CA2047203A1 - Fibroblast growth factor for use in the prevention and treatment of viral infections - Google Patents

Abstract

(57) Abstract This invention refers to fibroblast growth factors useful in the prevention and treatment of viral infections caused by enve-loped viruses such as, for example, type 2 herpes simplex virus (HSV2), human respiratory syncytial virus (HRSV), Semliki Forest virus (SFV), the virus responsible for the acquired immuno deficiency syndrome (HIV) and the virus responsible for the Moloney Sarcoma (MSV).

Description

WO 91/09610 PCl`~EP90/02231 Ti ~ l e: FIBROBLAST GROWTH FACTOR FOR USE IN TE~3 PR~V~ION AND
TR~ATI~T OF VIRAL IN~?~CTIONS

The invention refers to fibroblast growth factors (FGF) for use in the prevention and treatment of viral infections caused by viruses known as enveloped viruses, for example" alpha type herpes viruses such as, e.g., type 2 herpes simplex virus (HSV2) and herpes varicella/zoster; beta ox gamma type herpes viruses, e.g., cy~omegalovirus; orthomyxovirus, e.g. influenza virus; or paramy~ovirus, e.g. human respiratory syncytial virus ~H~SV); tropical viruses responsible for exa~thematous fevers and/or encephalitis, e.g., Semliki Forest virus (SFV), or other tropical diseases, belonging, e.g., to the "Alpha", "Flavi" and "Arena" groups of viruses; or retroviruses, e.g. the HIV virus responsible for the ac~ùired immuno deEiciency syndrome, and the virus responsible for the Moloney Saxcoma (MSV).
Although much effort has been made to fight the infections caused by the aforementioned viruses, in particular, or example, HSV2, no really effective drug has yet been identified.
Fibroblast growth factors are, for example, the human and bovine basic fibroblast growth factor (bFGF) and the human and bovine acidic fibroblast growth factor (aFGF).
Said factors are known to be powerful mitogens for a large variety of cells, including fibroblasts and endothelial W0~ 961~ ! `. ` PCT/EP9~/02231 ! ~.3 vascular cells. I~ particular, rnention is made of their angiogenic effect, related precisely to their proliferative action on endothelial cells of blood vessels, and their use in cicatrizing wounds and repairing tissues, including bone and nerve tissues.
Recently, Kaner et al. ~Science, 1990, 248, 1410-1413) have described the activity of fibroblast growth factors and their analogues on the type l Herpes simplex Virus (H~Vl) h~lding that this activity is highly specific so that it is found only on this HSVl type of virus.
The attachment of the growth factor to its specific cellular receptors could prevent the viruses from binding to their receptors which enable them to be internalized.
Since the HSV1 and HSV2 receptors are reported to be different (A. VAHLNE et al., J.Gen. Virology, vol. 44, pp. 217-225 (1979), it is not predictable whether the antiviral action exerted by a growth factor on HSVl type viruses can also be exerted on HSV2 type viruses. This is confirmed~by the fact that one drug can act differently on HSV1 and HSV2 (N.
LANGELAND et al. Biochem. Bioph. Res. Comm. 1986, 141 t PP-198-203).
It has now been found that fibr~blast growth faetors (FGFs) and their analogues are capable of preventing the growth and infectiousness of certain viruses.
This invention refers to a fibroblast growth factor (FGF) for use in the prevention and treatment of viral infections caused by enveloped viruses liXe alpha type herpes viruses, such as type 2 herpes simplex virus (HSV2) and herpes varicella/7osteri W09l/09610 ~ PCT/EP90/0~231 } ~
beta or gamma type herpes viruses, e.g., cytomegalovirus;
orthomyxovirus, e.g. influenza virus; paramyxovirus, e.~. human respiratory syncytial virus (HRSV); tropical viruses responsible for exanthematous fevers and/or encephalitis, e.g.
Semliki Forest virus (SFV) or other tropical diseases, belonging, e.g. to the "Alpha"; "Flavi" and "Arena" groups of vixuses; or retroviruses, e.g. the HIV virus responsible for the acquired immuno deficiency syndrome, and~ the virus responsible for the Moloney Sarcoma (MSV).
Particular examples of viruses towards which the fibroblast growth factors accordins to the invention have proved to be effective are, e.g., the type 2 herpes simplex virus HSV2, the human respiratory syncytial virus (HRSV), the Semliki Forest virus (SFV), the virus responsible for the acquired immuno deficiency syndrome IHIV~ and the virus responsible for the Moloney Sarcoma (MSV).
A fibroblast growth factor (FGF) according to the invention can be either basic FGF (bFGF), human or bovine, or acidic FGF
(aFGF), human or bovine, of an analogue of the aioresaid bFGF
and aFGF.
The aforementioned growth factors, namely human and bovine bFGF
and human and bovine aFGF, are known factors, which are de-scribed for example in the published international patent applications PCT W086/07595 and PCT W087/01728, and in the published European patent applications No. 226181, No. 237966 and No. 259953, as well as in various scientific articles such as, for example, Science vol. 233, pp. 565-548, August 1st, WO91~09610 ~ PCT/EP90/02231 l~86; Embo Journal Vol. 5, No. 10, pp. 2523-2528, lg86;
Biochemical and Biophysical Res. Communications vol. 140, No.
, pp. 874-880, 1986; Biochemical and Biophysical Res.
Communications vol. 133, No. 2, pp. 554-562, 138~, Science vol.
230, pp. 1385-1388, December 20th, 198~.
The nomencla~ure used to define the FGF factors referred to in the invention is the one indicated by Schroder and Lubke, "The Peptides", .~cademic Press (1365), ~here, accor~ding to a conventional representation, the residue having the free a amino group a~ the N-terminal is on the left while the residue having the ~-carboxy group at the C-terminal is on the right.
Thus, for example, hl~an bFGF is a polypeptide with 146 aminoacids, having the sequence shown hereunder:

