Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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
120
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
100 110
yr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys
120
Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys
130 140
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
100 110
yr Ile Ser Lys Lys His Ala Glu Lys His Trp Phe Val Gly Leu Lys
120
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.
O91/09610 PCT/EP90/02231
17
~ 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.
