cytokine is monocytes/macrophages. TNF induce a number of proinflammatory changes in endothelial cells, including cytokine production, expression of 10 adhesion molecules, and release of procoagulatory substances. These alterations may lead to septic shock. The actions of TNFa are known to promote asthmatic conditions; therefore, different groups have determined that blocking the action of soluble circulating TNFa provides means for relieving these conditions. This issue has 15 been addressed by different approaches in published patent literature. For example, WO 97/41895 relates to the treatment of asthma by administering a preparation of a chimeric protein comprising the soluble portion of TNFRI(p55) fused to IgG. This composition is claimed to block the action of TNFa and reverse inflammation in the lung. However, prolonged exogenous 20 administration of the soluble portion of TNFRI receptor could cause an alteration in the immune response of the organism. From Grell et al.(Cell, 83, 793-802, 1995) it is known that membrane-bound TNF (mTNF) is superior to soluble TNFc in triggering p75 receptor in various systems, such as thymocyte proliferation. In particular, the 25 authors provide evidence that mTNF is the prime physiological activator of TNF RII, which implies that TNF-RII controls the local response pattern in specific microenvironments. The proposed model is that, being mTNF the physiological precursor of sTNF, it could be an early and very effective stimulus because it could induce independent signal pathways at each of the TNFRs. 30 Yoshida et al.(Clin. Exp. Immunol., 106(1) 73-78, 1996) shows that the levels of soluble TNFRs having specific inhibitory activity for TNFc are higher in the serum of patients with bronchial asthma during asthma attacks and may WO 99/59632 PCT/IB98/00761 3 contribute to regulating TNFa production and the development of allergic inflammation. The respective roles of p55 and p75 receptors in mediating and modulating the biological effects of TNF have been studied in vivo by generating 5 and examining mice genetically deficient in these receptors (Peschon J. et al., J. Immunol., 160, 943-952, 1998). Pulmonary inflammation after intranasal instillation of heat-inactivated actinomycete M. faeni Ags - representing a Th1 inflammatory pathway model - has been examined in mice deficient in either p55 or p75, or both. Selective deficits in several inflammatory responses are 10 observed in mice lacking p55 or both p75 and p55, but not in mice lacking p75. In the latter model, the activity of p55 is not impaired by the absence of p75, arguing against a physiological role for p75 as an essential element of p55 mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased serum TNFa levels in mice lacking p75 suggest a 15 dominant role for p75 in suppressing TNF-mediated inflammatory responses. Accordingly, p55 and p75 TNF receptors seem to represent a balancing system for TNF action. A deficit in p55 activation or in p55 receptor leads to deficits in several inflammatory responses. Conversely, a deficit in p75 activation or in p75 receptor leads to exacerbated pulmonary inflammation. These results suggest a 20 role for endogenous p75 in down-modulating pulmonary inflammatory responses mediated by Th1 type inflammation pathway. DESCRIPTION OF THE INVENTION The present application is based on the assumption that the binding 25 of TNF-RIl(p75) receptor with an agonist triggers a TNF-RI(p55)-independent signal to the cell which causes a decrease in Th2-mediated inflammation, notably in allergic bronchial asthma and other allergic conditions. This assumption is based on the findings reported in the examples that selective TNF-RIl(p75) activation has a protective role in lung inflammation and hyperreactivity in a 30 murine model of allergic bronchial asthma.

