WO1993020108A1

WO1993020108A1 - Use of certain interferons for the treatment of disorders related to a misproportion of gamma-interferon producing t helper type 1 cells and interleukin-4 producing t helper type 2 cells

Info

Publication number: WO1993020108A1
Application number: PCT/EP1993/000744
Authority: WO
Inventors: Volker Brinkmann; Christoph Heusser; Sefik Alkan; Thomas Geiger
Original assignee: Ciba-Geigy Ag
Priority date: 1992-04-07
Filing date: 1993-03-26
Publication date: 1993-10-14
Also published as: ZA932441B; TW218846B; IL105316A0; AU3889893A

Abstract

The invention relates to the use of certain interferons for the treatment of disorders related to a misproportion of gamma-interferon producing T helper type 1 cells and interleukin-4 producing T helper 2 cells, especially allergic or parasitic diseases.

Description

Use of certain interferons for the treatment of disorders related to a misproportion of gamma-interferon producing T helper type 1 cells and interleukin-4 producing T helper type 2 cells

Human alpha-interferon (IFN-α) is belonging to a family of polypeptides secreted by a large variety of eukaryotic cells upon activation. IFN-α is coded for by a gene family comprising at least 15 non-allelic genes. The primary translation product of IFN-α comprises 189 amino acids (except IFN-α2: 188 amino acids), from which a signal peptide of 23 amino acids is removed by post-translational modification. The amino acid sequence homology among IFN-α subtypes is 80-85 .

The present invention relates to the use of certain human interferons (for the manufacture of a pharmaceutical composition) for the treatment of disorders where a disturbed ratio of gamma-interferon (IFN-γ) producing T helper type 1 cells and interleukin-4 (IL-4) producing T helper type 2 cells in mammals including man is of pathogenic importance.

In the present context, a "disturbed ratio" of IFN-γ producing T helper type 1 (THl) cells and IL-4 producing T helper type 2 (TH2) cells means that the ratio is smaller than normal, i.e. that the number of IFN-γ producing T helper type 1 cells is decreased and/or the number of IL-4 producing T helper type 2 cells is increased.

Disorders which are associated with a decreased production of IFN-γ and an increased production of IL-4 are, for example: allergic (including atopic) disorders, e.g. atopic dermatitis, hyper IgE syndrome; asthma, e.g. atopic (intrinsic) or extrinsic asthma; hypereosinophilia, allergic rhinitis or hay-fever. Furthermore, a decreased IFN-γ production has been observed with facultative intracellular bacterial infections and some parasitic diseases. Diseases caused by facultative intracellular bacteria include, for example: tuberculosis (Mycobacterium tuberculosis), brucellosis (Brucella abortus) and leprosy (Mycobacterium leprae). Parasitic disorders include, for example: malaria (Plasmodia), Chagas disease (Trypanosoma cruzi), toxoplasmosis (Toxoplasma gondii) and sleeping sickness (Trypanosomes). In the European patent EP-B-76489, inter alia the two human lymphoblastoid interferons LyIFN-α-2 and LyIFN-α-3 arc disclosed. These two interferons are also mentioned in US patent 4885 166. US patent 4885 166 further discloses, inter alia, the IFN hybrid B₁D₂B₃B₄. In the following, the IFN hybrid 6^6₃6₄ is designated as "IFN BDBB".

LyIFN-α-2 has the following sequence: see SEQ ID No. 1.

LyIFN-α-3 has the following sequence: see SEQ ID No. 2.

IFN BDBB has the following sequence: see SEQ ID No.3.

Surprisingly, it has now been found thatLyIFN-α-2 and IFN BDBB - but not, for example, yIFN-α-3 - increase the amount of IFN-γ producing T helper type 1 cells but do not increase the amount of IL-4 producing T helper type 2 cells in mammals including man. To demonstrate these findings, it was necessary to develop a new ELISA spot assay that detects single IL-4 or IFN-γ producing TH2 or THl cells, respectively [see under (4) below].

Furthermore, it has been found that, surprisingly, addition of exogenous IFN-γ to T cell cultures and/or neutralization of IL-4 in those cultures increases the number of IFN-γ producing T helper type 1 cells. In contrast thereto, addition of exogenous IL-4, surprisingly, reduces IFN-γ production.

