The present invention is in the area of pharmaceutical compositions and methoαs of treatment of viral diseases in humans. More particularly the invention relates to pharmaceutical compositions for the treatment of diseases mduced by the respiratory-syncytiaJ virus (RSV).

It is well known that recurrent respiratory tract viral infections are followed by rapid sensi izauon to one or several antigens with increased levels of immunoglobukns E, see e.g. Oscar L. FRICK in J. Allergy Clin. Immunol. (November 1986), pp. 1013-1018. Further, one dominant virus causmg wheezmg in humans and more especially in children is respiratory syncytial virus. The latter is especially observed m children below 2 years of age in whom it causes bronchiolitis and pneumonia.

T. CHONMAITREE et al. In Journal of Infections Diseases, vol 164 (3), pp. 592-594 (1991) discloses that mononuciear leukocytes from normal individuals produce a histamine- releasing factor (HRF) in response to exposure to respiratory viruses, suggesting that this cytokine may play a role m the mechanism of virus-mduced bronchospasm. However these authors have also shown that this HRF appears to be distinct from most other cytokines such as ιnterleuiαns-1-6, 8 and 9 or granulocytes.

R. C. WELLIVER et al. In New England Journal of Medicine vol. 305 (15), pp. 841-846 (1981) discloses that respiratory syncytial virus (RSV) -specific lmmunoglobulines E together with histamine are detectable in a majority of infants w th various forms of respiratorv illness due to RSV and showing wheezmg. However a direct correlauon of the titers of RSV-IgE with the quantity of histamine released could not be determined.

There are many theoretical mechanisms whereby viruses might mduce or exacerbate an inflammation in the lower airways. In addition to alveolar macrophages, the peπbroncnioiar infiltrauon with neutrophilic granulocytes is observed after an infection with respiratory syncyual virus. Neutrophils are not only cells capable of phagocytosis and low molecular weight mflammatory mediator release, but they also have the potential to secrete multiple promfJammatory cytokines. Recently, new cytokines named chemokmes were described as activating inflammatory cells, see for example Piotr KUNA in Pharmacia Allergy Research

Foundauon Award Book (1995) pp. 23-31. In this family of chemokmes, ιnterieukιn-8 (IL-8) is a very potent chemotacnc factor for polymorphonuciear cells. This chemokme, according to B KONIG et al. in Journal of Leukocyte Biology (July 1996), is produced ugh amounts by human polymorphonuciear cells dunng exposure to respiratory syncytial virus. In another study published by R. ARNOLD at al. in Immunology, 85, 364-372 (1995), evidence is presented that peripheral blood mononuciear cells synthesize and secrete the proirmammatory cytokine IL-8 following mfecuon with the respiratory syncyual virus (RSV) even at low doses. The authors of this study suggest that the release of the potent chemotoxm IL-8 from

peripheral blood mononuciear cells might be responsible for the pronounced accumulation of polymorphonuciear granulocytes the alveolar spaces dunng RSV-mduceα bronchiolitis.

H. P. HECKERT et al. In Berliner Mϋnchner Tierarzi Wschr. Vol. 106 (7), pp. 230-235 ( 1993) discusses the treatment of Bovine RSV-infecαon. In addition to antibiotic therapy, the effect of treatment with the antihistamine diphenylhydramm was evaluated by measure of the internal body temperature. With the additional daily application of the antihistamine to the antibiotic therapy, the animals were significantly faster with fever. However the teachmg of this document is stπctly limited to bovmes ana. on the other hand, it fails to explain the respective mechanisms of action of each constituent of the prescnbed combination From a therapeutic point of view, it must be pointed out that there is no specific treatment for cunng respiratory syncytial virus infections. Moreover, it is well known that several drugs used for the treatment of allergy and asthma (comcosteroids, theophylline. ketotifen) αispiav inhibiting effects on ceils directly involved in the immune defense mechanisms, thereby increasing the nsk of microbial and viral infections. Thus an objective of the present invention is to provide useful pharmaceutical compositions for treating diseases induced by the respiratory-syncytial virus in humans.

The present invention is based on the unexpected recognition that 2-(2-{4-[(4-chiorophenyl)phenylmethyl]-l-pιperazmvi]ethoxy| -aceαc acid, an individual optical isomer or a pharmaceutically acceptable salt thereof, displays a significant inhibiting effect on viral replication together with an inhibiting effect of RSV-mduced cell modifications (IL-8 production) Moreover this pharmacological effect takes place without lowenng the immune system of the patient.