ro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His ~0 30 he Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg lle His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp ro His lle Lys Leu 51n Leu Gln Ala Glu Glu Arg Gly Val Val Ser lle Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys ~he Phe Phe Glu 10~ 1 -Arg Leu Glu Ser ~sn Asn Tyr Asn Thr Tvr Ara Ser Arg L~s Tyr ~hr WO9]/096l0 (;~ PCT~EP90/0~231 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser 130 1~0 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala ~46 Lys Ser.
Bovine bFGF has the same sequence, the only difference being that the Thr aminoacid in position 112 of hu~an bFGF is replaced by Ser in bovine bFGF and the Ser aminoacid in position 128 of human bFGF is replaced by Pro in bovine bFGF.
The molecule of human bFGF, as well as that of bovine bFGF, can have an N-terminal extension that can contain all or part of the sequence of the following 11 aminoacids:
~i) Gly-Thr-Met-Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu, for example, in paxticular, the following 9 aminoacids (ii) Met-Ala-Ala-Gly-Ser-lle-Thr Thr-Leu, or the following 8 aminoacids (iii) Ala-Ala-Gly-Ser-Ile-Thr-Thr~Leu, or the following 7 aminoacids (iv) Ala-Gly-Ser-Ile-Thr-Thr-Leu.
The molecules of 146 aminoacids of human and bovine bFGF can also be lacking in one or more aminoacid residues at the N-terminal.
Human aFGF is a polypeptide wi~h 140 aminoacids, having the sequence shown hereunder:
1~
Phe Asn Leu Pro Pro ~ly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys WO91/~1961(~ PCT/EP90/0~231

2~ 30 er Asn Gly Gly ~is Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly I~hr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala lu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr eu Ala Met Asp Thr Asp ~ly Leu Le~ Tyr Gly Ser Gln~Thr Pro Asn snlu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr yr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp.
Lovine aFGF is also a polypeptide with 140 aminoacids, having the sequence shown hereunder and characterized by a high degree of holomogy with that of human aFGF;

he Asn Leu Pro Leu Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys er Asn Gly Gly Tyr Phe Leu Arg lle Leu Pro Asp Gly Thr Val Asp ~0 ly Thr ~ys Asp Arg Ser Asp Gln His l.e Gln Leu Gln Lea Gys ~la jO ~0 Glu Ser Ile Gly Glu Val Tyr Ile Lys Ser Thr ,lu Thr Gl-~ Gln Phe V91~096l0 PCT/EP90/02231 6 ~ `I a ~