Thus, administration of LyIFN-α-2 or IFN BDBB to mammals suffering from disorders which are associated with a disturbed ratio in the production of IFN-γ and IL-4 may increase IFN-γ production and thus bring back the IFN-γ/E -4 ratio to normal and ameliorate and/or cure the disorders connected therewith.

Therefore, the invention relates to the use of LyIFN-α-2 or IFN BDBB (for the manufacture of a pharmaceutical composition) for the treatment of disorders which are associated with a disturbed ratio of IFN-γ producing T helper type 1 cells and IL-4 producing T helper type 2 cells in mammals including man.

One embodiment of the invention concerns the use of IFN BDBB, and another embodiment of the invention concerns the use of LyIFN-α-2. The beneficial effects of said two interferons are determined, for example, by means of the following experimental methods. For comparison, the closely related LyIFN-α-3 is also included into the test methods:

(1) Induction of IL-4 and IFN-γ production by human T cells

Human PBMC (2xl0⁶/ml) are stimulated for 24h by anti-CD3 antibody coated to the culture plates (coating 24 h with anti-human CD3 antibody OKT3, 50μg/ml). Cultures are performed in the presence of IL-2 (50U/ml), IL-4 (50ng/ml), and various concentrations of test compound. IFN-γ is determined in culture supernatants after 24 hours, or the number of single IFN-γ or IL-4 producing cells is determined in spot ELISAs after 24 h.

(2) Determination of IFN-γ in culture supernatants

Human PBMC (2 x 10⁶/ml) are stimulated for 24h by plate bound anti-CD3 antibody in the presence of IL-2 (50U/ml), IL-4 (50ng/ml), and various concentrations of test compound. IFN-γ is determined in culture supernatants by ELISA: 96 well microtiter plates (Costar) are coated with mouse anti-human-IFN-γ MAb 23.9 (lμg ml). All plates are blocked with 2% BSA in PBS containing 0.05% NaN₃ (1 h, 37°C, 150μl well). After washing with PBS, test samples or control human IFN-γ are added in blocking buffer (lOOμl well) and incubated for 16 h at RT. After washing, mouse-anti-human IFN-γ MAb 76.18 is added (0.2μg lO0μl/well, 2 h 37°C). Plates are washed and incubated with goat-anti- mouse Ig coupled to alkaline phosphatase (Tago, Burlingame, CA; O.lμg/lOOμl/well). Plates are washed and phosphatase substrat added (0.1mg/150μl, Sigma).

Results: Concentration IFN-γ secretion induced by:

(Representative experiment; means of triplicate cultures; SD are <15% and have been omitted).

The results show that, surprisingly, LyIFN-α-2 increases IFN-γ production by a factor of 19 and IFN BDBB even by a factor of 22, whereas LyIFN-α-3 induces a 6-fold increase only, in each case at a concentration of 10 ng/ml.

(3) Determination of mRNA for IL-4 and IFN-γ in human T cells

Human PBMC (5xl0⁶Λnl) are stimulated for 24 h by anti-CD3 antibody coated to the culture plates (coating 24 h with anti-human CD3 antibody OKT3, 50μg/ml). Cultures are performed in the presence of IL-2 (50U/ml), IL-4 (lOOng ml), and the test compound at a concentration of 10 ng ml. IL-4 and IFN-γ mRNA is determined as follows: Preactivated cells are lysed with 600 μl of guanidinium thiocyanate buffer (4M guanidinium thiocyanate, 25mM sodium citrate, pH7, 0.5% sacrosyl, 0.1M mercaptoethanol) and the cellular RNA is isolated by 2 cycles of acidic phenol extractions. The RNA pellet is dissolved in diethylpyrocarbonate-treated water and the concentration is determined by measuring the OD₂₆o ^mM. The PCR amplifications are performed with the Gene Amp PCR it (Peridn-Elmer Cetus, Norwalk, USA) and commercially available primers for IL-4, IFN-γ, and beta-Aktin (Clontech, Palo Alto, USA) according to the manufacturer's instructions. Briefly, 100 ng of total RNA are reverse-transcribed with 5U of reverse transcriptase at 42°C for 20 min, using as primers the specific PCR primers. Amplifications are performed for 30 cycles (1 min 60°C, 1 min 72°C, 1 min 94°C) using a thermal reactor from Hybaid®, Middlesex, UK. The amplified DNA is separated on a 1 % agarose gel together with DNA size markers. The gel is stained with ethidium bromide and the bands are visualized by UV transillumination at 366 nM. The relative intensities of the bands are determined by scanning the polaroid picture of the gel with a video densitometer (Bio Rad, Richmond, USA) in the reflection mode. The values obtained with IL-4 and IFN-γ are standardized with respect to beta actin RNA levels, to correct for variations in RNA amount.

