MXPA98009301A - Tnfr-ig for the treatment of a - Google Patents

Abstract

The present invention relates to a method for combating asthma with a composition containing an effective amount of a chimeric TNF-alpha binding protein that includes the soluble portion of the p55 TNF receptor and all domains except the first domain of the region heavy chain constant of human IgG1 or IgG3, and the use of said chimeric TNF-alpha binding protein for the preparation of a medicament for the treatment of ace

Description

TREATMENT OF ASTHMA WITH TNFR-Ig Field of Invention The invention is directed to a method for combating asthma in patients suffering from an asthmatic state, by administering to the patient a composition containing a preparation composed of one or more chimeric TNFa binding proteins.

Background of the Invention Asthma is a chronic inflammatory disease of the respiratory tract that is characterized by recurrent exacerbations due to exposure to specific allergens (response mediated by IgE), exercise, cold air or stress. The distinctive aspects of inflammation associated with asthmatic disease are the presence of activated eosinophils, an increased sensitivity of the respiratory tract with respect to non-specific stimuli (airway hyperresponsiveness: AHR), edema, mucus hypersecretion and cough. It is believed that this process REF. : 28763 inflammatory is mediated in part by the generation and activation of Th2-type lymphocytes, which secrete a variety of cytokines. The cytokine TNFa is a cytokine involved in the appearance of asthmatic states.

Numerous therapeutic agents are used for the treatment of asthma, being the inhaled β2 agonists (relief of acute bronchospasm) and the steroids (inflammatory a t i) the drugs of choice. However, none of these regimens is considered adequate, since the mortality associated with the disease is increasing. One of the most urgent medical needs that is yet to be achieved is the availability of an agent that can rapidly reverse the severe progressive lung inflammation that occurs in severe or acute asthma. Additionally, none of the therapies can be classified as a disease modifying agent. There is certainly a need for an agent that can rapidly reverse the inflammatory response observed in severe or acute asthma, as well as an agent that actually modifies the disease. The actions of TNFa stimulate the appearance of asthmatic states, and the applicants have determined that blocking the action of TNFa provides a means to alleviate such conditions.

Description of the invention.

Applicants have discovered here a method for combating asthma in patients suffering from an asthmatic state, which method includes administering to said patient a composition containing a preparation composed of one or more chimeric TNAa binding proteins, each of which proteins in said preparation is composed of the soluble portion of the p55 TNF receptor protein fused to an IgG, wherein said fused IgG contains all the IgG domains except the first IgG domain of the chain constant region of the IgG, said composition containing a therapeutically inert pharmaceutical vector, and said preparation being administered to said patient for the purpose of supplying said patient with an effective amount of said chimeric protein preparation, with the objective of combating said asthmatic state.

The composition can be effectively administered to a patient suffering from an asthmatic attack, so that the chimeric protein preparations are administered in an amount sufficient to alleviate the effects of such an attack. The composition can also be administered to an asthmatic patient before the onset of the asthmatic attack, in an amount effective to prevent or delay the onset of said asthma.

The invention is directed to a method for combating asthma in patients suffering from an asthmatic state, by administering to the patient a composition containing a preparation composed of one or more chimeric TNFa binding proteins. The proteins in the preparation are composed of the soluble portion of the p55 TNF receptor protein fused to an IgG (immunoglobulin G), which contains all the domains except the first domain of the heavy chain constant region of the IgG (TNFR-). Ig). The composition also contains a therapeutically inert pharmaceutical vector. The composition is administered to the patient in order to provide the patient with an effective amount of the chimeric protein preparation, with the objective of combating said asthmatic state.

The use of a chimeric TNFa binding protein, composed of the soluble portion of the p55 TNF receptor protein fused to an IgG, wherein said fused IgG contains all the IgG domains except the first IgG domain of the constant region of the heavy chain of IgG, for the preparation of a medicament for the treatment of asthma constitutes a further object of the present invention.

The composition can be effectively administered to a patient suffering from an asthmatic attack, in a sufficient amount of protein preparation to alleviate the effects of the asthma attack. The composition can also be administered to an asthmatic patient before the onset of the asthmatic attack, in a quantity of protein preparation effective to prevent or delay the onset of the attack.

Any TNFR-Ig, that is, any chimeric TNFa binding protein, composed of the soluble portion of the p55 TNF receptor protein fused to an IgG, of which said fused IgG contains all of the IgG domains except the first IgG domain. IgG of the constant region of the heavy chain of the IgG, can be used in a preparation of this invention to combat asthma in patients with an asthmatic state as described in the previous paragraph. IgG can be human IgG1 or IgG3, IgG1 being preferred in this invention.

Examples of such TNFR-Igs include the proteins discovered in (EP 417 563, U.S. Patent Nos: 5,447,851 and 5,395,760, Losslauer et al., Eur. J. Immunol., 21 (11): 2883, 1991; Loetscher et al., J Biol. Chem. 266 (27): 18324, 1991; Ashkenazi et al Proc. Nati, Acad. Sci. (USA) 88: 10535, 1991), which can be obtained by the methods also discovered in these publications.

A preferred preparation of TNFR-Ig molecules of this invention is made with IgGl, and they contain proteins that possess a complex oligosarcharide terminated in one or more residues of sialic acid, and that have N-acetylglucosamine exposed, the molar ratio of acid residues being sialic in the preparation of about 4 to about 7 moles of sialic acid per mole of protein, in particular from about 5 to about 6, with the molar ratio of N-acetylglucosamine being exposed in the preparation of about 1 to about 2 moles of N-acetyl glucosamine per mole of protein, and the mole ratio of sialic acid residues to N-acetylglucosamine residues in the preparation being from about 0.35 to about 0.5, in particular from about 0.4 to about 0.45. The preparation has an isoelectric point (pl) of about 5.5 to about 7.5, which can be determined by chromatofocusing, and which is sensitive to treatment with neuraminidase.

The TNFR-Ig of this invention can be obtained by conventional methods of recombinant technology or by protein synthesis. The DNA encoding the p55 TNF receptor, the DNA encoding all the domains except the first of the constant region of the heavy chain of an IgG1 or IgG3, as well as the methods of ligation of said sequences together for expression in a suitable vector , are part of the knowledge of an expert and are described in the literature. Suitable cloning vectors and host cells are well known and can be selected by a skilled person, and suitable culture conditions can be determined (see Animal Cell Culture: A Practical Approach, 2nd Ed., Rick O'od and Hames eds., Oxford University Press, NY 1992).

TNFR-Ig can be purified using known methods of protein recovery and purification. The TNFR-Ig is preferably recovered from culture in the form of a secreted polypeptide, although it can also be recovered from lysates of host cells. The culture medium or lysate can be centrifuged to remove particulate cellular debris. Next, the TNFR-Ig is purified from contaminating soluble proteins and polypeptides, the following procedure being examples of suitable purification procedures: by fractionation in immunoaffinity columns or ion interchange; precipitation with ethanol; Reverse phase HPLC; chromatography on silica or a cationic-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; precipitation with ammonium sulfate; gel filtration using for example Sephadez G-75; and Protein A-Sepharose columns to remove contaminants such as IgG. It may also be useful to include a protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) to inhibit proteolytic degradation during purification. A person skilled in the art will appreciate that suitable purification methods for the polypeptide of interest may require a modification depending on the character of the polypeptide upon expression in the culture of recombinant cells.

The TNFR-Ig of this invention with the proportions of sialic acid and N-acetylglucosamine and the specific pl can be obtained by using recombinant mammalian cells expressing TNFR-Ig, and controlling the culture conditions of mammalian cells - under which produce TNFR-Ig as described, for example, in WO 96/39488. The selected host cells should be able to bind N- or O- linked carbohydrates, including sialic acid, to the proteins they will express. An example of such a host cell is a CHO cell. Suitable culture conditions are well known, and depend on the selected host cell. The increase in the specific productivity of the cell during the production phase of glycoproteins results in a decrease in the sialic acid content of the mature protein, and vice versa. Factors that affect the specific productivity of the cell are well known in the art, and include, but are not limited to, factors that affect the number of DNA / RNA copies, factors that affect RNA, such as which stabilize the RNA, nutrients and other supplements of the medium, as well as the concentration of transcription stimulators, the osmolality of the culture medium, the temperature and pH of the cell culture, and the like. These factors can be adjusted by well-known methods to obtain a preferred content of glycoprotein such as sialic acid. The variation in cell productivity specifies the production phase of the cell culture by the addition of an alkanoic acid such as sodium butyrate or a salt derived to the cell culture at a concentration of about 0.1 mM to about 20 mM, or 5 to 20 mM, and maintaining an osmolality of the cell culture of approximately between 250 and 600 mOsm, optionally by combining the one and the other method during the transition phase produces a protein with different amounts of sialic acid. A concentration of about 6.0 mM sodium butyrate and conditions of 350 to 400 mOsm provide a TNF-Ig of this invention highly sialylated, while a concentration of about 12 mM sodium butyrate provides, a TNFR-Ig of this invention less sialylated The latter concentration can be combined with conditions of 450 to 550 mOsm.