WO 99/59632 PCT/IB98/00761 4 Therefore, the main object of the present invention is a method for treating allergic bronchial asthma by administering an effective amount of a product having TNF-RII(p75) agonist properties. A further object of the invention is a method for treating other allergic 5 conditions such as rhinoconjuctivitis, conjuctivitis, rhinitis, dermatitis, urticaria, anaphylaxis, and others by administering an effective amount of a product having TNF-RII(p75) agonist properties. A still further object of the invention is a method for treating Th2 mediated inflammation conditions by administering an effective amount of a 10 product having TNF-RII(p75) agonist properties. The term "product having TNF-RII(p75) agonist properties" means any molecule that is able to bind and activate the receptor, for example a monoclonal antibody, such as those described by the group of Prof. Wallach in Bigda et al.(J. Exp. Med., 180, 445-460, 1994), and, in particular, those which 15 bind to epitopes A, B and C of the p75 receptor , as well as those described in WO 94/09137. This term also encompasses polyclonal antibodies, proteins, peptides, peptoids, nucleic acids, chemical analogues of peptides and nucleic acids, as well as any organic molecule having a molecular weight similar to other organic molecules having pharmaceutical properties, and preferably between 20 100 and 30000 daltons. This term also encompasses prokaryotic or eukaryotic living cells, viruses, or any other TNF-RII(p75) agonist particles. Preferably, the TNF-RII(p75) agonist used in the present invention has essentially no TNF-RI(p55) agonist properties. An "effective amount" refers to an amount of the TNF-RII(p75) 25 agonist that is sufficient to affect the course and the severity of the allergic condition, bronchial asthma or Th2-mediated inflammation and to improve the patient's conditions, leading to the reduction or remission of the disease. The effective amount will depend on the route of administration and the condition of the patient.

WO 99/59632 PCT/IB98/00761 5 In the present invention, administration of the product having TNF Ril(p75) agonist properties can be enteral, parenteral, or preferably topical such as in aerosol formulations. In a first embodiment of the invention, the product having TNF 5 RII(p75) agonist properties is administered during an asthmatic, allergic, or Th2 mediated inflammation attack or exacerbation and said product is administered in an amount sufficient to alleviate the effects of said attack or exacerbation. In another embodiment of the invention, the product having TNF RII(p75) agonist properties is administered to a human or animal having an 10 underlying asthmatic, allergic, or Th2-mediated inflammation condition prior to the onset of an attack or exacerbation of the condition, in an amount effective to prevent or retard the onset of said attack or exacerbation. A further object of the present invention is a method for treating an asthmatic, allergic, or Th2-mediated inflammation condition in a human or an 15 animal, comprising modifying the balance between TNF-RII(p75) and TNF RI(p55) effectors. A still further object of the present invention is the use of the product having TNF-RII(p75) agonist properties together with a pharmaceutically acceptable carrier in the preparation of pharmaceutical compositions for the 20 treatment of allergic bronchial asthma, allergic conditions, and Th2-mediated inflammation conditions. "Pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered. For 25 example, for parenteral administration, the product having TNF-RII(p75) agonist properties may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution. Any mode of parenteral administration may be suitable, including intravenous intramuscular and subcutaneous administration.

WO 99/59632 PCT/IB98/00761 6 Besides the pharmaceutically acceptable carrier, the compositions of the invention can also comprise minor amounts of additives, such as stabilizers, excipients, buffers and preservatives. The combination with one or more pharmaceutically active products 5 is also possible. A further object of the present invention relates to the use of a product having TNF-RII(p75) agonist properties for modifying the balance between TNF-RII(p75) and TNF-RI(p55) effectors. A still further object of the present invention relates to a method for 10 screening products having TNF-RlI(p75) agonist properties comprising monitoring the inhibition of pulmonary inflammation occurring in humans or animals having pulmonary inflammation. Inhibition of pulmonary inflammation can be assessed by determining the level of cellular (eosinophils) and soluble (leukotriene) markers in broncho-alveolar lavage or in any other suitable body 15 fluid, by indirect measures of airway resistance, or any other suitable means. In addition, the determination of the levels of cellular (eosinophils) and soluble (leukotriene) markers in broncho-alveolar lavage or in any other suitable body fluid of the human or the animal may help to establish a suitable dosage of products having TNF-RII(p75) agonist properties for said human or 20 animal. The invention will now be illustrated by the following examples which make reference to the figures as described below. DESCRIPTION OF THE FIGURES 25 Figure 1 shows the lung hyperreactivity of OVA-treated BALB/c mice receiving increasing doses of MCh via a nebulizer. Figure 2 shows that no lung hyperreactivity can be demonstrated in p55TNFR~ ~ and WT mice after OVA-sensitization.

