Note: Descriptions are shown in the official language in which they were submitted.
~ 33773 1
INHIBITORY TETRAPEPTIDE OF ENTRY INTO CYCLE OF
HEMOPOIETIC STEM CELLS, PROCESSES FOR ITS PREPARATION,
AND ITS USES.
The invention relates to a novel tetrapeptide
acting as an inhibitor of entry into cycle of hemopo-
ietic stem cells as well as its usual derivatives in the
field of peptides with biological activity.
The invention also relates to a process for the
extraction of this tetrapeptide from biological subst-
ances, particularly from fetal calf marrow and a process
for its synthesis, as well as of its substitution deriv-
atives, by the chemical route.
It also relates to the uses of this peptide and
of its substitution derivatives in biology and in
medicine, particularly in the protection of bone marrow
in the course of anti-cancer treatments by chemotherapy.
The use of medicines in the treatment of cancers
is limited by their toxic effects on healthy tissues,
and in particular, on the hemopoietic tissue. The
repeated use of these drugs results in a large number of
cases either in lethal marrow aplasia, or a secondary
leukaemia, or less serious hematological sequelae.
As the majority of anti-cancer medicines only
act on proliferating cells, Applicants have thought that
it would be possible to prevent hematological damages by
protecting pluripotent stem cells by proliferation
inhibitors.
Studies carried out by Applicants and previously
published [see particularly E. Frindel and M. Guignon,
Exp. Hemat., 5 (1977), 74-76, M. Guignon and E. Frindel,
Bull. Cancer 68(2), (1981), 150-153, M. Guigon, J.Y. Ma-
ry, J. Enouf and E. Frindel, Cancer Res. 42 (1982) 638,
J. Wdzieczak-Bakala, M. Lenfant and M. Guigon, IRCS Med.
Sci. 12 (1984) 868-869, J. Wdzieczak-Bakala, M. Guigon,
M. Lenfant and E. Frindel, Biomed. Pharm. 37 (1983),
~,
~,
2 l 337731
467-471 and M. Guigon, J. Wdzieczak-Bakala, J.Y. Mary
and M. Lenfant, Cell Tissue Kinet. 17 (1984), 49-55]
have shown that it was possible to reduce significantly,
by administration of a specific inhibitor of stem cells,
the lethality observed in animals in the course of
treatments with cytosine arabinoside (Ara-C), a drug
currently used for chemotherapy of cancer, whose use is
limited by its injurious effect on bone marrow. That
specific inhibitor which is extracted from certain
biological substances, particularly from fetal calf bone
marrow or fetal calf liver, protects the stem cells
which are at the origin of all blood lines.
In fact, biological studies carried out show
that the increase in survival observed can be attributed
to a protection of the hemopoietic stem cells, CFU-S
(Colony Forming Units in the Spleen) that is to say
pluripotent stem cells capable of giving rise to clones
in the spleen of mice irradiated at a lethal dose, this
protection being due to the maintenance of said cells
outside of the cellular cycle, by the inhibitor.
Published documents describe techniques enabling
more or less purified extracts containing the specific
inhibitor to be obtained, but in all cases the product
finally obtained is not homogeneous and the yields are
unsatisfactory. It has therefore not been possible up to
now to isolate the active principle and to establish its
structure.
Now, Applicants have now developed a novel ex-
traction process which enables a homogeneous active
fraction to be obtained from a biological material,
particularly from fetal calf marrow.
This process comprises essentially the steps
consisting of:
- grinding the starting material with delipid-
ation if necessary,
- suspending the product obtained in a buffer at
3 1 337731
a pH close to 7, in the presence of a sulphur-containing
reducing reagent, particularly dithioerythritol or pref-
erably mercaptoethanol,
- centrifuging the homogenizate at about 15 OOOg
for at least one hour,
- subjecting the supernatant to ultrafiltration
on a membrane having an exclusion limit close to 10 OOO
daltons,
- subjecting the ultrafiltrate obtained to
chromatography on a molecular sieve of the polyacryl-
amide gel type, with elution by a dilute solution of
acetic acid,
- subjecting the active fraction collected to
fractionation on a reverse phase support of silica
grafted with aliphatic residues, particularly with 18C,
repeating this step preferably at least once; and
- subjecting the novel active fraction collected
to high pressure liquid chromatography on a reverse
phase column of silica grafted with aliphatic residues,
particularly with 18C, to obtain a homogeneous active
fraction.