The experienced practitioner will recognize that the osmolality of the medium depends on the concentration of osmotically active particles in the culture fluid, and that various variables involved in a culture medium of complex mammalian cells have an impact on osmolality. The initial osmolality of the culture medium is determined by the composition of the culture medium. Osmolality can be measured using an osmometer, such as that marketed by Fisher Scientific, Pittsburg, Pennsylvania, under the trade designation OSMETTE (or Osmette model 2007, supplied by Precision Systems, Inc., Natick, MA), for example. In order to achieve an osmolality in the desired range, the concentration of various ingredients of the culture medium can be adjusted. The solutes that can be added to the culture medium with the aim of increasing the osmolality thereof include proteins, peptides, amino acids, hydrolyzed animal proteins such as peptones, non-metabolized polymers, vitamins, ions, salts, sugars (in particular glucose), metabolites , organic acids, lipids and the like. In one aspect, osmolality is controlled by the addition of a peptone to the cell culture together with other components of the culture medium during a batch culture procedure.

Three phases of cell culture can be employed, a growth phase during which cell growth in a mammalian host cell is maximized, followed by a transition phase in which cell culture parameters as described above for the content desired in sialic acid from the mature glycoprotein are selected and applied, followed by a production phase in which the parameters selected in the transition phase are maintained and the product glycoprotein is produced and recovered. In relation to the preferred purification in the context of the present invention are the purification processes and techniques that select the carbohydrates of the invention. The desired glycoforms of the present invention can be enriched in molecules containing sialic acid by, for example, ion-exchange gel-gel chromatography or HPLC using cationic or anionic exchange resins, in which the most acidic fraction is collected.

The preferred TNFR-Igs of this invention possess one or more of the following characteristics in the purified composition: the pl range of the TNFR-Ig composition is between about 5.5 and 7.5, the molar ratio of sialic acid with respect to protein is from about 4 to about 7 and especially from about 5 to about 6, having from about the 2 moles of exposed residues of N-acetylglucosamine per mole of protein, having a molar ratio of sialic acid to N-acetylglucosamine from about 0.35 to about 0.5 and more preferably about 0.4 to about 0.45. The most suitable conditions for obtaining said preferred TNFR-Ig employ culture conditions using sodium butyrate at a concentration of about 0.1 mM to about 6 mM, the osmolality being maintained between about 300 and 450 mOsm, and a temperature between about 30 ° C and 37 ° C.

The determination of the above characteristics of a TNFR-Ig can be carried out by an experienced person using well-known techniques. For example, the complex carbohydrate portion of the glycoprotein produced by the processes of the present invention can be easily analyzed, if desired, by conventional carbohydrate analysis techniques. Thus, for example, techniques such as the transfer of lectins, well known in the field, reveal proportions of terminal masses or other sugars such as galactose. The termination of mono-, bi-, tri-, or tert-aninated oligosaccharides with sialic acids can be confirmed by the release of sugars from the protein using anhydrous hydrazine or enzymatic methods, and by fractionation of the oligosaccharides by chromatography of ion exchange or size exclusion, or by other methods well known in the field. The Pi of the glycoprotein can also be measured before and after neuraminidase treatment to eliminate sialic acids. An increase in the pl after treatment with neuraminidase indicates the presence of sialic acids in the glycoprotein.

The structures of the carbohydrates of the present invention appear in the protein in the form of carbohydrates linked by N- or by 0-. The carbohydrates bound by N- or by O- differ mainly in the structures of their nuclei. N-linked glycosylation refers to the binding of the carbohydrate moiety via GlcNac to an asparagine residue in the peptide chain. All the carbohydrates bound by N-contain a common nuclear structure of Manl-6 (Man 1-3) Manßl-4GlcNacßl-4GlcNacß-R. Accordingly, in the nuclear structure described, R represents an asparagine residue of the protein produced. The peptide sequence of the produced protein will contain an asparagine-X-serine, asparagine-X-threonine or asparagine-X-cis-tein, in which X is any amino acid except proline. On the other hand, the carbohydrates bound by O- are characterized by a common nuclear structure, with a GalNAc linked to the hydroxyl group of a threonine or a serine. Among the carbohydrates bound by N- or by O-, the most important are the complex carbohydrates bound by N- or by O-. Such complex carbohydrates will contain various aerial structures. The mono-, bi-, tri- and tetra-antennas external structures are important for the addition of terminal sialic acids. Such external chain structures provide additional sites for specific sugars and linkers that comprise the carbohydrates of the present invention.

The resulting carbohydrates can be analyzed by any method known in the field, including the methods described herein. Various methods are known in the field for the analysis of glycosylation, which are useful in the context of the present invention. Such methods provide information regarding the identity and composition of the oligosaccharide attached to the peptide. Methods for the analysis of carbohydrates useful in the present invention include, but are not limited to, lectin chromatography; HPAEC-PAD, which uses an anion exchange chromatography at high pH to separate the oligosaccharides based on their charge; NMR; mass spectrometry, HPLC; GC; analysis of the composition of monosaccharides; Sequential enzymatic digestion.

Additionally, methods for cleavage of oligosaccharides are known. These methods include: 1) enzymatic, which are normally carried out using peptido-N-glycosidase F / endo-β-galactosidase; 2) elimination using a strong alkaline medium to release mainly the structures bonded by 0-; and 3) chemical methods that employ anhydrous hydrazine to release both the O-linked oligosaccharides and those bound by N-.

The analyzes can be carried out following the following stages: 1. Dialysis of the sample against deionized water, to eliminate all buffering salts, followed by lyophilization. 2. Release of intact oligosaccharide chains with anhydrous hydrazine. 3. Treatment of the intact oligosaccharide chains with anhydrous methanolic HCl to release the individual monosaccharides as O-methyl derivatives. 4. N-acetylation of all primary amino groups.

. Derivatization to yield the 0-trimethylsilyl methyl glycosides. 6. Separation of these derivatives by capillary GLC (gas-liquid chromatography) on a CP-S IL8 column. 7. Identification of the individual glycoside derivatives by the retention time from the GLC and mass spectroscopy, in comparison with known patterns.

Quantification of individual derivatives by FID with an internal standard (13-0-methyl-D-glucose).

The neutral and aminic sugars can be determined by high resolution anion exchange chromatography in combination with pulsed amperometric detection (HPAE-PAD Carbohydrate System, Dionex Corp.). for example, sugars can be released by hydrolysis in 20% (v / v) trifluoroacetic acid at 100 ° C for 6 hours. The hydrolysates are they are then dried by lyophilization or by a Speed-Vac (Savant Instruments). The residues are then dissolved in a 1% sodium acetate trihydrate solution and analyzed on an HPLC-AS6 column as described in Anumula et al. (Anal. Biochem. 195: 269-280 (1991)).

The sialic acids can be determined separately by the direct colorimetric method of Yao et al. (Anal. Biochem. 179: 332-335 (1989)) in samples in triplicate. In a preferred aspect, thyric thiobarbi acid (TBA) is used from Warren, L., J. Biol. Chem. 238 (8) (1959).

Alternatively, a carbohydrate analysis can be carried out by immuno transfer. • According to this method, protein-bound carbohydrates are detected using a commercial glycan detection system (Boehringer), which is based on the oxidative immunoblot procedure described by Haselbeck and Hosel (Haselbeck et al., Glycoconj ugate J., 7: 63 (1990)). The staining protocol recommended by the manufacturer is followed, except that the protein is transferred to a polyvinylidene difluoride membrane instead of a nitrocellulose membrane, and that the blocking buffers contain 5% bovine serum albumin. in 10 mM Tris-HCl buffer, pH 7.4, with 0.9% sodium chloride. Detection is carried out with anti-digoxigenin antibodies bound to an alkaline phosphatase conjugate (Boehringer), at a 1: 1000 dilution in tris-buffered saline, employing the phosphatase substrates of 4-nitroblue tetrazolium chloride, 0, 03% (w / v) and 0.03% (w / v) 5-bromo-4-chloro-3-indolyl-phosphate in 100 mM tris buffer, pH 9.5, containing 100 M sodium chloride and magnesium chloride 50 mM. Protein bands containing carbohydrate are normally visualized in approximately 10 to 15 minutes.

The carbohydrates can also be analyzed by digestion with peptido-N-glycosidase F. According to this procedure, the residue is suspended in 14 μl of a buffer containing 0.18% SDS, 10 mM beta-mercaptoethanol, 90 mM phosphate and EDTA 3, 6 mM, at pH 8.6, and heating at 100 ° C for 3 minutes. After cooling to room temperature, the sample is divided into two equal aliquots. An aliquot is not treated anymore and is used as a control. The second fraction is adjusted to a detergent concentration of about 1% NP-40, followed by 0.2 units of peptido-N-glycosidase F (Boehringer). Both samples are heated at 37 ° C for 2 hours, and then analyzed by polyacrylamide-SDS gel electrophoresis.