WO 99/59632 PCT/IB98/00761 7 Figure 3A (Total cell counts in BAL) and 3B (Differencial cell counts in BAL) show the total and differential cell counts in BAL fluid in p55TNFR' and WT mice after OVA-sensitization. Figure 4 shows lung hyperreactivity in p75TNFR~ ~ and WT mice after 5 OVA-sensitization. Figure 5A (Total cell counts in the BAL fluid) and 5B (Differencial cell counts in BAL) show the total and differential cell counts in BAL from p75TNFR' and C57BL/6 WT mice after OVA-sensitization. Table 1 presents a summary of the data obtained using TNFa -/- and 10 tmTNFk/l mice tested for lung hyperreactivity and inflammation. 0l IDC TITI ITr- IF irrT /IlhI f-t r n\ WO 99/59632 PCT/IB98/00761 8 EXAMPLES Materials and Methods: Animals. Female BALB/c or C57BL/6 mice (6-8 wk of age) were 5 purchased from Centre d'Elevage Janvier, Le Genest Saint-Isle, France. P55 deficient mice were obtained from Dr. K. Pfeffer (Munich); p75-deficient mice were purchased from Jackson laboratory. TNF&o and tmTNFk/l mice were obtained from G. Kollias (Athens). Wild-type mice (C57BL/6) of the same genetic background, sex and age, were used as controls. 10 Sensitization and intratracheal challenge with ovalbumin. Sensitization was performed using the method described by Hessel et al. {Hessel, van Oosterhout, et al. 1993 ID: 171. Mice were immunized by intraperitoneal (i.p.) injection of 10 mg ovalbumin (OVA; A-5503, Sigma Chemical Corp., St Louis, MO) in 0.1 ml NaCl (0.9% wt/vol) every other day for fourteen 15 days. Sham-sensitized mice received 0.1 ml of NaCl using the same protocol. On day 40 after the beginning of parenteral sensitization, mice were challenged by intranasal instillation of OVA, 50 ml of a 0.3 mg/ml solution. For this, mice were anaesthetized by inhaled 2% FORENE TM (isofluran, Abbott, Cham, Switzerland). Control mice were treated with 50 [i of PBS alone. This 20 procedure was repeated daily for 5 days. Lung hyperreactivity was assessed 24h after the last provocation, bronchoalveolar lavage (BAL) were done 2 days later. Bronchoalveolar lavages and differential cell counts. Three days after the third antigen challenge, mice were anesthetized with urethane and the tracheas cannulated. BAL fluid was collected by four repeated lavages with 0.4 25 ml of saline injected into the lung via the trachea. Differential cell counts were (TM) performed on cytospin preparations stained with Diff-Quick (Baxter Diagnostics, DOdingen, Switzerland). Two hundred cells were counted per field, with three fields per sample. Evaluation of allergen-induced airway hyperresponsiveness. 30 Airway responsiveness was measured by recording respiratory pressure curves by whole body plethysmography (Buxco@, EMKA Technologies, Paris, France) WO 99/59632 PCT/IB98/00761 9 in response to inhaled MCh (Aldrich-Chemie, Steinhein, Germany) at concentrations indicated in the figure legends. The period of nebulization was 20s. After each dose lung hyperreactivity was allowed to returned to baseline. This was typically in the order of 15 min. This method allowed the measurement 5 of spontaneous breathing in a non-restrained mouse. The airway reactivity was expressed in enhanced pause (Penh), a calculated value, which correlated with the measurement of airway resistance, impedance and intrapleural pressure in the same mouse. Penh = (Te/Tr-1) x Pef/Pif (Te = expiration time, Tr = relaxation time, Pef = peak expiratory flow, Pif = peak inspiratory flow) X 0.67 = coefficient 10 established by the manufacturer. Tr = relaxation time. The time it took for the box pressure to change from a maximum to a user defined percentage of the maximum. In this study, Tr measurement began at the maximum box pressure and ended at 40% {Chvatchko, Kosco-Vilbois, et al. 1996 ID: 3311. Results: 15 A) TNFa NEUTRALIZATION Figure 1 shows the lung reactivity of OVA-treated BALB/c mice receiving increasing doses of MCh via a nebulizer. Three groups are shown: 1) The control group: Balb/C without antibody treatment 20 2) Experimental group receiving 50mg anti-TNFca antibody 2 hours before each challenge 3) Experimental group receiving 50mg of isotype control. Hyperresponsiveness was monitored by plethismography and was expressed as maximum Penh values over 15 min after Mch exposure. p<0.05. 25 The curves show that Penh values were significantly increased in the anti-TNFc treated group in comparison to the non-treated and isotype control treated mice at doses from 1.5 to 6 x 10-2 M Mch, p<0.05.