The first five steps are advantageously carried
out at +4-C.
The "active fraction" is identified by biologic-
al tests, particularly as described in the experimental
part which follows.
A detailed example, which is not limiting, of
the practice of this extraction process is given in the
experimental part of the present specification.
The combination of different analysis techni-
ques, particularly NMR, mass spectrometry and analysis
of the amino acid content by high pressure liquid
chromatography (HPLC) has enabled the determination of
the structure of the homogeneous product so isolated.
This product is an acetylated tetrapeptide of molecular
weight 487, corresponding to the formula
4 l 33773 1
Ser(N-Ac)-Asp-Lys-Pro-OH
or
N-acetyl-seryl-aspartyl-lysyl-proline.
The results previously described did not permit
one to think that the activity observed of the more or
less purified extracts obtained could be due to a tetra-
peptide of the above-identified type.
In fact, there had especially been observed a
protective effect of mercaptoethanol on the inhibitory
fraction which seemed due to the presence of thlol
groups in the inhibitor molecule, the presence of such
groups having been established in the relatively purif-
ied inhibitory fraction obtained previously (see IRCS
Med. Sci. 12, (1984), 868-869).
In the same way, sugar residues having been
detected, it was thought that the active principle could
be of the glycopeptide type, this supposition being sup-
ported by the fact that the inhibitor concerned can be
classified among the chalones or anthormones and that
the majority of substances of this type are glycosylat-
ed.
The invention therefore relates to the tetra-
peptide
Ser-Asp-Lys-Pro-OH
and its substitution derivatives by one or several
groups, identical or different, currently used in the
chemistry of peptides for biological use. Advantageous-
ly, these groups are selected from among acetyl, benzyl,
methyl, and phenyl. The derivative monoacetylated on the
terminal nitrogen is most particularly preferred.
The invention also relates to the pharmaceutic-
ally acceptable salts of these compounds.
` 5 l 33773~
The tetrapeptide Ser-Asp-Lys-Pro-OH may be ob-
tained by extraction from biological materials, part-
icularly from fetal calf liver or marrow.
It can particularly be obtained by the process
described generally above and in more detail in the
experimental part. It is then in the acetylated (in the
N-terminal position) form and may be used as such or be
deacetylated according to conventional techniques in
peptide chemistry.
The peptide according to the invention and its
substitution derivatives can also be obtained by peptide
synthesis, especially in the liquid phase.
The liquid phase peptide synthesis advantageous-
ly takes place by successive additions, from the termin-
al C end, of the appropriate amino acid groups, suitablysubstituted or protected on their reactive groups, with,
when necessary, removal of the protective groups.
The inhibitor obtained according to the invent-
ion does not seem to have species specificity (in the
trials described here it is extracted from fetal calf
marrow and is active in the mouse), it is therefore
possible to contemplate using it, as well as its sub-
stitution derivatives, in the same way as their pharmac-
eutically acceptable salts, in numerous medical and
biological applications in man and in the animal.
These compounds may be used for the protection
of the bone marrow during anti-cancer treatments by
chemotherapy.
In this case, they are advantageously formulated
as a powder state and administered, in the presence of
usual adjuvants and/or excipients, i.v. or s.c., in the
course of the chemotherapeutic treatment. The frequency
of the administrations and the amount of product admin-
istered depend essentially on the kinetics of the stem
cells which can vary according to the therapeutic agent,
its posology and the protocol of its use.
6 l 3~7731
Other methods of "administration" may be en-
visaged, for example that consisting of causing the
inhibitor to be produced in the organism of the patient
by resorting to techniques of genetic engineering, for
example by means of bacteria.