The TNFR-Ig of the present invention comprises PEGylated TNFR-γg, which designates a molecule of TNFR-Ig which has been covalently conjugated to a polymer such as polyalkylene glycol (substituted or unsubstituted), in particular a polyethylene glycol. The conjugation can be direct, although it is preferably carried out by various coupling agents known in the field, such as, for example, the agents discovered in PE 510346, PE 593838, United States Patent Numbers 4,766,106, 4,917,888, 4,902,502, 4,847,325, 4,179,337, 5,832,657, Veronese et al., Applied Biochem. And Biotech. 11: 141 (1985), and Monfardini et al., Bioconj ugate Chem. 6:62 (1995). PEGylated TNFR-Ig can be used in the treatment and prevention of asthma in the same way as described for TNFR-Ig.

The TNFR-Ig preparations of this invention are useful for combating asthma in asthmatic patients. In the treatment of patients suffering from an asthma attack, these preparations can alleviate the effects of the attack, reversing the inflammation that has already occurred, and preventing the appearance of new inflammations. When administered prophylactically, these preparations can delay or prevent the onset of an asthma attack, or, in the event of an attack, ensure that the attack is of lesser severity.

In accordance with this invention, the preparations can be administered for the treatment or prophylaxis of a patient in any conventional manner. In this way the preparation of this invention can be administered in conventional pharmaceutical compositions. said compositions including any conventional therapeutically inert pharmaceutical vector. The pharmaceutical compositions may contain inert additives as well as pharmacodynamically active additives. The liquid compositions may be in the form for example of a sterile solution that is miscible with water. Additionally, substances conventionally employed as preservatives, stabilizers, moisture retainers, and emulsifiers, as well as substances such as salts for varying the osmotic pressure, pH-varying substances such as buffers, and other additives may also be present. If desired, an antioxidant such as tocopherol, N-methyl-gamma-tocophermin, butylated sun hydroxyani may be included in the pharmaceutical compositions. Pharmaceutically acceptable excipients or pharmaceutical vectors for the compositions include saline, buffered saline, glucose or water. The compositions may also contain stabilizing agents such as sugars, including mannose and mannitol, and local anesthetics for injectable compositions, including for example lidocaine. The substances and diluents as pharmaceutical vectors mentioned above can be organic or inorganic substances, such as for example water, gelatin, lactose, starch, magnesium stearate, talc, gum arabic, polyalkylene glycol and the like. A prerequisite is that all adjuvants and substances used in the manufacture of the pharmaceutical compositions are non-toxic.

The preparations of this invention can be administered parenterally (for example, by subcutaneous, intravenous, intramuscular, intharboric, t racapsular, or intimate injection), or by spray inhalation. Any conventional pharmaceutical preparation for parenteral administration or spray inhalation may be employed. Examples of suitable pharmaceutical compositions are injection or infusion solutions. A preferred mode of administration is intravenous injection. Another preferred mode of administration is aerosol.

Any effective amount of the preparations of this invention for combating asthma can be used to alleviate or reverse the effects of an attack or to prevent or delay the onset of an attack. A preferred dose of a composition of TNFR-Ig for parenteral administration (injection) of this invention for the treatment, prevention or reversal of asthma provides from about 0.1 to about 5.0 mg of preparation of TNFR-Ig per kg of body weight (mg / kg) per patient. A particularly preferred dose from about 1.0 to about 3.0 mg / kg. A particularly preferred dose of a TNFR-Tg composition for the same purpose and in spray administration provides from 0.03% to 5.0% by weight of TNFR-Ig preparation. In either mode of administration, the treatment dose may be administered once per day of treatment, or it may be divided into smaller doses administered over a period of time of approximately 24 hours to achieve the total dose to be administered.

For the immediate treatment of an asthma attack by parenteral administration, a single dose of 0.1 to 5.0, especially 1.0 to 3.0 mg / kg per patient per day may be administered. For the prophylaxis of asthma by parenteral administration, such a dose of 0.1 to 5.0, especially 1.0 to 3.0 mg / kg, would be preferably delivered at a frequency of daily, but preferably twice a week. once a week, once a month, or another intermediate frequency, depending on the patient and his condition. Similarly, for the immediate treatment of an asthma attack by spray administration, a single dose per day can be inhaled. For prophylaxis, said dose could be inhaled daily, but preferably from twice a week to once a week, once a month, or another intermediate frequency, depending on the patient and his condition. The exact doses in which the composition is administered may vary depending on the type of use, the mode of use and the requirements of the patient, as determined by an experienced practitioner. The exact dose for a patient can be adapted specifically by an experienced person depending on the severity of the condition, the specific formulation used and other drugs that may be involved.

The spray compositions of this invention may be liquid or powdered. They can be administered through the nose or mouth (nasally or orally). An example of a spray composition is an atomized spray which can be directed directly to the nose or mouth, producing from a spray atomizer by mechanical action on a solution in water or in saline of preparations of TNFR-Ig. Another example is a nebulized spray, whereby a vapor is produced from a solution such that it is then actively inhaled by the patient (instead of being sprayed directly onto the nose or mouth). If the TNFR-Ig is in powder form, the particles must be large enough to be deposited in the respiratory tract instead of exhaled by the patient after inhalation. Depending on the medical regimen, immediate treatment or prophylaxis, such a spray can be inhaled once or more times a day, or less frequently as recommended to the patient. For spray administration, the patient is administered by inhalation an amount of a TNFR-Ig of this invention effective for the treatment or prevention of asthma as described above. The affected portions of the respiratory tract (nasal cavities, trachea, bronchi, lower airways, or bronchioles) receive an adequate amount of TNFR-Ig by this means for asthma.

The spray formulations of this invention preferably provide from about 0.03% to 5.0% by weight of the preparation, more preferably from about 0.03% to 0.5%, especially from about 0.1. % to 0.3%. A preferred spray formulation is an aerosol formulation. In general, lower dosing ranges are preferred for sprays such as fogging formulations, whereas larger dosage intervals are preferred for aerosols. Any conventional aerosol formulation can be employed in this invention to deliver an effective amount of the TNFR-Ig preparation, which preferably provides the preparation amounts described above. The preparation is most effectively administered by releasing a measured dose of aerosol vapor into the patient's mouth while inhaling, taking the vapor into the mouth and through the airways to the lungs. As described above, the effective amount can be administered orally or nasally at one time or in smaller doses throughout the day, and be administered in a daily dose for immediate treatment, or at weekly or monthly intervals for prophylaxis.

A suitable aerosol formulation includes a suitable dose of the pharmaceutically active compound (e.g. TNFR-Ig preparation) and an effective amount of any conventional aerosol propellant. Any conventional agent with surface activity can also be included. The TNFR-Ig can be combined with the propellant in the form of a liquid in solution or as a powder (for example in lyophilized form by means of well known methods). The propellant can be liquefied properly.

Those agents with surface activity that are soluble or dispersible in the propellant are more effective. The agents with the most soluble surface activity in the propellant are the most effective. It is also important that the agent with surface activity is neither irritating nor toxic.

Any conventional propellant acceptable for use in a pharmaceutical composition known to an experienced person may be employed, liquefied or in any other way. The propellant may be one suitable for use with an active ingredient in powder form or suitable for use with a liquid active ingredient, depending on whether the TNFR-Ig is in powder or liquid form. The liquefied propellant used can be one that is in the form of gas at room temperature (18 ° C) and at atmospheric pressure (760 mm of mercury), that is, it can have a boiling point below 18 ° C at atmospheric pressure, and it is not toxic. Among the suitable liquefied propellants that can be employed are lower alkanes containing up to five carbons, such as butane and pentane. Fluorinated and f 1-chlorinated lower alkanes such as those sold under the trade name "Freon" are examples of liquefied propellants. Mixtures of the propellants mentioned above can be used suitably.

Having described the invention in general, it will be more readily understood by reference to the following examples, which are presented by way of illustration, and are not intended to limit the present invention, unless otherwise specified.

In the Example 1: Plasmid coding for TNFR-Ig for use in the production of TNFR-Ig Cellphone line The Chinese hamster ovary (CHO) cell line used as the mammalian host cell line was derived from CH0-K1 (ATCC Number CCL61 CHO-K1). A CHO-K1 mutant cell line deficient in dihydrofolate reductase (DHFR ") called CHO-Kl DUX-Bll (DHFR") (obtained from Dr. L. Chasin of Columbia University; Simonsen, CC, and Levinson, AD, (1983) Proc. Nati, Acad. Sci. USA 80: 2495-2499, Uriaub G., and Chasin, L., (1980) Proc. Nati, Acad. Sci. USA 77: 4216-4220) was used below to obtain a cell line with reduced insulin requirement by transfection with the vector containing the cDNA of preproinsulin (Sures et al., (1980) Science, 208: 57-59). The selected clone, called dpl2.CHO, requires glycine, hypoxanthine and thymidine for its growth, which verifies its DHFR genotype ".