WO 99/59632 PCT/IB98/00761 10 B) ANALYSIS OF LUNG HYPERREACTIVITY AND LUNG INFLAMMATION IN P55TNFR' AND P75TNFR~ ~ MICE Figure 2 shows lung reactivity in p55TNFR~ and WT mice after either NaCI or OVA-sensitization and challenge. Penh was monitored as 5 described in material and methods in response to 6 x10-2 M Mch. Results show that p55TNFR~ ~ animals do not develop lung hyperreactivity to MCh challenge. Figure 3 shows total and differential cell counts in BAL fluid in p55TNFR~ ~ and WT mice after either NaCl or OVA-sensitization and challenge. In Figure 3, legends are the following: 10 MAC, macrophages; EOS, eosinophils; LYM, lymphocytes; NEUT, neutrophils. NaCI challenge animals did not have eosinophilic infiltrates, in their lung. Eosinophils were present in large number in the lungs of C57BL/6 WT mice after challenge, but were absent in p55TNFR~ ~ animals lavage fluid. Results suggests that p55TNFR~ ~ animals do not respond to the 15 MCh challenge due to the absence of eosinophils in their BAL. Figure 4 shows lung reactivity in p75TNFR~ and C57BL/6 WT mice after either NaCl or OVA-sensitization and challenge. Penh was monitored as described in material and methods in response to indicated doses of MCh. Results show that p75TNFR'~ mice developed lung hyperreactivity in 20 the model of allergic asthma. Figure 5 shows total and differential cell counts in BAL from p75TNFR' and C57BL/6 WT mice after either NaCl or OVA-sensitization and challenge. Cells were identified on cytospin preparations after Diff-Quick staining 25 as macrophages (MAC), eosinophils (EOS), lymphocytes (LYM) and neutrophils (NEUT). Bars show mean +/- S.E.M. of 5 mice per group. Results show that there are more eosinophils in BAL from p75TNFR when compared to C57BL/6 WT mice indicating that there is more inflammation. The p75 TNF-R may have a protective role for lung inflammation.

WO 99/59632 PCT/IB98/00761 11 Table 1: Summary of data obtained using TNFa -/- and tmTNFk/l mice Wild-type, TNFa~ ~ and knock in mice for the membrane TNFa in 5 C57BI/6-129sv background were treated as indicated in Materials and Methods and tested for lung hyperreactivity in response to methacholine and for lung inflammation. OVA- BHR BAL sensitized Group Penh Eos Macro Lymp Mch 6x 10-2M Control 0.8 0.2 x 105 2.4 x 105 0.01 3x10 5 TNFa-/- 1.7 0 0.2x10 5 0.1 x10 5 tmTNF k/i 0.7 ND ND ND OVA- BHR BAL sensitized and Challenged Group Penh Total cells Eos Macro Lymp Mch 6x 10-2 M Control 3.4 0.5 x 105 0.4 x 105 0.2 x 105 0.041x10 5 TNFa-/- 4.8 1.2 x 105 0.4 x 105 0.7x1 0 0.096 x10 tmTNF k/i 1 0.77x 105 0.14x 105 0.5 x 105 0.1 x 105 WO 99/59632 PCT/IB98/00761 12 Conclusion : in C57BL/6 x 129sv over-expression of the membrane form of TNFca (and absence of soluble TNFa) protects the animal against MCh induced lung hyperreactivity and reduces lung inflammation, suggesting a 5 dominant role of p75 in suppressing TNF-mediated lung inflammation in this murine model of allergic asthma.