The tetrapeptide and its substitution derivat-
ives according to the invention can besides be used for
obtaining specific antibodies for the determination of
the level of inhibitor circulating in the patients and
the determination of its role in certain diseases of the
hemopoietic system, as well as in the course of bone
marrow graftings.
The use of these antibodies, in the case of
hyperproduction of the inhibitor associated with patho-
logies, may also be envisaged.
ExPerimental Part.
I. Extraction of the PePtide N-acetvl-serYl-asPartvl-
lvsYl-Proline.
The raw material is constituted by fetal calf
bone marrow preserved frozen. At each of the steps
described below, the fractions isolated are supplemented
with mercaptoethanol at the final concentration 10 2M.
5kg of tissue are ground by means of a homogen-
izer of the Waring blender type (3 times, 60 sec.) in
40l of 10 2M phosphate buffer, pH 7.2 in the presence of
10 2M mercaptoethanol at 4-C. The suspension is centrif-
uged at 15 OOOg for lh30.
The supernatant collected is ultrafiltered on
membrane of the Sartorius 121 36 type which enables the
separation of the molecules according to their molecular
weight and the exclusion limit of which is 104 daltons.
The ultrafiltrate is concentrated 20 times by
evaporation under vacuum by means of a "flash evapor-
ator" (LUWA) type evaporator. The concentrate is then
lyophilized. The yield is 16.5g/kg and the fraction is
7 1 337731
active in the mouse at the dosage level of about 20mg/-
mouse (activity determined according to one or the other
of the protocols that are described below). The lyo-
philized powder (SOg) is then redissolved in 200ml of a
10 2M acetic acid solution (pH 3) then centrifuged (10
min., lS OOOg). The supernatant is chromato~raphed on a
column of molecular sieve of the Bioge~P-2 type, having
about 0.07 to 0.04mm "mesh opening" (200-400 mesh) (BIO-
RAD), of dimensions 12.5 x 100cm, and eluted with 201 of
10 2M acetic acid (flow rate 400ml/hour).
The active fraction is eluted with the elution
ratio Ve/Yo 1.2-1.8, Ve being the elution volume and Vo
the void volume of the column. The yield is 250mg/kg of
starting material and the fraction is active in the
mouse at the dosage level of about 5~g/mouse.
The active fraction (lOmg) is then dissolved in
lml water, then fractionated successively onto two cart-
ridges of in reverse phase support of silica grafted
with aliphatic residues with 18C of the Sep-pa~ C-18
type (WATERS). The cartridges are eluted in order with:
- 2ml of H20
- 2ml of a mixture H20/CH30H (50/50)
- 2ml of CH30H
The active fraction eluted with the mixture
H20/CH30H (50/50) is then concentrated in vacuum, at a
temperature of about 20 C on an apparatus of the Speed
Vac type, then lyophilized.
The purification is then continued by high pres-
sure liquid chromatography on a reverse phase analytical
column of octadecyl-silica of the ODS-Hypersil~C-18
type. (250 x 4.5mm) 5~ ~SFCC). The elution is done with
the mixture H20, 0.1% CF3COOH - MeOH (80-20) or the mix-
ture H20, 0.1% CF3COOH - CH3CN (95-5) with a flow rate
of lml/min. The detection of the fractions is carried
out by measurement of the absorption at 215nm.
The results obtained are as follows:
'P'~
8 1 33773~
1. Solvent (H20, 0.1% CF3COOH)/CH30H, (80/20)
flow rate lml/min.; retention time: 6 min.: one homogen-
eous peak.
2. Solvent (H20, 0.1% CF3COOH/CH3CN, (95/5) flow
rate lml/min.; retention time: 18 min: one homogeneous
peak.
It is observed that this process enables the
obtaining of a homogeneous product whose structure is
determined as indicated below.
The overall yield is 60~g/kg of starting mater-
ial.
The fraction is active in the mouse at the dos-
age of about 100ng/mouse.
II. Determination of the structure of the active Princ-
iPle.