Results: Relative amounts of mRNA after 24 h

aCD3 aCD3 + yIFN-α-2 (10 ng/ml)

100% 350% .

The results show that, surprisingly, LyIFN-α-2 increases IFN-γ mRNA expression by anti-CD3 stimulated T cells more than 3-fold.

(4) Detection of single IL-4 or IFN-v secreting cells in ELISA spot assays

An assay to detect single IL-4 or IFN-γ producing cells has been developed by us which is performed as follows: ELISA spot plates (96 well plates, Costar) are coated with mouse-anti-human-IL-4 MAb 8F12 or with mouse anti-human-IFN-γ MAb 23.9 (lμg/ml). PBMC ar activated for 24h with plate bound anti-CD3 (50μg ml) in the presence of IL-4 (lOOng/ml) and IL-2 (50U/ml), washed and added in appropriate dilutions (IL-4: 2xl0⁵/100μl; IFN-γ: 2xl0⁴/100μl) into the coated ELISA spot plates. ELISA spot plates are centrifuged (3 min 40 x g) and incubated for 3h at 37°C. Cells are removed and developing antibodies added to the plates (IL-4: biotin-labelled mouse-anti-human IL-4 MAb 3H4; 0.2μg 100μl well, 2h 37°C, IFN-γ: biotin-labelled mouse-anti-human IFN-γ MAb 76.18; 0.2μg/100μl/well, 2h 37°C). Plates are washed, and avidin-AP added (Zymed, 0.2μg/100μl well, 2h 37°C). After additional washing, 5-bromo-4-chloro-3-indolyl phosphate (Sigma, O.lμg/lOOμl/well) is added as substrate, and the development of visible ELISA spots followed microscopically. After 30-40 min, plates are rinsed with H₂O and air dried. ELISA spots are counted either by microscopy or by using an automated ELISA spot counter. Results: Concentration IFN-γ secreters/10e4 PBMC in the presence of [ng/ml] LyIFN-α-2 IFN BDBB LyIFN-α-3

0 39

0.01 91 0.1 101 1 243

Results: Concentration IL-4 secreters 10e5 PBMC in the presence of [ng ml] LyIFN-α-2 IFN BDBB LyIFN-α-3

0 19 19 19

0.01 19 18 17 0.1 17 11 8 1 15 9 13

The results show that, surprisingly, LyIFN-α-2 as well as IFN BDBB increase the number of IFN-γ producing cells about 6-fold, whereas LyIFN-α-3 only induces a 2-3 fold increase. IL-4 producing cells are about 20-fold lower within PBMC, and it is important to note that they are not increased in the presence of LyIFN-α-2, IFN BDBB and LyIFN-α-3.

(5) Above, it is shown that IFN-α increases the amount of IFN-γ produced by THl cells. i contrast thereto, a slight and non-significant suppressive effect on the secretion of IL-4 is observed under the applied culture conditions. To determine whether higher concentrations of IFN-γ would interfere with IL-4 production, human PBMC (10⁶/ml) are stimulated 24h by anti-CD3 antibody coated to the culture plates (coating 24 h with anti-human CD3 antibody OKT3, 50μg/ml) in the presence or absence of either IFN-γ (10 and lOOng/ml) or IL-4 (10 and lOOng ml), and the IL-4 and IFN-γ secreting cells are determined as described above.

Results: IFN-γ added IL-4-secreters IFN-γ-secreters

(ng ml) /10e5 PBMC /10e4 PBMC

20 58

64

The data show that, surprisingly, addition of IFN-γ to the cultures reduces the number of IL-4 producing TH2 cells about 3-fold, but increases the number of IFN-γ producing THl cells about 3-fold, and changes the ratio of TH2 vs THl cells from 1:10 to 1:100. Therefore, the IL-4 production by TH2 cells is under the tight control of IFN-γ produced by THl cells. An increase of the IFN-γ production by IFN-α in vivo may therefore lead to a reduced IL-4 production and decreased IL-4 mediated immune responses.