Claims (22)

1. A method for treating an allergic condition in a human or an animal, comprising administering to said human or animal an effective amount of a 5 product having TNF-RII(p75) agonist properties.

2. A method for treating a Th2-mediated inflammation condition in a human or an animal, comprising administering to said human or animal an effective amount of a product having TNF-RIl(p75) agonist properties.

3. A method for treating bronchial asthma or a pathophysiological equivalent 10 of bronchial asthma in a human or an animal, comprising administering to said human or animal an effective amount of a product having TNF-RII(p75) agonist properties.

4. The method of anyone of claims 1 to 3, in which said product having TNF RII(p75) agonist properties has essentially no TNF-RI(p55) agonist properties. 15

5. The method of anyone of claims 1 to 4, wherein said human or animal is suffering an asthmatic, allergic, or Th2-mediated inflammation attack or exacerbation and said product is administered in an amount sufficient to alleviate the effects of said attack or exacerbation.

6. The method of anyone of claims 1 to 4, wherein said product is 20 administered to a human or animal having an underlying asthmatic, allergic, or Th2-mediated inflammation condition prior to the onset of an attack or exacerbation of the condition in an amount effective to prevent or retard the onset of said attack or exacerbation.

7. The method of anyone of claims 1 to 6, in which said product is 25 administered enterally.

8. The method of anyone of claims 1 to 6, in which said product is administered parenterally. WO 99/59632 PCT/IB98/00761 14

9. The method of anyone of claims 1 to 6, in which said product is administered topically.

10. The method of anyone of claims 1 to 9, in which said product is a monoclonal or a polyclonal antibody. 5

11. The method of anyone of claims 1 to 9, in which said product is a protein, a peptide, a peptoid, or a nucleic acid.

12. The method of anyone of claims 1 to 9, in which said product is a chemical analogue of a peptide or a chemical analogue of a nucleic acid.

13. The method of anyone of claims 1 to 9, in which said product is an 10 organic molecule having a molecular weight similar to other organic molecules having pharmaceutical properties.

14. The method of anyone of claims 1 to 9, in which said product is an organic molecule having a molecular weight between 100 and 30000 daltons.

15. The method of anyone of claims 1 to 9, in which said product is a 15 prokaryotic or eukaryotic living cell, a virus, or any other TNF-RII(p75) agonist particle.

16. A method for treating an asthmatic, allergic, or Th2-mediated inflammation condition in a human or an animal, comprising modifying the balance between TNF-RIl(p75) and TNF-RI(p55) effectors. 20

17. The use of a product having TNF-RII(p75) agonist properties together with a pharmaceutically acceptable carrier for the preparation of a pharmaceutical composition for the treatment of an allergic condition in a human or an animal.

18. The use of a product having TNF-RIl(p75) agonist properties together 25 with a pharmaceutically acceptable carrier for the preparation of a pharmaceutical composition for the treatment of a Th2-mediated inflammation condition in a human or an animal, comprising administering to said human or animal an effective amount of a product having TNF-RII(p75) agonist properties. WO 99/59632 PCT/IB98/00761 15

19. The use of a product having TNF-RII(p75) agonist properties for the preparation of a pharmaceutical composition for the treatment of bronchial asthma or of a pathophysiological equivalent of bronchial asthma in a human or an animal. 5

20. The use according to anyone of claims 17 to 19, in which said product having TNF-RII(p75)agonist properties has essentially no TNF-RI(p55) agonist properties.

21. The use of a product having TNF-RII(p75) agonist properties for modifying the balance between TNF-RIl(p75) and TNF-RI(p55) effectors. 10

22. A method for screening products having TNF-RII(p75) agonist properties comprising monitoring the inhibition of pulmonary inflammation occurring in humans or animals having pulmonary inflammation.