eu Ala Me~ Asp ~hr Asp ~ly Leu Leu Tyr Gly Ser Gln Thr Pro Asn gO

lu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr yr Ile Ser Lys Lys His Ala Glu Lys His Trp Phe Val Gly Leu Lys L-~s Asn Gly Arg Ser Lys Leu Gly Pro ~rg Thr His PhelGly Gln Lys 130 1~0 Ala Ile Leu Phe ~eu Pro Leu Pro Val Ser Ser Asp.
The molecules of both human and bovine aFGF can also have the same extensions and deletions at the N-terminal indicated above with reference to bFGF molecules.
The fibroblast growth factors according to the invention can also be amidated at the C-terminal.
An analogue of the aforementioned fibroblast growth factors can be, for example, according to the invention, any fragment, even in amidated form, of the entire FGF molecule, which retains its capacity to bind itself to the receptor.
Examples of such fragments are described in the published European patent application No. 246753. They may be, for example, in particular, the polypeptides composed of the aminoacid sequences 93-120; 97-120; 100-120i 103-120; 103-146;
106-~15; 106-lla; 106-120: 106-12~; 106-130; 106-1~5: 106-140;
106-146 and 107-110 of both human and bovine bFGF, both in free form and in amidated form.

wogI/os6l0 PCTIEP90/0223]

An analogue of ~he fibroblast growth f actors o~ the invention can also be a mutein deriving from the aforementioned FGF
polypeptides or their analogues, both in amidated and non amidated fonm, by replacement and/or deletion of one or more aminoacids, which retains e~uivalent properties, in particular an equivalent capacity to bind itself to the FGF
receptor. Thus, for example, the aminoacid in position 112 can be either Thr or Ser; the aminoacid in position~128 can be either Ser or Pro; the aminoacid in ~osition 113 can be either Ala or Ser; the aminoacid in position 114 can be either Met or Trp and the aminoacid in position 115 can be either Phe or Tyr.
Mixtures of different forms of FGF, for example, of the forms referred to previously which derive from different types of extensions or deletions at the N-terminal, are also to be considered as being within the scope of this invention. The term "fibroblast growth factor" refers also to such mixtures, as well as to all the analogues indicated above.
As mentioned previously, the growth fact:ors used according to the invention are known factors and can therefore be prepared by known methods, for example by recombinant DNA, e.g. by procedures similar to those described in the published international patent applications PCT Wo 86/07595 and PCT
WO87/01728 and in the published European patent applications No. 226181, No. 237966 and NO. 259953, mentioned previously.
Xnown techniques may also be used to obtain the FGF analogues, e.g. the fragments of FGF envisaged by the invention. For example; it is possible to use procedures similar to those WO91/09610 9 ~ ;~ PCT/EP90/02231 described in the aforesaid published European patent applicati~n No. 246753 and in the refexences mentioned therein, for example by means of solid phase synthesis as described in JACS 8~, pp 2149 (1963).
Chemical synthesis is preferred for fragments composed of short aminoacid sequences, e.g. sequences shorter than 40 aminoacids, whereas preparation by recombinant DNA is preferred, for example, for preparing full-lenght native FGF molecules or their analogues containing, e.g., more ~han 40 ~minoacids.
In order to demonstrate the antiviral activity of the fibroblast growth faotors according to the invention, tests were carried out using viruses belonging to different types and families, of a DNA and RNA nature. The experiments carried out to ascertain the activity of several fibroblast growth factors according to the invention on HSV2, SFV, HRSV, HIV and MSV
viruses are given by way of example. The following strains were used in these experiments: the diploid strain of human smooth artery muscle cells (A6l7), the heteroploid strain of epithelloid carcinoma Hep~2, a culture of human primary lymphocytes and-a culture of mice Balb C primary fibroblasts.
The ~ormer two strains show an identical permissivity towards the type 2 strain of herpes simplex viruses HSV2 and towards the Semliki Forest virus ~SFV) used; this was observed both in monolayers stabilized for 24 hours, and in cell suspensions obtained by txypsinization, by titration in pla.es with 96 wells per tissue/culture and observation or the cytopathic effect end point (CEP).