B. Construction of soluble TNFR-IgGj type 1 chimera A soluble type 1 TNFR-IgGi chimera was constructed by gene fusion of the extracellular domain of human type 1 TNFR with the hinge region and the CH2 and CH3 domains of the heavy chain IgG? (hereinafter referred to as TNFRl-IgGi). Alternatively, the hinge region and the CH2 and CH3 domains of the IgG3 heavy chain can also be used.

The DNA sequence encoding human type-1 TNFR (see Loetscher et al., Supra) was obtained from the pRK-TNF-R plasmid (Schall et al., Cell 61, 361 (1990)). To construct this starting plasmid, a 2.1 kb placental cDNA clone (Schall et al., Supra) was inserted into the mammalian expression vector pRK5, the construction of which is described in EP Publication Number 307,247 . This DNA is initiated at the nucleotide position 64 of the sequence described by Loetscher et al., With the start methionine 118 bp downstream.

The source of the IgGi coding sequence was the CD4-IgG expression plasmid Prkcd42Fc? (Capón, DJ et al., Nature 337, 525 (1989); Byrn et al., Nature 344, 667 (1990)) which contains the cDNA sequence encoding a hybrid polypeptide consisting of residues 1-180 of the Mature human CD4 protein (two N-terminal variable domains of CD4) fused to IgG1 sequences that start in aspartic acid 216 (considering amino acid 114 as the first residue of the constant region of the heavy chain (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition (1987), which is the first residue of the IgGl hinge after the cysteine residue involved in the binding of the heavy and light chains), and which ends in Reissue 441, to include the Fc CH2 and CH3 domains of IgGi. See also PE 227110. Alternatively, the vector PCD4-HY3 (DSM 5523, PE 394,827) can be used. The CD4 cDNA is removed by cleavage with SstI to obtain the desired IgG3 sequences.

TNFR1-IgGi was constructed by generating restriction fragments from the plasmids pRK-TNF-R and pRKCD42Fc ?, and ligating them, using deletion mutagenesis, so that the threonine residue 171 of the mature TNFR is superimposed on the acid residue Aspartic 216 of the IgGi heavy chain (Kabat et al., supra). The resulting plasmid pRKTNFR-IgG contained the complete coding sequence of TNFRi-IgGi. This plasmid can then be transfected into a host cell such as a CHO cell by conventional methods such as calcium phosphate precipitation. The resulting cells can be cultured by conventional methods to express TNFR-Ig, which can be isolated by conventional methods.

Example 2: Production of TNFR-Ig having approximately: from 5 to 6 m of sialic acid per protein, from 0.4 to 0.45 M of N-acetylglucosamine per protein, pl from 5.5 to 7.5 Cell culture The gene of Example 1 encoding soluble TNFR-IgGi type 1 was introduced into dpl2.CHO cells by transfection. This was carried out by the calcium phosphate technique to introduce DNA into mammalian cells. Two days after transfection, the cells were rinsed and replated in selective medium (Ham DM F-12 free of glycine-hypoxantine and thymidine, 1: 1 with 2% v / v of dialyzed serum). . Subsequent isolates were screened for the secretion of TNFRl-IgG ?. Clones expressing TMFR1-IgGi were amplified in methotrexate, obtaining high expression clones, which were subsequently adapted to medium without serum. The cells were maintained under continuous selective pressure until transfer to a nonselective medium for growth and expansion of the inoculum.

In order to provide cells for TNFRl-IgGi production cultures, the cell population described above was expanded from media containing methotrexate by serial subcultures in recipients of increasing volumes to growth medium without methotrexate. For these stages of the process, the non-selective growth medium was a formulation based on DMEM / HAM F-12 (see US Pat. No. 5,122,496, for example), with modified concentrations of some components, such as glucose, amino acids, salts, sugar, vitamins, glycine, hypoxanthine and thymidine; recombinant human insulin, hydrolyzed peptone (Primatone HS or Primatone RL), a cellular protective agent such as Pluronic F68 (pluronic polyol) or equivalent; gentamicin; Lipids and trace elements.

The cultures were controlled at pH 7.2 ± 0.4 using the C02 (acid) and / or Na2CO3 (base) gas. The temperature was controlled at approximately 37 ° C during the growth period. The dissolved oxygen was maintained above 5% of the air saturation by direct injection of air and / or gaseous oxygen. The osmolality during the expansion phase of the inoculum was maintained between approximately 250 mOsm and 350 mOsm.

The growth phase for each crop was followed by a second phase or transition phase, in which the crop parameters were changed from optimal growth to production conditions. During this transition phase the temperature of the culture system was lowered, generally to approximately between 30 and 35 ° C. Butyrate was added, and a certain range of osmolality was maintained. The product accumulated during this production phase was analyzed for its sialic acid content.

In a typical production program, approximately 1.2 x 106 cells derived from the inoculum expansion of the selection stage were grown in a growth phase with an initial osmolality of 300-450 mOsm, preferably around 300. The medium of growth was supplemented with trace elements, recombinant human insulin and hydrolysed peptone. The cells were grown under these conditions for 2 days. At the beginning of the third day the temperature of the cell culture was lowered. Simultaneously to, or after, the temperature drop, approximately 1 to 6 mmol of sodium butyrate, preferably 6 mmol, were added to the cell culture, and the desired production osmolality was maintained by the addition of various components of the medium. The cells were grown under these conditions with nutrient supply for 9 to 10 days. Various components of the medium were delivered to the cells when necessary.

The pl of a highly sialylated composition is less than the pl of a poorly sialylated composition. An isoelectric focus was made for the composition. The isoelectric focusing gels separate the glycoproteins from the composition according to their isoelectric point, pl, using a pH gradient created with ampholytes of different pH. The analysis of the composition was carried out using a pH gradient of 10 to 4.

The above composition shows an isoelectric point range of about 5.5 to about 7.5, determined by isoelectric focusing, in which the pl is sensitive to the neuraminidase treatment.

Recovery of TNFR-IgG As described in Capón et al., Nature 337: 525, 1989, the IgG fusion proteins can be purified by centrifugation of the cell culture and processing of the supernatant of the resulting culture by a protein A column. Therefore, culture The cell obtained previously was centrifuged and the supernatant was applied to a protein A column. The preparation of TNFRl-Ig was purified to a homogeneity greater than 95% by this affinity chromatography on immobilized Protein A of Staphylococcus aureus, as described in Capón et al., supra.

Carbohydrate analysis A. Sialic acid content can be analyzed by the method of Warren, L. (1959), J. Biol. Chem. 234: 1971-1975.

B. The release of amino-sugars and neutral infected sugars can be determined by high-anion exchange chromatography at high pH in combination with pulsed amperometric detection. The analysis was carried out by the following steps: 1. A buffer change was made for TNFRl-Igd (approximately 50 μg / ml) and the appropriate reference samples, so that the final sample was contained in 1% acetic acid. 2. Approximately 90 μg of TNFRl-IgGx together with samples of the reference materials were frozen in a dry ice and alcohol bath, and the frozen sample was lyophilized overnight. The lyophilized samples were reconstituted in 500 μl of trifluoroacetic acid and incubated at 120 ° C for 1 hour. After acid hydrolysis, the samples of TNFRl-IgGi and the reference samples were cooled and evaporated to dryness. The samples were reconstituted in water to a final concentration of approximately 0.6 mg / ml. The separation of the monosaccharides was carried out at room temperature by anion exchange chromatography at high pH with pulsed amperometric detection using a Dionex CarboPac PAl column. (4 x 250 mm) (Dionex Corp., Sunnyvale, CA, USA UU ). The quantification of the individual monosaccharides was performed by comparison with reference monosaccharides.

