NERVE GROWTH FACTOR FOR USE IN THE PREVENTION AND TREATMENT OF VIRAL INFECTIONS

Abstract: This invention refers to nerve growth factors useful in the prevention and treatment of viral infections caused by enveloped viruses such as, for example, type 1 herpes simplex virus (HSV-1), type 2 herpes simplex virus (HSV-2), herpes varicella/zoster virus, cytomegalovirus the virus responsible for the acquired immunodeficiency syndrome (HIV) and the virus responsible for the Moloney Sarcoma (MSV).

Description: NERVE GROWTH FACTOR FOR USE IN THE PREVENTION .AND TREATMENT OF VIRAL INFECTIONS The invention refers to a nerve growth factors (NGF) for use in the prevention and treatment of viral 5 infections caused by viruses known as enveloped viruses, for example, alpha type herpes viruses such as, e.g., type 1 herpes simplex virus (HSV-1), type 2 herpes simplex virus (HSV-2) and herpes varicel¬ la/zoster; beta or gamma type herpes viruses, e.g., 0 cytomegalovirus; or retroviruses, e.g. a human immu¬ nodeficiency virus, which is responsible for the acquired immunodeficiency syndrome and the virus responsible for the Moloney Sarcoma (MSV) . Although much effort has been made to fight the infe- 15 ctions caused by the aforementioned viruses, in part¬ icular, for example, HSV-1 and HSV-2, no really ef¬ fective drug has yet been identified. A nerve growth factors (NGF) was first observed in mouse sarcoma tumors (Levi-Montalcini R. et al.: 20 J.Exp.Zool. ,116:321, 1951; Ann. N.Y.Acad. Sci 55, 330,1952). It was purified to homogeneity from ' sub- mandibular salivary glands of male mice (Varon S. et al: Biochemistry 6 : 2202, 1967) and from snake veπvi (Angeletti R.H. : Proc. Nat.Acad. Sci USA 65 : 668, 25 1970). It was also found in trace amounts in a variety of mammalian tissues and body fluids. The primary structure of a mammalian NGF (mouse NGF) was first elucidated by Angeletti et al.: Proc.Nat. Sci. USA 68:2417 (1971). NGF is composed of subunits but only the β subunit (β-NGF) is responsible for the biological activity as nerve growth factor. Human β NGF production by DNA-recombinant technology is disclosed in European Patent Application EP-A-121 338. A method for the preparation of β FGF from human placenta is disclosed in European Patent Application EP 333 574. NGF is thought to play a key role in the survival of cholinergic neurons in the central nervous system and this has led to propose its use in the treatment of Alzheimer's disease. It has now been found that nerve growth factors are capable of preventing the growth and the infectiousness of certain viruses. This invention therefore provides use of a nerve growth factor in the preparation of a medicament for use in the prevention or treatment of viral infection caused by an enveloped virus. A NGF can therefore be used in accordance with the invention in the prevention or treatment of viral infections caused by enveloped viruses like alpha type herpes viruses, such as HSV-l,HSV-2 and herpes vari¬ cella/zoster; beta or gamma type herpes viruses, e.g., cytomegalovirus; or retroviruses, e.g. a HIV such as HIV-1 or HIV-2, and MSV. Particular examples of viruses towards which a NGF has proved to be effective accor¬ ding to the invention are, e.g. HSV-1, HSV-2 and MSV. A (NGF) for use according to the invention can be a mammalian NGF. The NGF may be, human NGF or mouse NGF or Vipera lebetina NGF, or a subunit or an analogue of the aforesaid NGFs. A subunit of a NGF for use according to the invention may be α, β or Vsubunit, preferably the β-subunit. An analogue of the nerve growth factors according to the invention can also be e.g., a mutein deriving from the aforementioned NGF polypeptides, subunits and analogues, both in amidated and non-amidated form, by replacement and/or deletion of one or more aminoacids, which retains equivalent properties, in particular an equivalent capacity to bind itself to the NGF receptor. A specific example of NGF for use according to the invention is a NGF which is obtained from Vipera lebetina and which is a commercially available product. in order to demonstrate the antiviral activity according to the invention of a NGF, tests were carried out on cell suspensions obtained by trypsinization. HSV-1 and HSV-2 were grown on the heteroploid cellular strain of epithelioid carcinoma Hep # 2. The cell suspensions in Eagle's minimum essential medium (MEM) supplemented with 5% foetal calf serum, were treated directly with twofold serial dilutions (in duplicate) of NGF in 96 well plates. After 2 hours of incubation at 37° C in 5% C0 2 , 50-100 50% infective doses (ID 5D ) of HSV-1 or HSV-2 were added. The degree of virus-induced cytopathogenicity (CPE), observed under an inverted microscope (enlargement 35x), was recorded at the day CPE reached 90% in