W~9l/09~l0 ' ~ ~ ~ PCT/E~90/02231 ,.
The strain A617 is not permissi~e towards the human respiratory syncytial virus (HRSV) and therefore only the Hep~2 cells were used with this infection, both in monolayer and in suspension.
In the experiments with monolayers, scalar concentrations of the substances under examination were incubated in the cultures, grown in the 96 wells after having discarded the culture medium.
After 2 hours of incubation at 37 DC in 5% CO2, 5~-100 plaque forming units ~PF~) of HSV2, or 50-lO0 50% infective units (ID50) of SFV or HRSV were added.
In other experiments the cell suspensions were treated directly with scalar concentrations of the substances under examination in the 96-wells plates.
After 2 hours of incubation at 37~C in ~% CO2, the lnfection was induced as described above~
The degree of CP~ was determined after a period of incubation ranging from 24 to 96 hours according to the type of virus used, by assiyning scores to the fcesh preparations when observed under an inverted microscope ~enlargement 35x). The percentage of reduction o~ CPE in the treated cultures compared to the infection controls was transferred onto semilogarithmic coordinate paper.
The same cultures were then frozen in order to titrate the viral contents under the different experimental conditions (I.V. ) .
The cryolysates were titrated according to one of the standard techniques for determining plaques on confluent mono1ayers o, WO91/09610 ~ PCT/EP90/0223l he~ 2 cells ~rown on lamellas in Leighton tubes, using methylcellulose culture medium.
The I.V. titre under the various experimental conditions was given as PFU and the signiflcance of the differences between the controls and treated cultures was evaluated by means of the Dunnett test (JASA, December 1955, pp. 1096-1121).
The activity on HIV virus was tested in primary cultures of human peripheral lymphocytes, isolated b-~ gradient centrifugation and stimulated by mitogens.
The infection was performed by an HIV standard preparation and the infected cultures were incubated 4 days in presence of the drug.
The evaluation of the results was performed by measuring, at the third and forth day from the infection, the amount of the viral protein of the P24 core (by Elisa) and the total amount of synthetized RNA ~by the molecular hybridation technique with nucleic acid) in comparison to the control~
The activity on Moloney Sarcoma Vlrus ~MSV) was tested in primary cultures of embryonal fibroblasts of Balb C mice infected with a s~andard amount of MSV virus. The results were evaluated on the basis of the reduction of the transformation foci in the treated cultures, in comparison to the control cultures.
The results of the a~oresaid experiments are given in the Figures l to 9 attached hereto.

WO91/09610 12 ~ PCTtEP90/02Z31 Leqenda_to the rigures With reference to the figures, the compound FCF 26184 (compound 1) represents a human bFGF of 153-154 aminoacids, precisely an approximately 50:50 mixture of a) a 1~3 aminoacid molecule having the sequence of 146 aminoacids shown previously for human bFGF and a N-terminal extension of 7 aminoacids as indicated in point (iv) on page 5, and b) a 154 aminoacid molecule having the sequence of 146 aminoacids shown previously for human bFGF and a N-terminal extension of 8 aminoacids as indicated in point (iii) on page 5.
~he preparation of this compound is described hereinafter in the examples section.
The compound "89-092~`' (compound 2) represents a human bFGF of 145 aminoacids, i.e. a compound having the sequence of 146 aminoacids shown previously for human bFGF but without the aminoacid Pro at the N-terminal end.
The preparation o~ this compound may be performed according to the procedure described in the publi.shed European patent application No. 36367~
The compound shown as "~mersh~m bovine`' ~compound 3) is a commercial recombinant bovine bFGF of 146 aminoacids (supplied by the ~irm Amersham).
The compound shown as "~Serval) bovine" (~ompound 3') is a commercial bovine bFGF extracted ~rom the pituitary gland (supplied by the firm Serva).
Figures lA and lB sh3w the percenta~e of inhibiti3n 3f CPE of the compounds 1 (continous line -~) and 2 (discontinous line ~ ) on the 'riSV2 (592 strain) virus infectiGn obtained in a WO9]/096l0 ; ~ PCT/EPsO/0223l cell suspension of A617 cells ~ig. lA) and Hep~2 cells lFig.
13): percent values of CPE reduction are reported in ordinate and b:~GF concentration values are reported in abscissa.
Figures 2A and 2B show the percentage O r inhibition of CPE of compo~nd 1 on the HSV2 l592 strain) virus infection obtained in a cell suspension (discontinous line --C'-) and, parallelly, on a monolayer (continous line -~), o~ A617 cells (~ig. 2A) and Hep~2 cells (Fig. 2B): percent values of CPE r~duetion are reported in ordinate and bFGF concentration values are reported in abscissa.
Figures 3A and 3B show the percentage OI inhibition of CPE of compound 2 on the HSV2 (592 strain) virus infection obtained in a cell suspension (discontinous line --CL-~ and, parallelly, on a monolayer (c~ntinous line ~3-) of A617 cells (Fig. 3A) and Hep~2 cells (Fig. 3B)o percent values of CPE reduction are reported in ordinate and bFGF concentration values are reported in abscissa.
Figure 4 shows the activity of the compounds 1 ~middle histogram) and 2 (histogram on the right) in comparison to control (histogram on the left) estimated as a decrease in the production of HSV2 ~592 strains) infectant virus in Hep~2 cell cultures: the histograms give, in ordinate, the viral index in 104 PFU/mL in the various experimental groups and, in abscissa, the bFGF concentration values.
Figure 5 shows the percentage of inhibition of CPE of the compounds 1 (line _~L), 2 (line ~ -) and 3' (line ~ .n the WO91/09610 19 t~ ., PCT/EP90/OZ23J