(6) Above, it is shown that IFN-γ is able to suppress IL-4 production and to increase the number of IFN-γ producing T cells. To further analyse whether the IL-4 is able to block IFN-γ production by THl cells, similar cultures are performed in the presence of IL-4 (1 and lOng ml), or neutralizing antibodies to IL-4 (8F12, 1 and lOμg/ml). After 24 h, secreted IFN-γ in culture supernatants is determined by ELISA [see under (2) above].

The data show that, surprisingly, neutralization of endogenously produced IL-4 increases the amount of IFN-γ produced about 7-fold, whereas addition of exogenous D -4 strongly reduces IFN-γ production up to 7-fold. These data show that IFN-γ production by THl cells is under the tight control of IL-4 produced by TH2 cells. Excess production of IL-4 in the case of allergy therefore will block protective IFN-γ mediated responses.

A further major advantage of the two interferons used according to the present invention, LyIFN-α-2 and IFN BDBB, is that the typical side effects for IFN-α are surprisingly mild as compared to classical^'recombinant human IFN-α subtypes, such as the commercially available alpha-interferons Roferon A® and Intron A®. Furthermore, LyIFN-α-2 as well as IFN BDBB do not induce antibodies in vivo in contrast to, for example, IFN-α2a (= Roferon A®) [see e.g. J. Biol. Standard 15 (1987) 231-244] or IFN-α2b (= Intron A®) [see e.g. Dianzani etaL, J. Interferon Res.9 (1989) 33-36].

These advantages of LyIFN-α-2 and IFN BDBB make it possible for the first time to envisage long term treatment of diseases which are not as severe as those classically being treated with IFN-α, such as neoplastic diseases. Disorders which are associated with a disturbed ratio in the production of IFN-γ and IL-4, for example atopic dermatitis, asthma or allergic rhinitis, can now be treated in the long term with LyIFN-α-2 and IFN BDBB without causing unacceptable side effects.

With IFN BDBB, two Phase II trials have been performed in patients with malignant disease.

In the first trial, 27 patients were treated thrice weekly subcutaneously with increasing doses (inter-patient) of IFN BDBB. The Maximum Tolerated Dose (MTD) was found to be between 64-96 x 10⁶ units. In contrast thereto, Roferon A® and Intron A® are reported to have a MTD for similar longterm treatment between 15-20 x 10⁶ units only. The side effects observed in this trial-were very mild, also over long periods of treatment No antibodies were formed against IFN BDBB, also not in patients who were under IFN BDBB treatment for over two years.

In a second trial, under daily longterm treatment (subcutaneously) the MTD was found to be around 25 x 10⁶ units for IFN BDBB. In this trial, 22 patients were treated with IFN BDBB. IFN BDBB was similarly well tolerated as in the first trial. Also in this trial no anti-IFN BDBB antibodies were formed. In contrast thereto, the MTD for Roferon A® and Intron A® at daily treatment schedule is below 10 x 10⁶ units for longterm treatment Since subcutaneous treatment of IFN BDBB at doses over 16 x 10⁶ units leads to prolonged pharmacokinetics, thrice weekly administration is sufficient to maintain significant serum levels.

LyIFN-α-2 and IFN BDBB are preferably used in the form of pharmaceutical preparations that contain a therapeutically effective amount of the active ingredient optionally together with or in admixture with inorganic or organic, solid or liquid, pharmaceutically acceptable carriers which are suitable for administration.

The pharmaceutical compositions may be, for example, compositions for enteral, such as oral administration, compositions for parenteral administration, such as intravenous or subcutaneous administration, compositions for local administration and, especially, compositions for administration by inhalation to mammals, in particular humans, the compositions comprising the pharmacologically active interferon on its own or together with customary pharmaceutical excipients.

The present pharmaceutical preparations, which may, if desired, contain further pharmacologically valuable substances, are produced in a manner known per se, for example by means of conventional dissolving of lyophilising processes, and contain from approximately 0.1 % to 100 %, especially from approximately 1 % to approximately 50 %, and in the case of lyophilisates up to 100 %, of the active ingredient It is worthy of note that the.two interferons used according to the invention can be lyophilised and redissolved without any loss of activity.

The particular mode of administration and the dosage will be selected by the attending physician taking into account the particulars of the patient, the disease and the disease state involved.