controls, by assigning scores to the fresh prepara- tions. The percentage of reduction of CPE in the treated cultures compared to the infected controls was transferred onto semilogarithmic coordinate paper. The same cultures were then frozen in order to titrate the viral contents (I.V.). The cryolysates were then titrated according to one of the standard techniques for determining plaques on confluent monolayers of Hep # 2 cells grown on lamellas in Leighton tubes, using methylcellulose culture medium. The I.V. titre under the various experimental conditions was given as PFU/ml. The significance of differences between the controls and treated cultures was ^ evaluated by means of the Dunnet test (*p < 0.01). Moloney sarcoma virus (MVS), prepared from virus- induced tumors in newborn Balb/c mice was tested on murine embryo fibroblasts Balb/3T3 grown on MEM cont¬ aining 10% foetal calf serum. NU-Serum IV° (Eurobio) , 10% instead of foetal bovine serum was used in the test medium. The effect on NGF on MSV-induced transformation of Balb/3T3 cells was tested in 48 well microplates. Briefly, 4 x 10 cellls/ml were seeded into each well (in duplicate). Four hours later, the growth medium was replaced by 1 ml of test medium containing fourfold serial dilutions of NGF. After 1 hour incubation, cell cultures were infected with 40 focus-forming units of MSV in 0.2 ml of test medium. After 3 days, the foci of transfor¬ mation in cell cultures were counted at 35x magnifi¬ cation and the percentage of reduction in the treated cultures conmpared to the infected controls was plotted on a semilogaritmic scale. In all experiments mock-infected cells were treated to test cytotoxicity (as modification of cellular growth and morphology) . The toxicity of NGF on Hep = 2 and on Bolb/3T3 was more than lOOOnM. The results of the aforesaid experiments are given in the Figures 1 to 5 of the accompanying drawings, in which : Figure 1 shows the percentage of inhibition of CPE of vipera lebetina commercial NGF obtained from Serva, on the HSV-1 (HF strain) virus infection obtained in a cell suspension of HEp # 2 cells : percent values of CPE reduction (ordinate) are plotted against NGF conc¬ entration values (abscissa). Figure 2 shows the percentage of inhibition of CPE of the same NGF on the HSV-2 (G strain) virus infection obtained in a cell suspension of Hep # 2 cells : percent values of CPE reduction (ordinate) are plotted against NGF concentration values (abscissa). Figure 3 shows the activity of NGF in comparison to control (histogram of the left) estimated as a decrease in the production of HSV-1 (HF strain) infectant virus in Hep # 2 cell cultures : the histograms give, as the ordinate, the viral index in 10 4 PFU/ml in the various experimental groups and, as the abscissa, the NGF concentration values. Figure 4 shows the activity of NGF in comparison to control (histogram on the left) estimated as a decrease in the production of HSV-2 (G strain) infectant virus in Hep # 2 cell cultures : the histograms give, as the ordinate, the viral index in 10 2 PFU/ml in the various experimental groups and, as the abscissa, the NGF concentration values. Figure 5 shows the inhibitory effect of NGF on the MSV induced transformation of Bolb/3T3 cells : percent valus of Foci reduction (ordinate) are plotted against NGF concentration values (abscissa). In view of their antiviral activity, the NGFs according to the invention can be useful, as already said, in the prevention and treatment of the viral infections caused by enveloped viruses, particularly those mentioned before in the present specification. A NGF can be used to alleviate a viral infection. The NGFs according to the invention can be administered in the form of pharmaceutical compositions containing one or more of said factors, as such or in the form of phar a- ceutically acceptable salts, as the active principle, and one or more excipients, e.g. pharmaceutically acceptable carriers and/or diluents and/or binders. Examples of pharmaceutically acceptable salts can be salts with pharmaceutically acceptable inorganic acids, for example hydrochloric, hydrobromic, sulphuric and phosphoric acid, salts with pharmaceutically acceptable organic acids, e.g. acetic, citric, maleic, malic, succinic, ascorbic and tartaric acid, salts with pharmaceutically acceptable inorganic bases, e.g. sodium hydroxide and potassium hydroxide, and salts with pharmaceutically acceptable organic bases, e.g. diethylamine, triethylamine and dicyclohexylamine. They can be administered, for example, by the topical, parenteral, intravenous, intrathecal or oral route. One particularly preferred route of administration is the topical route which is used, for example, in the treatment of genital infections caused, e.g. by HSV-1 