~RSv virus infection obtained in a monolayer of Hep~2 cells:
percent values of CPE reduction are reported in ordinate and bFGF concentration values are reported in abscissa.
Figure 6 shows the percentage of inhibition of CPE of the compounds 1 ~line -~), 2 (line ~-C~-) and 3' (line ~ -) on the SFv virus infection obtained in a monolayer of A617 cells:
percent values of CPE reduction are reported in ordinate and bFGF concentration values are reported in abscissa.
Figure 7 shows, in ordinate, the percent inhibition of the compound l on the synthesis of viral protein P24 at t~e third day (white spaces) and forth day (dotted spaces) after infections with the HIV virus: in abscissa bFGF concentrations are reported.
Figure 8 shows, in ordinate, the percent inhibition of the compound l on the synthesis of viral RNA at the third day (white spaces) and forth day (dotted spaces) after infection with the HIV virus: in abscissa bFGF concentrations are reported.
Figure 9 shows the percent inhibition of the compound l on the transformation foci induced by MSV (Moloney Sarcoma Virus):
percent inhi~ition values are reported in ordinate and bFGF
concentration values are reported in abscissa~
The growth factors (FGF) 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 pharmaceutically acceptable salts, as the active principle, and one or more excipients, e.g. pharmaceutically acceptable carriers and~or diluents and/or binders.

W091/09610 pcT/Epso/o2231 Example5Of pharmaceutically acceptable salts can be salts with pharmaceutically acceptable inorganic acids, for example hydrochloric, hydrobxomic, sulphuric and phosphoric acid, and salts with pharmaceutically acceptable organic acids, e.g.
acetic, citric, maleic, malic, succinic, ascorbic and tartaric acid.
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 HSV2, or of respiratory infections caused by HRSV.
Compositions suitabl~ for topical administration can be for example, creams, pastes, ointments or lotions for dermatological treatment; suppositories or pessaries for the treatment of vaginal infections; collyr.ium for the treatment of ocular injections; or aerosols for the treatment of injections of the respiratory system, especially, for example, for treating HRSV infections in newborn babies.
These formulations can be prepared according to known techni~ùes; for example, creams, pastes, ointments and lotions can be obtained by mixing the active principle with conventional oleaginous or emulsifying excipients.
Compositions suitable for intravenous or intrathecal administration can be, for example, sterile aqueous solutions or sterile isotonic physiological saline solutions.