The dosage of the active ingredient may depend on various factors, such as effectiveness and duration of action of the active ingredient severity of the disease to be treated and its symptoms, respectively, mode of administration, warm-blooded species, and/or sex, age, weight and individual condition of the warm-blooded animal. The approximate daily dosage normally to be recommended for a warm-blooded animal weighing approximately 75 kg is from approximately 10⁴ to approximately 10⁸ units, especially from approximately 10⁵ to approximately 10⁷ units, which is optionally taken in several, optionally equal, partial doses. But also weekly regimens, for example 10⁵ to 10⁷ units once, twice or thrice weekly, come into consideration.

Pharmaceutical preparations for enteral and parenteral administration are, for example, those in dosage unit forms, such as dragέes, tablets or capsules and also ampoules. They are prepared in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilising processes. For example, pharmaceutical preparations for oral administration can be obtained by combining the active ingredient with solid carriers, where appropriate granulating a resulting mixture, and processing the mixture or granulate, if desired or necessary after the addition of suitable adjuncts, into tablets or drage cores.

Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starch pastes, using, for example, corn, wheat rice or potato starch, gelatin, tragacanth, methyl- cellulose and or polyvinylpyrrolidone and, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate. Adjuncts are especially flow-regulating agents and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and or polyethylene glycol. Drage cores are provided with suitable coatings that may be resistant to gastric juices, there being used, inter alia, concentrated'sugar solutions that optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or lacquer solutions in suitable organic solvents or solvent mixtures or, to produce coatings that are resistant to gastric juices, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Colouring substances or pigments may be added to the tablets or drage coatings, for example for the purpose of identification or to indicate different doses of active in 'gere* dient

Other orally administrable pharmaceutical preparations are dry-filled capsules made of gelatin, and also soft sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol. The dry-filled capsules may contain the active ingredient in the form of a granulate, for example in admixture with fillers, such as lactose, binders, such as starches, and/or glidants, such as talc or magnesium stearate, and, where appropriate, stabilisers. In soft capsules the active ingredient is preferably dissolved or suspended in suitable liquids, such as fatty oils, paraffin oil or liquid polyethylene glycols, it being possible also for stabilisers to be added.

Parenteral formulations are especially injectable fluids that are effective in various manners, such as intravenously, intramuscularly, intraperitoneally, intranasally, intradermally or subcutaneously. Such fluids are preferably isotonic aqueous solutions or suspensions which can be prepared before use, for example from lyophilised preparations which contain the active ingredient alone or together with a pharmaceutically acceptable carrier. The pharmaceutical preparations may be sterilised and/or contain adjuncts, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers.

Pharmaceutical compositions for local administration are, for example, for the topical treatment of the skin, lotions, creams and ointments, that is to say liquid or semi-solid oil-in-water or water-in-oil emulsions, fatty ointments which are anhydrous, pastes, that is to say creams and ointments with secretion-absorbing powder constituents, gels which are aqueous, of low water-content or anhydrous and consist of swellable, gel-forming materials, foams, that is to say liquid oil-in-water emulsions in aerosol form which are administered from pressurised containers, and tinctures which have an aqueous-ethanolic base, it being possible that these compositions in each case contain other customary pharmaceutical excipients, such as preservatives. Suitable for the local treatment of the eyes are, for example, eye drops which comprise the active ingredient in sterile aqueous or oily solution, and eye dintments which are also preferably manufactured in sterile form. Suitable for the local treatment of the nose are, for example, sprays, similar to those described below for the treatment of the respiratory tract, coarse powders which are administered by rapid inhalation through the nostrils, and especially nose drops which comprise the active ingredient in aqueous or oily solution. Suitable for the local treatment of the buccal cavity are, for example, lozenges and pastilles which comprise the active ingredient in an inert mass formed, for example, of sugar and gum arabic or tragacanth, to which flavourings may be added. The pharmaceutical compositions for local admini¬ stration are prepared in a manner known per se by mixing the active ingredient with the pharmaceutical excipients, for example by dissolving or suspending the active ingredient in the base material or in a portion thereof, if necessary. In order to prepare emulsions in which the active ingredient is dissolved in one of the liquid phases, the active ingredient is, as a rule, dissolved therein before the emulsification; in order to prepare suspensions in which the active ingredient is suspended in the emulsion, the active ingredient is mixed with a portion of the base material after the emulsification and then added to the remainder of the formulation.