1. AnalYsis of the amino acids:
After acid hydrolysis, the analysis by HPLC of
the derivatives formed by reaction with orthophthaldial-
dehyde enables the presence of lysine, aspartic acid and
serine to be observed. Analysis of the product by means
of an amino acid analyzer, after acid hydrolysis and
oxidation with sodium hypochlorite, enables the presence
of proline to be observed and the presence of aspartic
acid, serine and lysine to be confirmed.
2. NMR sPectroscoPv:
The one dimension and two dimension NMR (H20 and
D20) shows the presence of four amino acids (proline,
lysine, aspartic acid, serine) and of an acetyl group.
The NH2 of the side chain of the lysine and the OH of
the serine are free, the NH2 of the amino acid parts of
the serine, of the lysine and of the aspartic acid are
substituted once. The probable structure is that of a
peptide acetylated on the terminal NH2.
3. Mass sPectrometrY:
The mass spectrometry (MS) techniques in FAB
9 1 337731
(Fast Atom Bombardment) have shown that this peptide has
a molecular weight tM+1) of 488.
A study of the sequence of amino acids by a
modification of mass spectometry, namely the so-called
MS-MS technique, has enabled it to be established that
the complete primary structure of the peptide is:
Ser(N-Ac)-Asp-Lys-Pro-OH
III. DescriPtion of bioloqical tests:
The biological activity of the fractions is
measured by in vivo inhibition tests of the entry into
cycle of the CFU-S, induced by an injection of Ara-C.
The CFU-S of the mouse, normally quiescent,
enter into cycle after an injection of Ara-C. Applicants
have shown that when the inhibitor is injected 6 hours
after the drug, it prevents the entry of the CFU-S into
the synthesis of DNA -(S phase).
The number of CFU-S is determined by the tech-
nique of J.E. Tlll and E.A. McCulloch, Radiat. Res. 14,
tl961), 213-222, whose principle rests on the capacity
of CFU-S to form macroscopic colonies in the spleen.
These colonies are clones descended from one CFU-S.
The proportion of CFU-S in S phase is determined
by the method of A.J. Becker et al., Blood, 26. (1965),
296-308, based on the principle of cellular suicides.
The incubation of the cells with a tritiated thymidine
(precursor of DNA) solution leads to the selective death
of the cells in phase of DNA synthesis by integration of
a lethal dose of radioactivity (tritium) into the DNA
molecule.
The tests are performed on SPF (Specific Patho-
gen Free) mice of Balb/C or CBA strain, aged from 2 to 3
months.
1. The control groups receive by the i.p. route
the following treatment:
1 33773 1
Time 0: Ara-C (lOmg - protocol I or 20mg - protocol II)
dissolved in 0.2ml of saline:
Time 6h: 0.2ml of saline;
Time 8h: sacrifice (protocol I);
S Time 12h: sacrifice (protocol II).
2. The treated groups receive by the i.p. route:
Time 0: Ara-C (lOmg - protocol I or 20 mg - protocol II)
dissolved in 0.2ml of saline.
Time 6h: fraction to be tested dissolved in 0.2ml of
saline;
Time 8h: sacrifice (protocol I);
Time 12h: sacrifice (protocol II).
Protocol I Protocol II
0 6 8 12
hours
I I I I--
r\
t t
Ara-CInhibitor
Determination of
the number of CFU-S
in cycle
For each of the groups, the bone marrow of the
animals is collected and suspended in 2 ml of Medium 199
(EUROBIO). After the numbering and suitable dilution, 2
aliquots of 5 x 106 nucleated cells are incubated at
37-C for 20 min. either with lml of Medium 199 (Test A),
or with lml of tritiated thymidine (200 ~Ci) (Test B). A
cellular suspension of 0.2ml containing 8 x 104 to 1.2 x
105 nucleated cells is injected intravenously into the
retro-orbital sinus of the irradiated receiver animals
(9 Gy, 8 mice per experimental plot). Nine days later,
the receiver mice are sacrified, their spleen taken out
1 337731
11
and fixed in Bouin fluid (1% picric acid: 720g; formol:
240g: acetic acid: 4g, per 1l of fluid). After some
hours of fixation, the nodules visible macroscopically
are counted.