This recognition demonstrates the existence of an unexpected protective effect obtained in treating diseases, such as acute bronchioious or viral pneumonia, mduced by the respiratory syncytial virus in humans, by a method which compπses administering to a human in need of such therapy, a pharmaceutical composition compπsing as an active ingredient, an effective amount of at least one compound selected from

2-[2-(4-[(4-chiorophenyl)phenylmethyl]-l-pιperazmyl]etho xy]-acetιc acid, an individual optical isomer or a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salt" as used herein with respect to

2-{2-[4-[(4-chlorophenyl)phenylmethyl]-l-pιperazmyl]etho xy]-aceαc acid means not only its addition salts with non-toxic organic and inorganic acids, such as acetic, citnc, succinic, ascorbic, hydrochloπc, hydrobromic, sulfunc, and phosphoπc acids and the like, but also its metal salts (for example sodium or potassium salts), ammonium salts including quaternary ammonium salts and aminoacid salts.

The term 'individual optical isomer" as used here means the ievorotatory and the dextrorotatary enantiomers thereof. As is well known m the art, purification of such enantiomers is a rather difficult process dependmg upon the selected way of preparation of the

compounα and the opαcai punty of the starting matenai. Therefore the term ' individual optical isomer "as used herem means that the said compound compnses at least 90%, preferably at least 95%, by weight of the said individual (either dextro- or Ievorotatory) optical isomer and at most 10%, preferably at most 5%, by weight of the other individual (respectively levo- or dextrorotatary) optical isomer. Each individual optical isomer may be obtained from its racemic mixture by usmg conventional means such as disclosed in British patent application No. 2,225,321. Additionally, each individual optical isomer can be prepared from the racemic mixture by enzymatic biocatalvtic resolution, such as disclosed in U.S. Patents No. 4,800, 162 and 5,057,427. The most preferred active ingredients of the present invention are the racemate of

2-[2-[4-[(4-chlorophenyl)phenylmethyl]- l-pιperazmyl]ethoxyJ-acetιc acid and its dihydrochlonde salt which is a histamine H^ receptor antagonist well known as ceαπzine dihydrochlonde, and its Ievorotatory and dextrorotatory enantiomers.

For implementing the invention, the composition hereinabove descπbed should contain an effective amount of 2-(2-[4-{(4-chlorophenyl)phenylmethyl]-l-pιperazmyl]ethoxy| -aceαc acid, a pharmaceutically acceptable salt or individual optical isomer thereof. An effective amount can be readily determined by the use of conventional techmques and by observing results obtained under analogous circumstance. In determining the effective amount, a number of factors are considereα including, but not limited to: the species of patient; its size, age, and general health; the specific disease mvolved; the degree of or mvolvement or the seventy of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavaiiabύity charactenstics of the preparation administered; the dose regimen selected; and the use of concomitant medication. An effective amount of 2-[2-[4-[(4-cnlorophenyl)phenylmethyi]-l-piperazmyl)ethoxy)- acetic acid, its pharmaceutically acceptable salt or individual optical isomer thereof in the composition of the invention will generally vary from about 0.1 milligram per kilogram of body weight per day (mg/kg/day) to about 0.5 mg/kg/day. A posology (dose) of about 5 mg to about 50 mg, preferably once or twice per day, is preferred. A composition according to the invention can be administered to a patient in any form or mode which makes the composition bioavailable in effective amounts, namely the oral route. For examole, it can be administered orally, lntranasaily, or rectally. Oral administration is generally preferred. One skilled in the art of preparing formulations can readily select the proper form and mode of administration dependmg upon the particular charactenstics of the disease state to be treated, the stage of the disease, and other relevant circumstances. The compositions of the invention can compnse 2 -[2-(4-[(4-cnlorophenyl)phenylmethyl]-l-pιperazmyl]ethoxyl -acetic acid, its pharmaceutically acceptable salt or individual optical isomer thereof alone or in combination with at least one

pharmaceutically acceptable earner or excipient, the proportion and nature of which are determined by the solubility and chemical properties of the composition selected, the chosen route of administration, and standard pharmaceutical practice.