Example 3. Preparation of TNFR-Ig 1. The CHO DP12 cell line (PE 307.247) was transfected with the pSVI 6B plasmids. TNFR-IgG (which codes for TNFRl-IgGi) and pFDll (which codes for DHFR) using coprecipitation with calcium phosphate. 2. A cell clone derived from this transfection was scaled up to nine liter suspension cultures using a selective medium containing 2% diafiltered calf serum and 100 nmol / l methotrexate. Two nine-liter cultures were inoculated in a 100-liter fermenter using medium without serum and without methotrexate. The 100-liter culture was inoculated in a 400-liter culture using medium without serum, and then the cells were inoculated in a 1000-liter production fermenter after washing the cells with a 1000-fold volume of production medium to reduce components residual serum. The production medium consisted of a formulation based on DMEM / HAM F-12 supplemented with glucose, amino acids, glycine, hypoxanthine, thymidine, recombinant human insulin, hydrolyzed peptone, Pluronic F68, gentamicin, lipids and trace elements. 3. The production culture was maintained for 13 days at a pH of 7.2 + or - 0.2, using C02 gas and / or Na2C03. The culture temperature was changed from 37 ° C to 30 ° C after reaching a cell concentration of 5 x 106 cells per ml. The osmolality of the culture was adjusted to approximately 450 mOsm / kg by adding medium components on days 2 and 4. Sodium butyrate was added to a final concentration of 12 mM. The dissolved oxygen concentration was maintained at 60% air saturation by injecting air and / or oxygen into the crop. 4. The culture was harvested after 13 days, and the cells were separated from the supernatant by tangential flow filtration. The collected cell culture fluid (designated HCCF) was concentrated approximately 20 times using Maxisette 10 KD membranes. The retained material was clarified and conditioned for chromatography on Protein A by the addition of solid NaCl to a concentration of 1.0 M, followed by filtration through a Durapore filter of 0.22 microns.

. After filtration, the conditioned HCCF was subjected to chromatography on immobilized protein A. After loading the material, several washes were made before elution. The TNFR-IgG was eluted in one step with 0.05 M sodium citrate / 20% glycerol (w / v), pH 3.0. The eluted fraction was adjusted to pH 5.0 by the addition of 1 M sodium citrate. After pH adjustment, the Protein A fraction was diluted and loaded onto an S-Sepharose column.

Fast Flow. The loading was followed by washing, and then elution in one step with 0.05 M MOPS / 0.05 M NaCl, pH 7.2. 7. After elution in one step, the S-Sepharose Fast Flow fraction was diluted and loaded onto a Q-Sepharose Fast Flow column. The column was eluted using a linear gradient of 0.0125 M NaCl at 0.125 M NaCl in 0.125 M MOPS, pH 7.2. 8. After elution with linear gradient, the fraction of Q-Sepharose fast Flow was conditioned by the addition of 1 volume of 0.1 M sodium acetate / 4.0 M urea / 2.0 M ammonium sulfate, pH 5.0 , and was then loaded onto a Toyopearl 650M phenyl column that had been equilibrated with 0.05 M sodium acetate / 2.0 M urea / 1.0 M ammonium sulfate, pH 5.0. The TNFR-IgG goes through the column of these conditions. The load was followed by a wash with the equilibration buffer. The filtered liquid and the washing liquid were combined to build the Toyopearl phenyl fraction. 9. The two Toyopearl phenyl fractions were combined and concentrated using Filtron Centrassette 10 KD membranes. The retentate was passed through a G-25 column equilibrated with 0.01 M sodium citrate / 0.023 M glycine / 0.023 M mannitol, pH 6.0, to produce the composition of TNFR-IgG.

The following Examples from 4 to 10 describe the i_n vivo experiments performed to demonstrate that the TNFR-Ig of this invention relieves the effects of asthmatic attacks. The fusion protein of the TNF receptor called TNFR-Ig markedly reduced, and in some cases completely eliminated, the responses produced by the challenge with antigen in animal models of pulmonary allergic inflammation. The magnitude of the TNFR-Ig inhibitory effect was comparable to that obtained with the broad spectrum dexamethasone anti-inflammatory agent.

Abbreviations: TNFa, tumor necrosis factor alpha; TNFR-Ig, fusion protein of TNF receptor and recombinant soluble IgGl; BAL, bronchoalveolar lavage; OA, ovalbumin; RL, pulmonary resistance; HBSS, Hank's balanced salt solution; Substance P, a rodent peptide used to induce acute bronchospasm, has been referred to in the literature as substance P (see for example Selig and Tocker, Eur. J. Pharmacol. 213 (3): 331-336 (1992)).

An a 1 i s s of the da t os. The means ± S.E.M. for all values in each experiment. The statistical differences were determined by means of an analysis of the variance of two vias of repeated measures of ordered data, followed by a multiple comparison test using a Student Newman-Keul s t-test, or a Student's t-test. A p <value was considered statistically significant; 0.05. The calculations were made using the Microsoft EXCEL 5.0 software packages (Softmart, Exton, PA, USA) and sigmaStat (Jandel Scientific, San Rafael, CA, USA) on a PC.

Fama cos. All drugs were administered in a dosage volume of 1 ml / kg. TNFR-Ig was produced and purified as described in Example 3, and stock solutions were prepared in a buffer containing sodium citrate (10 mM), glycine (23 mM9 and mannitol (230 mM), at pH 6. Aliquots were preserved of the stock solution at -20 ° C before use, and the dilutions were made with sterile saline The following products were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and were prepared in sterile saline solution: ovalbumin, aluminum hydroxide, urethane, substance P acetate, (+/-) propanol hydrochloride, dexamethasone 21-phosphate and Evans blue.

Example 4: Asthma symptoms in guinea pigs (airway hyperreactivity) alleviated with TNFR-Ig This test employs guinea pigs converted to OA allergic, so that subsequent exposure to OA will induce the asthmatic symptom of airway hyperreactivity. This symptom is relieved by TNFR-Ig, which is also compared with dexamethasone, a stable steroid used in asthma relief.

Male guinea pigs were sensitized to OA (10 μg plus 1 mg of Al (OH) 3 in 0.5 ml of saline, sc) on day 0, receiving the same dose of OA as a booster dose on day 14. In On day 21, the animals were challenged with an aerosol containing 0.1% OA for 30 minutes. This sensitization causes symptoms analogous to asthmatics in guinea pigs, including airway hyperreactivity to substance P. In order to determine the effects of TNFR-Ig on this symtom, TNFR-Ig was administered before the challenge, comparing the symptoms with unchallenged animals, showing a reduction in symptoms. Comparable effects were obtained in airway hyperreactivity using dexamethasone (30 mg / kg, i.p., 1 hour before and 4 hours after challenge). Dexamethasone is a known treatment of asthma.

The guinea pigs were prepared for the experiments as follows, being sensitized, challenged and treated.

Sensitization . Male guinea pigs of Hartley strain (Charles River, Kingston, NY, USA) weighing between 250 and 300 g were actively sensitized to ovalbumin (OA) by s.c. single (10 μg of OA + 1 mg of aluminum hydroxide in 0.5 ml of sterile saline) on days 0 and 14 used for the study between days 21 and 28.

Challenge The protocol for antigen challenge has been previously described (O'Donnell, et al., 1994). The sensitized animals were placed in A Plexiglas chamber was challenged with a 0.1% OA aerosol (w / v in sterile saline) for 30 minutes.The aerosol was administered using an ultrasonic nebulizer De Vilbiss UltraNeb 100 (Breathing Services, Ephrata, PA, USA), using an air flow of 30 1 / min, generated by an internal fan.

In order to minimize the number of deaths due to anaphylaxis, the nebulizer was switched on and off for 60 seconds during the first 5 minutes of exposure. The mortality of the animals in these conditions was approximately 5%.

Pharmacotherapy . Groups of animals received either vehicle (see Drugs) or TNFR-Ig (1 or 3 mg / kg, i.p.) at 18 and 1 h before the OA aerosol. For experiments in which dexamethasone was used, separate groups of animals received either vehicle (saline) or dexamethasone (30 mg / kg, i.p.), 1 hour before and 4 hours after the OA spray. The dosing parameters for TNFR-Ig and dexamethasone were determined as optimal based on the results of preliminary observations.

The results of this example show that guinea pigs sensitized to OA by exposure to OA, exhibited a reactivity to the substance P of the airways increased (1-10 μg / kg, i.v.) at 6 hours after the challenge with OA. The increased reactivity to substance P is characteristic of asthmatic sensitization, and demonstrates that guinea pigs suffered the symptom of hyperreactivity produced by asthma induced by the experimental conditions described. Hyperreactivity was inhibited by TNFR-Ig (3 mg / kg, i.p., 18 and 1 hours before challenge).

The method employed was as follows: airway responses to substance P were evaluated in challenged and unchallenged sensitized guinea pigs 6 hours after exposure to the OA aerosol, in accordance with previously described methods (Seiling and Tocker, 1992). The animals were anesthetized with urethane (2 g / kg, i.p.) and cannulated with a PE-50 tube, the carotid artery (blood pressure) and the jugular vein (administration of the drug). The trachea was cannulated with a 15-gauge needle, and the animals were placed in a full body shape (Modular Instruments, Marven, PA, USA). spontaneous respiration was interrupted with succinylcholine chloride (1.2 mg / kg, iv) and the animals were mechanically ventilated (Haverd Apparatus Model 683, South Natick, MA, USA), using a tidal volume of 1 ml / 100 g of body weight and a frequency of 60 breaths / min. The plethysmograph had a total volume of 2 liters, and the volume of the ventilation circuit between the animal and the respirator was 10 ml. The plethysmograph was equipped with a Fleish pneumotachograph (model # 0000) connected to a Validyne differential pressure transducer (DP 45-14) for the measurement of air flow. Transpulmonary pressure was measured by a second Validyne transducer (DP 45-28) connected between a branch of the tracheal cannula and a 16-gauge intrapleural needle inserted between the fifth and sixth intercostal space. Airflow and transpulmonary pressure were recorded with a Modular Instruments M-3000 data acquisition system (Malvern, PA, USA) connected to an IBM 486DX2 PC. The computer used its own programs (BioReport, Modular Instruments) for the calculation of lung resistance (LR) based on the method of A dur and Mead (1958). The readings were taken continuously and averaged at 10 second intervals. The RL values were corrected for the internal resistance of the ventilation circuit (0.11 cm H20 / ml / sec). The animals received propanol (1 mg / kg, i.v.) and were allowed to stabilize before the start of administration of the substance P.