Pharmaceutical compositions for administration by inhalation are, for example, aerosols which are administered in the form of sprays or powders (powder aerosols). Aerosols administered in the form of sprays are solutions, suspensions or emulsions of the active ingredient in a suitable, pharmaceutically acceptable liquid phase, such as in ethanol or water or a corresponding mixture, which may, as necessary, also comprise other pharmaceutical excipients, such as non-ionic or anionic surface-active agents, emulsifiers and stabilisers, and/or active ingredients of otiier kinds, and which comprise a propellant for example an inert gas, such as butane, under elevated pressure or especially a readily volatile liquid, preferably a liquid that boils under normal pressure below customary room temperature (for example at from approximately -30°C to approximately +10°C), such as an at least partially fluorinated polyhalogenated lower alkane, or a fluorinated lower alkane, e.g. "134A" (= asymmetrical tetrafluoroethane) or "227" (= heptafluoropropane), or an extra-pure lower alkane, e.g. isobutane, or a mixture of such liquids. For the preparation of the pharmaceutical compositions in dosage forms finished for administration by inhalation, a corresponding pharmaceutical composition is introduced, together with the propellant into suitable containers, such as flacons or pressurised bottles, which are provided with a suitable spray device, for example a valve. The valve is preferably constructed in the form of a metering valve which on operation releases a predetermined amount of the contents of the container, corresponding to a predetermined dose of the active ingre'dient In the preparation of the finished pharmaceutical dosage form, it is also possible for appropriate amounts of the pharmaceutical composition and of the propellant to be introduced separately into the containers and to be mixed with one another only at that stage.

The following Examples illustrate the invention described hereinbefore. The term "active ingredient" is to be understood as being either LyIFN-α-2 or IFN BDBB.

Example 1: 100 vials each containing 3.33.10⁶ units of IFN BDBB for parenteral administration are manufactured as follows:

2 mg of IFN BDBB having a specific activity of 1.67«10⁸ units/mg on human WISH cells, are dissolved in 30 ml of 5N human serum albumin. The resulting solution is passed through a bacteriological filter and the filtered solution is subdivided under aseptic conditions into 100 vials each containing 3.33* 10⁶ units of IFN BDBB. The vials which are suitable for parenteral administration are preferably stored in the cold, for example between -20°C and +4°C.

In the same manner, vials containing 6.67* 10⁶ or 1.33* 10⁷ units may be prepared by using 4 or 8 mg, respectively, of IFN BDBB.

In an analogous manner; vials containing LyIFN-α-2 (specific activity 1.85»10⁸ units/mg) can be prepared.

Example 2: 100 g of an ointment comprising 0.005 % by weight of LyIFN-α-2 are manufactured as follows:

Composition

LyIFN-α-2 0.005 g¹⁾ vaseline 45.00 g paraffin oil 19.60 g cetyl alcohol 5.00 g beeswax 5.00 g sorbitan sesquioleate 5.00 g p-hydroxybenzoic acid ester 0.20 g water, demineralised 20.195 g

100.000 g

^1J As LyIFN-α-2 has a specific activity of 1.85»10⁸ units/mg on human WISH cells, 0.005 g of LyIFN-α-2 correspond to 9.25* 10⁸ units.

In the same manner, 100 g of an ointment containing 3.7«10⁹ units may be prepared by using 0.020 g of LyIFN-α-2 (and 20.180 g of demineralised water).

The fatty substances and emulsifiers are melted together. The preservative and the active ingredient are dissolved in water, and the solution is emulsified in the fatty melt at elevated temperature and then cooled.

In an analogous manner, an ointment containing IFN BDBB (specific activity 1.67»10⁸ units/mg) can be prepared. Example 3: An approximately 0.02 % aqueous solution, suitable for inhalation, of the active ingredient is manufactured as follows:

Composition active ingredient 20 mg disodium salt of ethylenediaminetetraacetic acid 10 mg benzalkonium chloride 10 mg water, freshly distilled ad 100 ml propellant as required

The active ingredient is dissolved in approximately 60 ml of freshly distilled water, and the stabiliser (disodium salt of ethylenediaminetetraacetic acid) and the preservative (benzalkonium chloride) are added. When all the components have completely dissolved, the resulting solution is made up to 100 ml and introduced into small pressurised bottles. The small bottles are sealed in gas-tight manner. The propellant is added, as required, in gaseous form under pressure or in liquid form.