The earner mateπal may be a solid, semi-solid, or liquid mateπal which can serve as a vehicle or medium for the active ingredient. Suitable earner matenals are well known in the art. The pharmaceutical compositions of the mvention may be adapted for oral use and may be administered to the patient m the form of tablets, capsules, powders, elixirs, syrups, solutions, susυensions, or the like. The pharmaceutical composition of the invention mav aiso be adapted for rectal use and may then be administered to the patient in the form of suppositoπes.

The earner matenal should be suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practice. For instance, for oral administration in the form of tablets or capsules, the therapeuticaily active drug components may be combined with any oral non-toxic pharmaceutically acceptable men earner such as lactose or starch. Optionally, the pharmaceutical composition of the invention aiso contain a binder such as microcrystailine cellulose, gum tragacanth or geiatine, a disintegrating agent such as alginic acid, a lubneant such as magnesium stearate, a gkdant such as colloidal silicon dioxide, a sweetenmg agent such as sucrose or saccharin, a coloring agent or a flavouπng agent such as peppermint or methvl salicylate. Because of their easy administration, tablets and capsules represent the most advantageous oral dosage unit form. If desired, tablets may be coated by standard aqueous or nonaqueous techniques with sugar, shellac or other entering coating agents. Desirably, each tablet or capsule contains from about 5 mg to about 50 mg of the active ingredient.

For the purpose of oral therapeutic administration, the compositions of the present mvention may be incorporated to a solution or suspension. These preparations should contain at least 0.1% by weight of the active ingredient of the composition of the invention.

Such solutions or suspensions may aiso include one or more of the following adjuvants: a stenle diluent such as water for injection, physiologic saline solution, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antobactenai agents such as benzyl alcohol, antioxidants such as ascorbic acid or sodium bisulfite; chelatmg agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting of tonicity such as sodium chlonde or dextrose. The preparation can be enclosed in ampoules, or multiple dose vials made of glass or plastic.

The mvention is further defined by reference to the following examples descnbing in detail the compositions of the present mvention, as well as their utility

While this invenuon has been desenbed and illustrated with reference to certain preferred embodiments thereof, those skilled m the art will appreciate that vanous changes, modifications and substitutions can be made therein without departing from the spint of the

invention. For example, effective dosages other than the preferred ranges set forth heremabove with respect to the active ingredients may be applicable as a consequence of vanauons of the responsiveness of the human treated, seventy of symptoms, dosage related adverse effects, if any observed and similar considerations. Accordingly, such expected vanations or differences in the practice of the present mvention and the results obtained are contemplated in accordance with the objects and practices of the present invention.

Matenals and Methods

Buffer The buffer used for washing the polymorphonuciear cells consisted of 137 mM NaCl,

8 mM Na HPO 3 mM KC1, and 3 mM KH9PO4, pH 7.4 (modified Dulbecco's phosphate-buffered saline). For stimulation assays the cells were suspended in RPMI 1640 medium (Gibco BRL, Eggenstein, Germany).

Preparation of polymorphonuciear (PMN) neutrophilic granulocytes

Human granulocytes were isolated from 200 ml of hepaπnized blood (15 U/ml) from healthy donors separated on a Ficoll-metnzoate gradient followed by dextran sedimentation and washed twice at 300 g. This method led to more than 95% pure PMN. The ceils were diluted to a final density of 1 10^ PMN.

Cell viability

Cell viability was studied by trypan blue exclusion, by analysis of lactate dehydrogenase (Boehπnger, Mannheim, Germany), as well as by determination of mitochondπal activity usmg WST- 1 (Boehnnger, Mannheim, Germany) in stimulated and non-sumulated cells. The assays were performed as descnbed by the manufacturer

(Boehnnger, Mannheim, Germany). All experiments were performed under conditions where the viability of the cell types in all three assays systems was greater than 80%.

Cell culture Hep-2 epithelial ceils, an epithelial tumor ceil line, were obtained from the Ameπcan

Type Culture Collection as ATCC CCL 23 and were cultured at 37°C in 5% carbon dioxide in Dulbecco's modified eagle containing 5% heat-inactivated fetal bovine serum, 4mM L-glutamine and 80 μg/ml gentamicin. The cells were subcultured twice weekly.

Virus preparation

Virus preparation was performed as descnbed by R. ARNOLD et al. m Immunology 82, 126-133 (1994). For crude preparation, respiratory syncytial virus (RSV), Long Strain (ATCC), was grown and titrated in HEP-2 cells. The RSV titre was determined in a plaque- orming unit

(PFU) assay The stock titre of the virus pool used m the study was 5 X 10 ⁶ PFU/ml. The stock solution was stored at -70°C until use. Interleukιn-8 (IL-8) levels were under the detection limit in the stock solution, as analysed by enzyme-linked immuno sorbent assay (ELISA). The absence of mycoplasma infection was venfϊed by microplasma-specifϊc PCR.