The dose-dependent effects of substance P (1, 3, 5 and 10 μg / kg, i.v.) were measured by administering bolus injections at intervals of about 5 to 10 minutes. The maximum changes in the RL for each dose were obtained, which were expressed as a percentage of the baseline value determined before the administration of the substance P. The ED200I values defined as the dose of substance P causing an increase in the 200% in the RL, were determined for each animal by linear regression of logarithmic dose-response curves.

The baseline values for RL were approximately 0.30 cm H20 / ml / sec, and were not significantly different between groups (p> 0.05). The unchallenged sensitized guinea pigs were relatively insensitive to substance P, requiring a dose of 30 μg / kg to increase the LR by at least 200% or more (Table 1). On the contrary, after the challenge with OA, a marked increase in the airways reactivity to the substance P was observed. The ED20o values increased by approximately 10 times (Table 1), with an increase of approximately 5 times in the Maximum R obtained with the highest dose of substance P (Table 1).

TABLE 1: Summary of the effects of TNFR-Ig and dexamethasone on the responses to the Pa substance of the airways of guinea pigs sensitized to OA.

Treatment -log ED20oc Maximum response 'n5 (% increase in RL) Not dull 4.86 ± 0.0. 139 ± 20 TNFR-Ig Vehicle 5.82 ± 0.04 1811 ± 228 7 1 mg / kg 5.87 ± 0.01 1344 ± 195 6 3 mg / kg 5.48 ± 0.05 * 714 ± 69 * 5 Dexamethasone Vehicle 5.87 ± 0.05 935 ± 91 5 mg / kg 5.35 ± 0.04 * 596 ± 66 * 6 a The dose-response effects of substance P were determined at 6 hours after a 30 minute challenge with a 0.1% OA aerosol. The animals were pretreated with propanol (1 mg / kg, i.v.) 10 minutes before the examination of the airway response to substance P.

The animals were previously treated with the respective vehicles, TNFR-Ig (18 and 1 hour before the challenge with OA) or dezametasone (1 hour before and 4 hours after the challenge with OA), ip, using a dose volume of 1 ml / kg. c The values represent the negative logarithms (mean ± S.E.M.) of the dose (in g / kg) of substance P required to increase lung resistance (LR) by 200% of the baseline value. d The values (mean ± S.E.M.) represent the% increase in the RL with respect to the baseline for the substance P (10 μg / kg, i.v.).

Number of animals per group. f The dose-response effects of substance P were examined in a group of sensitized animals that were not challenged with the OA aerosol.

* Statistically significant difference (p <0.005) in the values of the corresponding vehicle and the groups treated with the drug.

The administration of TNFR-Ig (3 mg / kg) to sensitized animals significantly inhibited (p <0.05) the hyperreactivity to the substance P of the airways induced by OA. A reduction of approximately 3-fold in the ED20o value for substance P, and a reduction of approximately 60% in the maximum RL (Table 1) was observed. A lower dose of TNFR-Ig (1 mg / kg) had no effect on the sensitivity to the substance (Table 1). Treatment with dexamethasone (30 mg / kg) also significantly inhibited (p <0.05) OA-induced airway substance P hyperreactivity in sensitized guinea pigs to a degree similar to that obtained with the higher dose of TNFR. -Ig (Table 1).

Example 5: Asthma symptoms in guinea pigs (influx of inflammatory cells) alleviated by TNFR-Ig This example uses the guinea pigs described in Example 4 converted to OA allergic, so that subsequent exposure to OA will induce an asthmatic symptom, in this case an influx of inflammatory cells. This symptom is alleviated by TNFR-Ig, which is also compared with dexama tasona, a steroid used in the relief of asthma.

As in Example 4, male guinea pigs were sensitized to OA and challenged as described to induce asthmatic symptoms, including the accumulation of inflammatory cells in the airways quantified at 6, 24, 48 and 72 hours after the challenge. As in Example 4, in order to determine the effects of TBFR-Ig on these symptoms, TNFR-Ig was administered before the challenge, and the symptoms were compared with those of the non-challenged animals, demonstrating a reduction in symptoms. TNFR-Ig was also administered before the challenge to measure its effect on inflammation, showing that it produced a reversal in the influx of inflammatory cells. Comparable effects were obtained regarding the accumulation of inflammatory cells at 6 and at 24 hours with dexamethasone (30 mg / kg, i.p., 1 hour before and 4 hours after the challenge).

The method used was as follows: the influx of inflammatory cells was evaluated at 6 and 24 hours post-challenge with OA by BAL, according to previously described methods (Seling and Tocker, 1992). Briefly, the guinea pigs were anesthetized with urethane (2 g / kg, ip), and tracheos were taken with a 15 gauge catheter. Lungs were washed 3 times with 5 ml of Hank's balanced salt solution (HBSS) without Ca2 + and without Mg2 + (Gibco, Grand Island, NY, USA). The samples were centrifuged at 200g for 10 minutes at 25 ° C and the red blood cells were used in the resulting pellet with distilled water (1 ml for 30 seconds), before restoring the osmolarity by the addition of 10 ml of HBSS. The samples were centrifuged again (220g, 10 minutes, 25 ° C) and the resulting pellet was resuspended in 1 ml of HBSS. The total number of cells was determined by exclusion with trypan blue (Sigma Chemical, St. Louis, MO, USA) from an aliquot of cell suspension using a hemocytometer. for differential cell counts, an aliquot of cell suspension was centrifuged using a Cytospin (5 minutes, 1300 rpm, Shandon Souther Instruments, Sewickey, PA, USA), and the slides were fixed and stained with an Wright modified (Leukostat, Fisher Scientific, Pittsburg, PA, USA). standard morphological criteria were used in the classification of at least 300 cells under optical microscopy. The data are expressed as BAL x 106 / animal cells.

Treatment with TNFR-Ig (1-3 mg / kg, ip, pretreatments at 18 and 1 hours for BAL at 6 and 24 hours, respectively) significantly inhibited (p <0.05) the accumulation of neutrophils and total cells in BAL at 6 and 24 hours post OA. TNFR-Ig (3 mg / kg, ip) also significantly reduced (p <0.05) the number of eosinophils in BAL at both time points, whereas a lower dose (1 mg / kg, ip) did not It had no effect. The results of the present study also show that the neutrophil component of the allergen-induced inflammatory response is mediated by TNF. It is noteworthy that TNFR-Ig abolished almost completely the neutrophil influx in the BAL of guinea pigs sensitized 24 hours after challenge with antigen, representing the amount of neutrophils approximately 2% of the total cells in the BAL. Additionally, treatment with TNFR-Ig, but not treatment with dexamethasone, produced a substantial reduction in guinea pig neutrophils 6 hours after the challenge. The blocking of TNF by TNFR-Ig or by similar antagonists can result in an indirect reduction in factors known to be involved in the recruitment of neutrophils to the lung.

The BAL cell composition of unchallenged sensitized guinea pigs has been previously described (Selig and Tocker, 1992). The total cell counts in these animals were on average 1 x 106 / animal, of which approximately 2-3% are eosinophils and neutrophils. At 6 hours post OA, the eosinophils and neutrophils constituted respectively at least 40% and 20% of the total cell count in animals treated with the vehicle for both TNFR-Ig and dexamethasone. The percentage of eosinophils in the BAL remained constant at 24 hours, while the neutrophil count fell to approximately 10% of the total cell count. Inflammatory cell counts and total cell counts were not different (p >; 0.05) between the respective vehicle groups for TNFR-Ig and dexamethasone.

The influx of inflammatory cells in the BAL of challenged sensitized guinea pigs was also inhibited by dexamethasone (30 mg / kg). At 6 hours post OA, a significant reduction (p <0.05) was observed in the number of eosinophils and total cells in BAL, which was similar to that produced by TNFR-Ig. No effect on the number of neutrophils was observed at the 6 o'clock point. At 24 hours post OA, the eosinophil, neutrophil and total cell counts in BAL were significantly inhibited (p <0.05) by dexamethasone. In comparison with TNFR-Ig, animals treated with dexamethasone had approximately 5 times less eosinophils (p <0.05). In contrast, although dexamethasone reduced the number of neutrophils present in the BAL, the proportion of these cells in the differential remained unchanged (p> 0.05) in approximately 8% of the total cell count.