In the same manner, an approximately 0.002 % aqueous solution, suitable for inhalation, may be prepared by using 2 mg of the active ingredient

Example 4: Tablets each comprising 1 x 10⁶ units of active ingredient are manufactured as follows:

Composition (10.000 tablets) active ingredient 10¹⁰ units lactose 500.0 g potato starch 352.0 g gelatin 8.0 g talc 60.0 g magnesium stearate 10.0 g silica (highly disperse) 20.0 g ethanol q.s.

The active ingredient is mixed with the lactose and 292 g of the potato starch, and the mixture is moistened with an ethanolic solution of the gelatin and granulated through a sieve. After drying, the remainder of the potato starch, the magnesium stearate, the talc and the silica are admixed and the mixture is compressed to give tablets each of weight 145 mg and active ingredient content 50 mg, which, if desired, can be provided with breaking notches for finer adjustment of the dosage.

Exam le 5: Film-coated tablets each com risin 1 x 10⁶ units of active ingredient are

The active ingredient, the lactose and 40 g of the cornflour are mixed. The mixture is moistened with a paste, prepared from 15 g of cornflour and water (with warming), and is granulated. The granules are dried, and the remainder of the cornflour, the talc and the calcium stearate are mixed with the granules. The mixture is compressed to give tablets (weight: 280 mg each) and these are coated with a solution of the hydroxypropylmethylcellulose and the shellac in dichloromethane (final weight of the film-coated tablets: 283 mg each).

Example 6: Hard gelatin capsules each comprising 1 x 10⁵ units of active ingredient are manufactured as follows:

Composition (1000 capsules) active ingredient 10⁸ units lactose 250.0 g microcrystalline cellulose 30.0 g sodium lauryl sulfate 2.0 g magnesium stearate 8.0 g The sodium lauryl sulfate is sieved into the lyophilised active ingredient through a sieve having a mesh width of 0.2 mm. Both components are intimately mixed. The lactose is then first sieved in through a sieve having a mesh width of 0.6 mm and subsequently the microcrystalline cellulose through a sieve having a mesh width of 0.9 mm. The four components are then intimately mixed for 10 minutes. Finally, the magnesium stearate is sieved in through a sieve having a mesh width of 0.8 mm. After further mixing (3 minutes), 390 mg each of the formulation obtained is filled into hard gelatin capsules of size O.

Example 7: An injection or infusion solution, comprising 5 x 10⁶ units of active ingredient per ampoule at 2.5 ml each, is manufactured as follows:

Composition (1000 ampoules) active ingredient 5 x 10⁹ units sodium chloride 22.5 g phosphate buffer solution (pH: 7.4) 300.0 g demineralised water ad 2500.0 ml

The active ingredient and the sodium chloride are dissolved in 1000 ml of demineralised water. The solution is filtered through a microfilter (0.22 μm). The filtrate is treated with the phosphate buffer solution, and the mixture is made up to 2500 ml with demineralised water. To prepare unit dose forms, 2.5 ml of the mixture each time are filled into glass ampoules, which then each contain 5 mg of active ingredient.

SEQUENCE LISTING

NUMBER OF SEQUENCES: 3

(iv) COMPUTER READABLE FORM: Not Applicable

(v) CURRENT APPLICATION DATA: APPLICATION NUMBER:

(2) INFORMATION FOR SEQ ID NO:l;

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 166 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu lie 1 5 10 15

Leu Leu Ala Gin Met Arg Arg ie Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30

Arg His Asp Phe Glu Phe Pro Gin Glu Glu Phe Asp Asp Lys Gin Phe 35 ' 40 45

Gin Lys Ala Gin Ala lie Ser Val Leu His Glu Met lie Gin Gin Thr 50 55 60

Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Leu Asp Glu Thr 65 70 75 80

Leu Leu Asp Glu Phe Tyr lie Glu Leu Asp Gin Gin Leu Asn Asp Leu

85 90 95

Glu Ser Cys Val Met Gin Glu Val Gly Val He Glu Ser Pro Leu Met 100 105 110

Tyr Glu Asp^' Ser He Leu Ala Val Arg Lys Tyr Phe Gin Arg He Thr 115 120 125

Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Ser Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu He Met Arg Ser Phe Ser Leu Ser He Asn Leu Gin Lys 145 150 155 160

Arg Leu Lys Ser Lys Glu

165

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 166 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met 1 5 10 15

Leu Leu Ala Gin Met Ser Arg He Ser Pro Ser Ser Cys Leu Met Asp 20 25 30

Arg His Asp Phe Gly Phe Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe 35 40 45