Stimulation experiments

If not stated otherwise, human PMN (1 x 10^/ml) are treated with vanous amounts of RSV (10^- 10 ' ) plaque forming units [PFU] corresponding to a multiplicity of infection jm.o.i.] of 0.001 up to 10 in a volume of 1 ml RPMI-1640 medium for the indicated time intervals. The incubation was performed m the absence or in the presence of cetinzme dihydrochlonde at the indicated concentrations. The cell supernatant of the stimulation experiments were collected by centrifugation and stored at -70°C until use for analysis. The cell supematants were used for IL-8 determination; ceil pellets were for RSVg _j - _j genomic nbonucieic acid (RNA) detection.

IL-8 assay

PMN were suspended RPMI medium at a concentration of 1 x 10°" /ml. The cells were cultured in the presence of the appropnate stimulus for up to 24 hours. Culture supematants were collected and analyzed for their IL-8 content. IL-8 release was determined usmg a sandwich ELISA accordmg to the method indicated hereinbefore. In bnef each weil of a 96 well plate (Nunc Maxisorb, Roskilde, Denmark) was coated overnight at 4"C with 100 μl of buffer/ polyoxyethylene sorbitan monolaurate (a product sold under the tradename TWEEN 20) (0.1%) containing anti IL-8 antibodies at a concentration of 5 μg/ml. The plates were washed three times with buffer/Tween, the appropnate samples of IL-8 standard (recombinant human IL-8; Calbiocnem, Bad Soden, Germany) were added and incubation proceeded for 2 hours at 37°C Thereafter, alkaline phosphatase-linked anti IL-8 antibody was added. After addition of p-nitrophenyiphosphate (15 mg/ml) for quantification, an ELISA reader and for calculation Mikrotek software (SLT Labinstruments, Crailsheim, Germany) were used.

Analysis of genomic RSV-RNA The analysis of RSV-specific genomic RNA was performed by coupled reverse transcnption and polymerase chain reaction PCR detection of RSV-genomic-RNA encoding for the small hydrophobic protein (SH) of RSV as descnbed previously by R. ARNOLD et al. in Immunology 82, 126-133 (1994). Total RNA from non-infected as well as from RSV-infected PMN (1 x 10^/ml) was extracted us g Tπzol (Gibco, Niedereggenstein, Germany). Total RNA was dissolved m 30 μl HoO. Expression of genomic RSVg _j _ _j RNA was analysed after reverse transcnption with sense primers and PCR amplification of the cDNA transcnpts. The reverse transcπpαon step mvolved a reaction mixture (final volume 20 μl) containing 10 mM Tns-HCl (pH 8.3), 50 mM KC1, 5 mM MgC-2, ImM desoxynucleoαdes, 100 pM sense pπ ers for RSVgjj,

10 U RNAse inhibitor, 10 μl RNA sample and 200 U reverse transcπptase from Moloney-Muπne-leukemia-virus (Gibco, Eggenstem, Germany). Reverse transcnption reactions were performed at 37 ^β C for 60 minutes. For PCR amplification of the cDNA products, reaction mixtures were mixed with 50 pM of sense and anαsense pnmers and 2 U Taq Polymerase (Gibco, Eggenstem, Germany). The products of 20, 25, and 30 cycles (1 mm, 94°C, 2 m . 53 ^β C, 3mm, 72°C) were analysed on an agarose gel and visualised by ethidium bromide staining. The respective pnmers for RSVg _H were sense: 5'-ACCAATGGAAAATACATCC-3', antisense: 5'-TGAATGCTATGTGTTG-3'. The predicted size of the amplified product was 204 base pairs for RSVg _j - _j accordmg to R. ARNOLD et al. already cited.

Statistical analysis

If not stated otherwise, all data show mean values of at least three individual experiments w th cells from different donors.