W~41/09610 16 ~ 1 iEP90/02231 Compositions suitable for parenteral administraticn can be, for example, suspensions or s~utions containing the active principle and a pharmaceutically acceptable carrier such as, for example, sterile water, olive oil, glycols, for eY.ample propylenic glycols and, if desired, an appropriate quantity of lidocaine hydrochloride.
~ormulations suitable for oral administration can be, e.g., tablets or capsules coated with a gas~ro- and enter~- resistant layer, in which the active principle can be mixed, for example, with diluen~s, e.g., lactose, dextrose and the like;
lubricants, e.g., silica, talcum, stearic acid and the like;
binders, e.g., starch; disaggregan~s, e.g., alginic acid and alginates; and other excipients commonly used for this type of formulation.
In general, the pharmaceutical compositions accordiny to the invention can be prepared with known techniques and according to procdures 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 example, for topical administrations, e.g., in the form of creams, pastes, ointments, lotions, vaginal pessaries, suppositories and collyriums, the growth factors according to the invention can be used at a concentration ranging from l micromole to l millimole.

~ inistration of the growth factors acor~ing ~o the invention can be useful both in preven'ing diffusion of the virus and in treating patients already infected.
Whenever there are also lesions requiring treatment, administration of bFGF or aFGF can be particularly desirable due to the known cicatrizing and tissue repairing properties of these factors.
The following examples show a non-restrictive method for preparing the FGF growth factors according to the invention.

Example 1 Preparation of b-FGF- FCE 26184 The construction of the synthetic DNA sequence for b-FG~ and of the expression plasmid carrying such sequence was performed according to the procedure described in EP-A-363675. The fermentation and purification process was carxied out as follows:
(a) Fermentation Process A bacterial strain, E. coli type B, from the Institute Pasteur collection, was transformed with a plasmid carrying both the human gene coding for b-FGF and the gene for tetracycline resistance. This transformed strain was used for the production of recombinant non-glycosylated h-b-FGF (numan b-FGF). A Master Cell Bank (15 freeze-dried vials) and a WorXing Cell Bank (W.C.B.) (70 vials stored in li~uid nitrogen at -190C) of this strain were prepared. The content of one vial of W.C.B. was used as the inoculum for the fermentation phase.

WO9~/09610 t8 ' ~ } PCT/EP90/02231 The fermentation process was carried out in lO l fermentors filled with 4 l of culture medium.
Tetracycline hydrochloride was added to the medium in order to maintain the conditions of strain selection.
After 20 hours of growth at 37C the final biomass was 42 + 2 g/l dry weight, an the production o bFGF was 2500 + 500 mg/l as measured by comparative gel electrophoresis.
Enrichment in pure oxygen was required during the f,ermentation phase in order to allow a large bacterial growth.
(b) Initial purification The cells (microorganisms) were separated from the total fermentation broth by centrifugation. The resulting pellet was resuspended in a sodium phosphate buffer containing sodium chloride.
A minimum of 3 passages through a high pressure homogeniæer were necessary for efficient cell breakage. The resulting cell lysate was clarified by centrifugation and the supernatant was collected for further processing.
(c~ Purification The clarified supernatant was loaded on a column of Sepharose (Trade Mark) S Fast Flow (cation exchanger) and the product was eluted from ~his column using a gradient of increasing sodium chloride concentrations in a phosphate buffer (Trade Mark).
The product was further purified on a column of Heparin Sepharose 6 B by eluting with a gradient of increasing sodium chloride concentration in a phosphate buffer. Finally a buffer exchange was made on a Sephadex (Trade Mar~) G2~ resin to WO91/096!0 PCT/EP~0/02~31 obtain the product in ~he bulk product buffer (Sodium phosphate -EDT~).
(d) Column sanitization Sepharose s Fast Flow and Sephadex G2~ columns were sanitized by washing with sodium hydroxide solutions.
Heparin Sepharose was washed alternatively with solutions at pH
8.5 and pH 5~5 containing 3M sodium chloride.
In this was, there was obtained b-FGF designate~ FCE 26184.
This is an pproximately 50:50 mixture of:
- a 154 amino acid human bFGF having the amino acid sequence of the l5~ amino acid form which is reported by Abraham et al but without the N-terminal Met residue, this 154 ~mino acid sequence also being shown in Figure l o~ the accompanying drawings; and - a 153 amino acid human bFGF consisting of the above 154 amino acid form without the Ala residue at position l.