Gin Lys Ala Pro Ala He Ser Val Leu His Glu Leu He Gin Gin He 50 55 60

Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp 65 70 75 80

Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu

85 90 95

Glu Ala Cys Val Met Gin Glu Glu Arg Val Gly Glu Thr Pro Leu Met 100 105 110

Asn Ala Asp Ser He Leu Ala Val Lys Lys Tyr Phe Arg Arg He Thr 115 120 125

Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140

Arg Ala Glu He Met Arg Ser Leu Ser Leu Ser Thr Asn Leu Gin Glu 145 150 155 160

Arg Leu Arg Arg Lys Glu

165 (2) INFORMATION FOR SEQ ID NO:3

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 166 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Cys- Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu He 1 5 10 15

Leu Leu Ala Gin Met Arg Arg He Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30

Arg His Asp Phe Glu Phe Pro Gin Glu Glu Phe Asp Asp Lys Gin Phe 35 40 45

Gin Lys Ala Gin Ala He Ser Val Leu His Glu Met He Gin Gin He 50 55 60

Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp 65 70 75 80

Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu

85 90 95

Glu Ser Cys Val Met Gin Glu Val Gly Val He Glu Ser Pro Leu Met 100 105 110

Tyr Glu Asp Ser He Leu Ala Val Arg Lys Tyr Phe Gin Arg He Thr 115 120 125

Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Ser Cys Ala Trp Glu Val Val 130 135 140

Arg Ala Glu He Met Arg Ser Phe Ser Leu Ser He Asn Leu Gin Lys 145 150 155 160

Arg Leu Lys Ser Lys Glu

165

Claims

1. Use of LyIFN-α-2 or IFN BDBB for the manufacture of a pharmaceutical composition for the treatment of disorders which are-associated with a disturbed ratio of gamma-interferon (IFN-γ) producing T helper type 1 cells and interleukin-4 (IL-4) producing T helper type 2 cells in mammals including man.

2. Use according to claim 1, characterized in that the pharmaceutical composition manufactured is used for the treatment of an allergic disorder.

3: Use according to claim 2, characterized in that the allergic disorder is atopic dermatitis.

4. Use according to claim 2, characterized in that the allergic disorder is asthma.

5. Use according to claim 2, characterized in that the allergic disorder is the hyper IgE syndrome.

6. Use according to claim 2, characterized in that the allergic disorder is allergic rhinitis.

7. Use according to claim 2, characterized in that the allergic disorder is hypereosinophilia.

8. Use according to claim 2, characterized in that the allergic disorder is a food allergy.

9. Use according to claim 2, characterized in that the allergic disorder is a contact allergy.

10. Use according to claim 1, characterized in that the pharmaceutical composition manufactured is used for the treatment of a parasitic disorder.

11. Use according to claim 1, characterized in that the pharmaceutical composition manufactured is used for the treatment of an infection caused by facultative intracellular bacteria.

12. Use according to claim 1, characterized in that the pharmaceutical composition manufactured is used for the treatment of a chronic viral infection.

13. Use according to any one of claims 1-12, characterized in that IFN BDBB is used for the manufacture of the pharmaceutical composition.

14. A method for the treatment of disorders which are associated with a disturbed ratio of gamma-interferon (IFN-γ) producing T helper type 1 cells and interleukin-4 (B -4) producing T helper type 2 cells in mammals including man, which method comprises administering to a mammal in need of such treatment a therapeutically effective amount of LyIFN-α-2 or IFN BDBB.

15. A method according to claim 14, wherein IFN BDBB is administered.

16. Use of LyIFN-α-2 or IFN BDBB for the treatment of disorders which are associated with a disturbed ratio of gamma-interferon (IFN-γ) producing T helper type 1 cells and interleukin-4 (IL-4) producing T helper type 2 cells in mammals including man.

17. A pharmaceutical composition for the treatment of disorders which are associated with a disturbed ratio of gamma-interferon (IFN-γ) producing T helper type 1 cells and interleukin-4 (IL-4) producing T helper type 2 cells comprising a therapeutically effective amount of LyIFN-α-2 or IFN BDBB together with a pharmaceutically acceptable carrier.

18. A pharmaceutical composition according to claim 17, which comprises a therapeutically effective amount of IFN BDBB together with a pharmaceutically acceptable carrier.