Effects of cetinzme on RSV-specific mRNA synthesis

Recently, we have shown that RSV-specific genomic RNA resides inside PMNs up to 24 hours . We analyzed the effects of cetinzme on RSV-specific mRNA expression after stimulation of human PMN with RSV. Therefore, in a first set of experiments human PMN (1 x 10 ⁶ /ml) were treated with RSV at a m.o.i. of 1-, 0.5-, 0.05-, 0.005-, for 2 hours as well as for 16 hours. The results of these experiments are shown in figure 1: Human PMN (1 x 10^/ml) were left untreated (lanes 1,2 and 7,8) or were treated with RSV (1 m.o.i.. lanes 3,9; 0.5 m.o.i.. lanes 4,10; 0.05 m.o.i.. lanes 5,11; 0.005 m.o.i.. lanes 6,12) for 2 hours (lanes 1-6) as well as for 16 hours (lanes 7-12) at 37 ^β C. Cell pellets were analyzed for RSV-specific mRNA expression of the SH gene. The arrow indicates the amplified PCR product. M: 123 base pairs ladder (Gibco BRL, Eggenstem, Germany). Figure 1 shows that RSV-specific mRNA mcreases inside PMN with prolonged incubation time.

Next, human PMN (1 x 10^ /ml) were treated with RSV at a m.o.i. of 1 in the absence or in the presence of cetinzme. The results of these experiments are shown in figure 2: human PMN were left untreated (lane 1) or were treated with RSV (1 m.o.i.) m the absence (lane 2) or in the presence of cetinzme (lanes 3-7; 100-, 10-, 1-, 0.1-, 0.01 μg/ 10 ⁶ PMN). Incubation proceeded for 2 hours (Fig. 2A) as well as for 16 hours (Fig. 2B) at 37" C. The cell pellets were analyzed for RSV-specific mRNA of the RSV-SH-gene by RT-PCR. The arrow indicates the amplified product of the appropnate size. M: 123 base pairs ladder (Gibco BRL, Eggenstem, Germany). Figure 2 shows that the addition of cetinzme inhibits RSV-specific mRNA expression of the small hydrophobic (SH) gene. A decrease in RSV-specific mRNA expression was observed over the whole concentration range of cetinzme. Moreover a decrease in RSV-specific mRNA is indicative for a decrease in repiicative virus. It should be noticed that such viral replication inhibition by an antihistamine was never observed before.

Effects of cetinzme dihydrochlonde on RSV-induced IL-8 release from human PMN Human PMN (1 x 10 ⁶ /ml) were treated with RSV at a multiplicity of infection (m.o.i.) of 1 in the absence or m the presence of cetinzme dihydrochlonde (100-, 10-, 1-, 0.1-, 0.01 μg/ml) for a total mcubation time of 2 hours at 37 ^β C. As control, cells were treated with cetinzme (100-, 10-, 1-, 0.1-, 0.01- μg/ml) or without cetinzme, in the absence of RSV (buffer control). Cell supematants were analyzed for IL-8 release by ELISA. The results of these experiments are shown in figure 3: data present mean and standard deviation values from 8 independent experiments. Figure 3 shows that cetinzme dihydrochlonde (hereinafter "cetinzine") down-regulated the RSV-mduced IL-8 release. However, the effects of ceαnzine were dose-dependent. In this regard, cetinzme at concentrations > 0.01 μg/rni but < 100 ug/ml led to a significant reduction in RSV-mduced IL-8 release. Similar results were obtained after an mcuDation time up to 24 hours as well as at RSV concentrations up to 5 m.o.i. It should be noticed that such interference between an antihistamine and an IL-8 induction by RSV was never observed before.

Effects of cetinzme on the ongomg IL-8 release induced by RSV

Further experiments were performed to analyze the effects of cetinzine on an ongoing IL-8 release mduced by RSV Therefore, human PMN (1 x 10 ⁶ /ml) were treated with RSV (1 m.o.i.) and cetinzine (100 ug, lOμg, 1 μg, 0.1 ug, 0.01 μg). Cetinzme was either added immediately (FIG. 4A), or 30 mmutes (Fig. 4B), or 60 mmutes (Fig. 4C), or 90 mmutes (Fig. 4D) after onset of RSV treatment; a total mcubation time of 2 hours at 37°C was chosen. Cell supematants were analyzed for IL-8 release by ELISA. Our data show that cetinzme down regulates an ongomg IL-8 release by RSV However, the most pronounced effects were observed when cetinzme was added at the onset of RSV treatment. Down regulation of

RSV-induced IL-8 release was most pronounced at cetinzme concentrations > 0.01 μg/ml and < 10 μg/ml.