ExamPle 2 Pre~aration of bFGF fra~ments The synthesis of fragment 93-120 of bFGF with the formula:
H-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-~eu-Lys-Arg-NH2 was carried out in consecutive stages using a peptide synthesizer and a MB~IA resin. The bond to the resin was obtained via BOC-Val according to the procedures described in the USA patent No. 4292313. After bonding to the resin the ~mino protective grouD was removed by treatment with trifluoroacetic acid at OC .

WO9]/096l0 ` PCT/EP90/02231 After deprotection and subsequent neutralization, the peptide chain was constructed step by step on the resin following the procedure described in the USA pa~ent No. 3,904,594.
A similar metnod can be used to prepare the following peptides:
1) fragment 97-120 of bFGF with the formula:
H-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NHz;
2) fragment 100-120 of bFGF with the formula:
H-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg=MHz.

3) fragment 103-120 witht the formula:
H-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-NH2 i

4) fragment 103-146 with the formula:
H-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-~yr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln-T~r-Lys-Leu-Gly-Pro-Lys-Thr-~,ly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-ser-NH2 i

5) fragment 106-115 with the formula:
H-Tyr-Arg-ser~Axg-Lys-Tyr-ser-5er-Trp-Tyr-NH2;

6) fragment 106-118 with the formula:
H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser~Ser-Trp-Tyr-Val-Ala-Leu-NH2:

7) fragment 106-120 with the formula:
H-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala Leu-Lys -Arg-NH2 i

8) amidated fragmen~ 106-125 of ~FGF;

9) amidated fragment 106-130 of bFGF;

c~
WO9l/096l0 21 PCT/EP90/02231

10~ amidated ra~ment ].06-;35 of bFGF;

11) ~midated fragment 106-140 of bFGF;

12) ~midated ~ragment 106-146 Gf bFGF;

13) amidated framgnet 107-110 OI bFGr.

Formulation exam~le A FGF eye drop formulation may comprise:
Compound 2 2 ~g Dextrane sulphate 600 ~g Water for injections 10 ml This solution may be lyophylised and reconstituted with 10 ml of a suitable sterile licuid diluent at the moment o, the use.

Claims (17)

1. l. Use of a fibroblast growth factor in the preparation of a medicament for the prevention and treatment of a viral infection caused by an enveloped virus chosen from an alpha type herpes virus, except type l Herpes Simplex Virus (HSV1); a beta or gamma type herpes virus; an orthomyxovirus or paramyxovirus; a tropical virus; and a retrovirus.
2. 2. Use according to claim l where the virus is an alpha type herpes virus, and is the type 2 herpes simplex virus (HSV2).
3. 3. Use according to claim l where the virus is an alpha type herpes virus, and is the herpes varicella/zoster.
4. 4. Use according to claim l where the virus is a beta or gamma type herpes virus, and is a cytomegalovirus.
5. Use according to claim l where the virus is an orthomyxovirus, and is an influenza virus.
6. 6. Use according to claim l where the virus is a paramyxovirus, and is the human respiratory syncytial virus (HRSV).
7. 7. Use according claim l where the virus is a tropical virus, and is the Semliki Forest virus (SFV).
8. 8. Use according to claim l where the virus is a retrovirus, and is the HIV virus.
9. 9. Use according to claim l where the virus is a retrovirus and is the Moloney Sarcoma Virus (MSV).
10. WO91/09610 23 PCT/EP90/02231 l0. Use according to each of the previous claims, where the factor is a basic fibroblastic growth factor (bFGF).
11. 11. Use according to claim 10, where the factor is human bFGF
    or an analogue thereof.
12. 12. Use according to claim 10, where the factor is bovine bFGF
    or an analogue thereof.
13. 13. Use according to each of the previous claims 1 to 9, where the factor is an acidic fibroblast growth factor (aFGF).
14. 14. Use according to claim 13, where the factor is human aFGF
    or an analogue thereof.
15. 15. Use according to claims 11, 12 or 14, where the analogue is a fragment of the whole molecule.
16. 16. A fibroblast growth factor, as defined in the previous claims, for use in the prevention and treatment of a viral infection caused by an enveloped virus as defined in claims l to 9.
17. 17. A method of preventing and treating a viral infection caused by an enveloped virus as defined in claims l to 9 in a patinet in need of it, which method comprises administering to the patient an effective amount of a fibroblast growth factor as defined in the preceeding claims.