and HSV-2. Compositions suitable for topical administration can be for example, creams, pastes, ointments or lotions for dermatological treatment; suppositories or pessaries for the treatment of vaginal infections; eyewashes for the treatment of ocular infections; or aerosols for the treatment of infections of the respiratory system. These formulations can be prepared according to known techniques; for examples, creams, pastes, ointments and lotions can be obtained by mixing the active principle with convention oleaginous or emulsifying excipients. Compositions suitable for intravenous or intrathecal administration can be, for example, sterile aqueous solutions or sterile isotonic physiological saline solutions. Compositions suitable for parenteral administration can be, for example, suspensions or solutions containing the active principle and a pharmaceutically acceptable carrier such as, for example, sterile water, olive oil, glyσols, for example propylenic glycols and, if desi¬ red, an appropriate quantity of lidocaine hydro- chloride. Formulations suitable for oral administration can be, e.g., tablets or capsules coated with a gastro- and entero- resistant layer, in which the active principle can be mixed, for example, with diluents, e.g., lacto¬ se, dextrose and the like; lubricants, e.g., silica, talcum, stearic acid and the like; binders, e.g., starch; disaggregants, e.g., alginic acid and algina- tes; and other excipients commonly used for this type of formulation. In general, the pharmaceutical compositions according to the invention can be prepared with known techniques and according to procedures commonly used in the field of galenic preparations. The beneficial dose depends upon the pathological conditions to be treated, the type of formulation used, the condition of the patient and the lenght of the treatment. For parenteral or oral administration, the growth factor according to the invention may be administered at a dose ranging from 1 ng to 100 μg, for example from 10 ng to 10 μg, per kg body weight. For topical administration, e.g., in the form of creams, pastes, ointments " , lotions, vaginal pessaries, suppositories and eyewashes, the growth factor accord¬ ing to the invention can be used at a concentration ranging from 1 ng to 100 μg, for example from 10 ng to 10 μg, per ml. Administration of the growth factors according to the invention can be useful both in preventing diffusion of the virus and in treating patients already infected. Formulation example A NGF eye drop formulation may comprise : Vipera lebetina commercial NGF (SERVA) 3 μg Dextrane sulphate 600 μg Water for injections 10 ml This solution may be lyophylised and reconstituted with 10 ml of a suitable sterile liquid diluent at the moment of the use.

Claims: CLAIMS 1. Use of a nerve growth factor in the preparation of a medicament for use in the prevention or treatment of a viral infection caused " by an enveloped virus. 2. Use according to claim 1 wherein the viral infect¬ ion is caused by an envepoled virus chosen from an alpha type herpes virus; a beta or gamma type herpes virus; and a retrovirus. 3. Use according to claim 2 where the virus is type 1 herpes simplex virus, type 2 herpes simplex virus or the herpes varicella/zoster virus. 4. Use according to claim 2 where the virus is a cytomegalovirus. 5. Use according to claim 2 where the virus is a human immunodeficiency virus or Monoley Sarcoma Virus. 6. Use according to any one of the preceding claims, where the nerve growth factor is a human nerve growth factor, mouse nerve growth factor , Vipera lebetina nerve growth factor or an analogue thereof. 7. Use according to any one of the preceding claims, where the nerve growth factor is a subunit thereof. 8. Use according to claim 7 where the subunit is the β-subunit. 9. A nerve growth factor as defined in any one of claims 1 or 6 to 8 for use in the prevention or treat¬ ment of a viral infection caused by an enveloped virus as defined in any one of claims 1 to 5. 10. A method for preventing or treating a viral infe¬ ction caused by an enveloped virus as defined in any one of claims 1 to 5, which method comprises admini¬ stering to a patient in need of it an effective amount of a nerve growth factor as defined in any one of claims 1 or 6 to 8. - lO - ll. An agent for the prevention or treatment of a viral infection caused by an enveloped virus as defined in any one of claims 1 to 5, comprising a nerve growth factor as defined in any one of claims 1 or 6 to 8.

Inventors: CARMINATI PAOLO, MAZUE GUY, RONCUCCI ROMEO, UNGHERI DOMENICO, CARMINATI, PAOLO, MAZUE', GUY, RONCUCCI, ROMEO, UNGHERI, DOMENICO
Applicants: ERBA CARLO SPA, FARMITALIA CARLO ERBA S.R.L.
Application year: 1992
Main classification: A61K37
IPC codes: A61K38, A61P31