The ability of TNFR-Ig to reverse the inflammatory response in process was examined in sensitized guinea pigs. The animals were sensitized to OA, and then challenged as described above. TNFR-Ig (3 mg / kg, i.p.) was administered 30 minutes after challenge with OA. Inflammation was determined by BAL at 24, 48 and 72 hours after the challenge. For the 48 and 72 hour time points, TNFR-Ig was administered daily. The treatment with TNFR-Ig after the challenge reversed markedly the influx of inflammatory cells in the BAL of sensitized guinea pigs.

Example 6: Asthmatic symptoms in guinea pigs (pulmonary edema) relieved by TNFR-Ig This example employs the guinea pigs described in Example 4 made allergic to OA, so that subsequent exposure to OA will induce an asthmatic symptom, in this case pulmonary edema. This symptom is alleviated by TNFR-Ig, which is also compared with dexamethasone, a steroid used to relieve asthma.

As Example 4, male guinea pigs were sensitized to OA, and challenged as described to induce asthmatic symptoms among which edema is included, quantified 6 hours after the challenge. As in Example 4, in order to determine the effects of TNFR-Ig on these symptoms, TNFR-Ig was administered before the challenge, and the symptoms were compared with those of unchallenged animals, showing to be reduced.

The method employed was as follows: microvascular hemorrhage of the airways, a marker of pulmonary edema, was quantified 6 hours post OA by extravasation of Evans blue dye using methods previously described (Wassermann et al., Adv.

Prostaglandin Thromboxane Leukotriene Res. 23: 271-273, 1995). The guinea pigs were anesthetized with urethane (2 g / kg, i.p.) and the jugular vein was catheterized. The animals were then given Evans blue dye (30 mg / kg, i.v.), administered for 60 seconds. After 10 minutes, the thorax was opened and a catheter (PE-240 tube) was introduced through the left ventricle into the aorta. The ventricle was clamped, the right aricula was cut and the blood was expelled by perfusing the animal with 100 ml of saline using a cartridge pump (Masterflex, Cole-Palmer, Chicago, IL, USA) at a flow rate of 100. ml / min. The pulmonary circulation was perfused with an additional 50 ml of saline solution by inserting the catheter into the pulmonary artery and cutting the right aricula. The lungs were removed en bloc and the trachea (5 mm distal) and the main bronchi were dissected, dried between filter paper and weighed. The Evans blue dye was extracted in formamide (37 ° C, 24 hours) and quantified by measuring the absorbance at 620 nm with a spectrophotometer (Beckman Instruments, Model DU-64, Somerst, NJ, USA). The dye content of the tissue was interpolated from a straight line of Evans blue concentrations (0.5 to 10 μg / ml) and expressed as ng / mg wet tissue weight.

Using Evans blue dye as a marker of microvascular airway haemorrhage, it was determined that the baseline levels in the trachea and main bronchi of unchallenged sensitized guinea pigs were 10 to 20 ng / mg wet tissue weight. Six hours after the challenge with OA, the content of Evans blue dye in the trachea and main bronchi increased 5 times. Treatment with either TNFR-Ig (1 or 3 mg / kg) or dexamethasone (30 mg / kg) attenuated (p <; 0.05) the airway hemorrhage induced by OA at 6 hours post challenge in both the trachea and the main bronchi.

TNFR-Ig (1 or 3 mg / kg, i.p.) eliminated the OA-induced airway edema (quantified by the Evans blue dye content of the tissue) in the trachea and major bronchi of sensitized guinea pigs.

Example 7: Asthma states in Brown Norway rats relieved with TNFR-Ig As in Example 4 for guinea pigs, accumulation of cells of inflammation, a symptom of asthma, was induced in rats, and treated with TNFR-Ig. This model differs from the guinea pig model in that the allergic responses of the Brown-Norway rat are measured by IgE.

The method used was as follows: Brown-Norway male rats were sensitized with OA (1 mg of OA plus 100 mg of Al (OH) 3 in 0.5 ml of saline, ip) on days .0, 1 and 2 On day 21, the animals were challenged with a 1% OA spray for 30 minutes. The accumulation of inflammation cells was quantified by BAL at 24 hours after the challenge. The protocol for sensitization, challenge and BAL was similar to that described above for the guinea pig, with the exception of the following. Brown-Norway male rats (Charles River, Kingston, NY), with weights between 200 and 250 g were actively sensitized to OA with a single i.p. injection. (1 mg of OA plus 100 mg of aluminum hydroxide in 1 ml of saline) on days 1, 2, and 3, and were used for the study on day 21 (Elwood et al., 1992). Separate groups of rats either received the corresponding vehicle, either TNFR-Ig (1 or 3 mg / kg, i.p.) or dexamethasone (0.3 mg / kg, i.p.), 1 hour before the OA spray.

On the day of the experiment, the rats were challenged with a spray of 1% OA for 30 minutes. BAL was performed 24 hours after the challenge by washing the lungs twice with 1 ml / 100 g of HBSS. The red blood cells are They were used with 0.5 ml of distilled water, and the osmolarity was restored with 5 ml of HBSS.

The treatment of these rats with TNFR-Ig (3 mg / kg, i.p., 1 hour before the challenge) virtually abolished the accumulation of neutrophils in the BAL, and caused a significant reduction in the number of eosinophils and the total cell count. Similar results were obtained with dexamethasone (0.3 mg / kg, i.p., 1 hour before the challenge). A lower dose of TNFR-Ig (1 mg / kg, i.p.) also significantly reduced the number of neutrophils in the BAL, although it did not exert any effect on the eosinophil and total cell count.

In greater detail, unchallenged Brown-Norway sensitized rats had a basal level of total cell counts in the BAL of approximately 1 x 106 / animal, of which a proportion of 1% to 2% were eosinophils and neutofibers. Twenty-four hours after challenge with OA, the total number of cells in the Bal of the vehicle-treated animals increased approximately 3-fold, with eosinophils and neutrophils representing at least 40 and 25% of the cell population, respectively. The cell counts of the inflammation and the total number of cells in the BAL were not different (p> 0.05) between the groups treated with the corresponding vehicle.

Treatment with either TNFR-Ig (3 mg / kg, ip) or with dexamethasone (0.3 mg / kg, ip) significantly reduced (p <0.05) the accumulation of eosinophils, neutrophils and total cells in BAL in similar degrees. Treatment with a lower dose of TNFR-Ig (1 mg / kg, ip) significantly inhibited the number of neutrophils in the BAL (p <0.05), although it did not exert any effect on eosinophil accumulation or the total cell count in the BAL.

Example 8: Reduction of neutrophilia in injured rat lungs.

The rat model of Sephadex-induced lung inflammation causes an increase in the number of eosinophils and neutrophils in the lung, as well as the formation of granulomas that resemble observers in chronic asthma. This model is used to demonstrate that TNFR-Ig reduces this chronic symptom.

The method employed was as follows: accumulation of inflammation cells in the lung was induced in male Sprague-Dawley rats by administration of a suspension of Sephadex particles (7.5 mg / kg, i.v.). at 24 and 72 hours after Sephadex, a significant increase in the total number of leukocytes in BAL fluid was observed. At 24 hours, the number of neutrophils accounted for about 50% of the total number of leukocytes, decreasing to approximately 10% of the total at 72 hours. The eosinophil count remained around 10% of the total number of leukocytes.

Pretreatment either with TNFR-Ig (1 and 3 mg / kg, ip, 1 hour before challenge) or dexamethasone (0.1 and 0.3 mg / kg, ip) inhibited neutrophilia at 24 hours after Sephadex, although TNFR-Ig did not have any significant effect on the total cell count. At 72 hours after Sephadex, TNFR-Ig (1 and 3 mg / kg, i.p., daily) significantly reduced the influx of neutrophils in BAL fluid, although it had no inhibitory effect on the number of eosinophils. In contrast, dexamethasone (0.1 and 0.3 mg <1 kg, i.p., daily) virtually abolished the infiltration of neutrophils and eosinophils in BAL. TNFR-Ig (1 and 3 mg / kg, i.p.) or 1 dexamethasone (0.1 and 0.3 mg / kg, i.p.) significantly reduced the total cell count at the 72-hour time point.

Example 9: Attenuation of atopic asthma in primates A model in primates that uses cynomolgus monkeys of wild origin in captivity that show a natural sensitivity of the airways to the As ea rissu um antigen was used to demonstrate that TNFR-Ig relieves the effects of one of the symptoms of asthma: hyperreactivity of the aerial roads. Repeated exposure to the antigen induces asthma symptoms in the airways, in particular an increase in lung resistance (LR). Monkeys treated with TNFR-Ig showed a reduction in the RL.

Cynomolgus monkeys of wild origin in captivity naturally sensitive to As ca ri s demonstrated an increased sensitivity of the airways to inhaled methacholine and an airway eosinophilia when subjected to repeated exposure to the antigen of A. s u um In this way, the antigen was used to induce an allergic reaction and the consequent symptom of asthma hyperreactivity of the airways in these monkeys. The following protocol was used to examine the effect of TNFR-Ig on hyperractivity of the airways.

Dia 1: Determination of the dose of methacholine (MCh), an inducer of bronchospasm similar to substance P, which produces a 100% increase (PC100) in lung resistance.

Day 3 Challenge the monkeys with an inhaled dose of Asca ri s antigen that produces at least one duplication in the RL.

Days 5 and Repetition of day 3 Day 10: Repeat the day 1. Determine the change in log PCioo values between days 1 and 10.

The administration of TNFR-Ig (3 mg / kg, i.v.) on days 1, 3, 5 and 8 attenuated airway hyperreactivity to MCh.

Example 10: Relief of allergic inflammation of the airways with TNFR-Ig in mice A murine model of OA-induced aerobic lung inflammation in mice was used to demonstrate that TNFR-Ig relieves asthmatic symptoms of inflammatory cell influx. Sensitized mice repeatedly exposed to OA gradually develop a hyperreactivity of the airways and an influx of eosinophils increasing in BAL. This model is associated with elevated levels of IgE, and shows a classical profile of Th2 cytokines (ie, high levels of IL-4 and IL-5). The TNFR-Ig was evaluated in this model in terms of its ability to attenuate an inflammatory allergic response in development.

The murine model of allergic inflammation of the lungs is similar to that studied in primates in that a multiple exposure of sensitized animals to the allergen is required to induce hyperreactivity and an influx of inflammatory cells in the airways. Female C57BL / 6 mice were sensitized with OA (10 μg with 1 mg of A1 (0H) gel 3, 0.1 ml, i.p.) on day 0, and then challenged daily from days 14-20 with a 1% OA aerosol for 30 minutes. The hyperreactivity of the airways to the MCh and BAL were investigated 24 hours after the last challenge with OA. Some lungs of sensitized and challenged animals were fixed for histological examination, while others were homogenized for the determination of TNF levels.

Peaks of inflammatory cell accumulation and TNF levels were observed in sensitized and challenged mice after the last challenge (day 20) with the OA aerosol. In the present study, TNFR-Ig (3 mg / kg, ip) was administered immediately after exposure to final OA to OA OA-induced hyperreactivity was attenuated by TNFR-Ig, although this did not exert any effect on the influx of inflammatory cells in the BAL. The daily administration of TNFR-Ig during the challenge period also had no effect on the cell count in BAL. However, administration of TNFR-Ig (3 mg / kg, i.p., day 20) significantly reduced the number of eosinophils in the lung tissue of challenged sensitized mice, and drastically reduced TNF levels in the lung tissue.

Example 11: Spray compositions The following are examples of aerosol compositions containing TNFR-Ig preparations of this invention: Example A - Aerosol (TNFR-Ig, particle size range from 1 to 5 microns) 3.0% Span® 85 (sorbitan trioleate) 1.0 Freon © 11 (trichloromonofluoromethane) 30.0 Freon © 114 (dichlorotetrafluoromethane) 41.0 Freon® 12 (dichlorodif luoromethane) 25.0 Example B - Aerosol TNFR-Ig 0.5% Span 85 0.5% Propellant B1 99.0% 1 Propellant B consists of Freon 11 at 10%, Freon 114 at 50.4%, Freon 12 at 31.5 & and 8.0% butane.

Example C - Aerosol TNFR-Ig 1. 00% Span 85 0.25% Freon 11 5, 0% Freon W 93, 75% 1 Freon W consists of Freon 114 at 61.5% and Freon 12 at 38.5%.

Example D - Aerosol TNFR-Ig 0, 50% Span 85 0, 50% Propellant (C) x 99, 0% 1 Propellant C consists of freon 11 to 30, 01 Freon W at 70%.

Example E - Aerosol TNFR-Ig 0.88% Sodium sulphate (anhydrous) micronized 0.88% Span 85 1.00% Propellant consisting of Freon 12 at 50%, Freon 11 at 25% and Freon 114 at 25% 97.24% Example F - Aerosol TNFR-Ig 0, 06% Span 85 0, 05% Freon 11 20, 0% Freon 12 / Freon 114 (20/80) 78, 9% Example 12: Injectable composition The following is an example of an injectable composition containing TNFR-Ig preparations of this invention.

Ingredient Each ml contains TNFR-IgGl * Citric acid, anhydrous, USP 1.92 mg Glycine, USP 1.70 mg Mannitol pyrogen-free, USP 41.90 mg Sodium hydroxide c.s.p. pH 6.0 Hydrochloric acid c.s.p. pH 6.0, Water for injection, USP c.s.p. 1.0 ml ** The concentration of TNFR-IgGl used in this formulation can be from 1.0 mg / ml to 20 mg / ml. The final amount of TNFR-IgGl in this formulation is selected based on the concentration of the formulation and the volume per vial. Eliminated by lyophilization. 1 mg vial (using 1 ml of a formulation at 1.0 mg / ml) 2.5 mg vial (using 1 ml of a formulation at 2.5 mg / ml) 5.0 mg vial (using 1 ml of a formulation at 5.0 mg / ml) 10 mg vial (using 2 ml of a formulation at 5.0 mg / ml) 10 mg vial (using 1 ml of a formulation at 10.0 mg / ml) Vial of 20 mg (using 2.5 ml of a formulation at 8.0 mg / ml) Vial of 20 mg (using 4 ml of a formulation at 5.0 mg / ml) Vial of 50 mg (using 2.5 ml of a formulation at 20 mg / ml) References Ackerman, V., Marini, M-, Vi ori, E., Bellini, A., Vassali, G. and attoli, S. Detecdon of cyto-? Nes and their cell sources in bronchial biopsy specimens from asthmatíc parients. Reladonship to atopic status, symptoms, and level of airway hyperresponsiveness. Chest 105 (3): 687-96, 1994.

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Having described the invention as above, the content of the following is claimed as property.

Claims (11)

Claims

1. A pharmaceutical composition containing a preparation composed of one or more chimeric TNF-ß-binding proteins, characterized by each of which proteins in said preparation is composed of the soluble portion of the TP receptor protein P55 -fused to an IqG , in the said I gG fused contains all the IgG domains except the first IqG domain of the constant region of the IgG heavy chain, said composition containing an inert pharmaceutic vector, and said preparation being administered to said patient with the Objective to provide said patient with an effective amount of said chimeric protein preparation, with the objective of combating said asthmatic state.

2- The composition according to claim 1, characterized in that said patient is suffering an asthmatic attack, and the chimeric protein preparations are administered in an amount sufficient to alleviate the effects of said attack.

3- The composition according to claim 1, characterized in that said composition is administered to an asthmatic patient before the onset of the asthmatic attack, in an amount effective to prevent or delay the onset of said attack.

4. The composition according to any of claims 1 to 3, characterized in that the fused IgG is human IgGi.

5. The composition according to any of claims 1 to 4, characterized in that the proteins in said protein preparations possess a complex oligo-saccharide terminated in one or more residues of sialic acid and have exposed N-acetylglucosamine, the molar ratio of residues being of sialic acid in said preparation from about 4 to about 7 mmoles of sialic acid per mole of protein, the molar ratio of N-acetylglucosamine being exposed in said preparation from about 1 to about 2 moles of N-acetylglucosamine per mole of protein, and the mole ratio of sialic acid residues to N-acetylglucosamine residues in said preparation being from about 0.35 to about 0.5, and said preparation having an isoelectric point of about 5.5 to about 7.5.

6. The composition according to claim 5, characterized in that the molar ratio of sialic acid to N-acetylglucosamine is from about 0.4 to about 0.45 and the molar ratio of sialic acid to protein is about 5, 0 to about 6.0.

7. The composition according to any of claims 1 to 7, characterized in that said preparation is administered to said patients by injection at a dose of 0.1 mg to 5.0 mg per kilogram of body weight of patient per day.

8. The composition according to claim 1, characterized in that the dose is 1.0 mg to 3.0 mg per kilogram of body weight per day.

9. The composition according to any of claims 1 to 8, characterized in that said preparation is administered by a nasal or buccal spray.

10. The composition according to claim 9, characterized in that said spray is a spray of a liquid composition containing from about 0.03% to 5.0% by weight of said preparation.

11. The use of a chimeric TNF-a binding protein composed of the soluble portion of the p55 TNF receptor protein fused to an IgG, in said fused IgG contains all the IgG domains except the first IgG domain of the constant region of the heavy chain of IgG, for the preparation of a medically for the treatment of asthma.