If N and N' are the average numbers of the
nodules obtained on the spleens of the receivers having
received cells coming from tests A and B respectively,
the proportion of CFU-S in phase of DNA synthesis is
calculated by the formula:
N-N'
% of CFU-S in S = ------ X 100
N
Results:
1. Inhibition of the entrv into cYcle of CFU-S.
In the course of each purification step, a
fraction active with respect to this test is isolated.
However, a particularly interesting activity is observed
with the homogeneous fraction obtained according to the
invention, as is shown by the following table:
12 l 33773t
Inhibition of the entrY into cYcles of CFU-S bY iniect-
ion of 100nq Per mouse of the homoqeneous fractlon ob-
tained accordinq to the invention, accordinq to Protocol
II.
TABLE I
Treatment % of CFU-S in
DNA synthesis
Ara-C (20mg) 43 + 9
Ara-C (20mg) 7 + 4
+ inhibitor (100 ng)
2. ActivitY on the survival of animals treated
with lethal doses of Ara-C.
The animals receive fractionated doses of Ara-C
(4 x 925mg/kg) at times 0, 7, 24 and 30 hours. The
tested fraction is administered at once 26 hours after
the first injection of Ara-C. The survival of the
animals is estimated 15-30 days after the treatment. In
this protocol comprising lethal, repeated injections of
Ara-C, it was shown that the concomitant administration
of the inhibitor enabled the definitive survival of a
~0 large number of animals. The results obtained are
comparable with those observed when an isogenic bone
marrow grafting is performed in the treated animals,
instead of using the inhibitor.
13 l 337731
Protocol:
O 7 24 26 30
hours
I I I I I
t t t t t
Ara-C Ara-C Ara-C Ara-C
Inhibitor
3. Absence of activitY of the inhibitorY fract-
ion on the reqression of EMT6 tumors.
The treatment of animal carriers of the EMT6
tumor by 4 successive injections of Ara-C at times O, 7,
24 and 30 hours followed by an injection of the inhibit-
or 26 hours after the first injection of Ara-C, does not
prevent the therapeutic action of Ara-C with respect to
tumor cells and permits the survival of the treated
animals comparably with the results obtained following a
bone marrow grafting.
4. Toxicitv
The inhibitory fraction is devoided of toxicity
with respect to CFU-S and does not result in any lethal-
ity in the treated mice, at the doses used.
IV. Liquid Phase sYnthesis of NAc-Ser-AsP-LYs-Pro-OH,
written conventionally abreviated AcSDKP.
a) SYnthesis of Boc-K(Z)-P-OH (I):
To a solution of Boc-K(Z)-OSu (4.19 mmoles) in
DMF is added an aqueous solution of P (8.38 mmoles) in
which 1.15ml of triethylamine (8.38 mmoles) have been
placed. The mixture is stirred overnight at room temper-
ature.
After evaporation of the solvent, the residue is
- 1 33773t
14
dissolved in ethyl acetate. The solution washed success-
ively with a 5% citric acid solution, a 5% sodium
carbonate solution and with water, is dried over MgS04.
After evaporation of the solvent, the product thus
obtained is characterized:
- yellowish oil: 3.98 mmoles; yield: 95%
~ []D = -38- (C=1.0; MeOH)
Rf: 0.5 (MeOH/CHCl3: 1/9); 0.24 (EtOAc/MeOH: 8/1)
The Rfs are determined by thin-layer chromatography on
silica.
- NMR performed in solution in CDCl3: in agreement.
Note: it is recalled that
Boc = tert-butyloxycarbonyl:
Z = benzyloxycarbonyl;
Su = sulfonamide;
DMF = dimethylformamide.
b) SYnthesis of Boc-D(OBzl)-K(Z)-P-OH (II):
To a solution of Boc-D(OBzl)-OSu (3.31 mmoles)
in DMF is added a solution of TFA.K(Z)-P-OH (3.15mmoles)
in DMF containing 915~1 of triéthylamine, namely 6.62
mmoles. The solution of TFA.K(Z)-P- OH is obtained by
treatment of product (I) with a solution of TFA (10
equivalents) and anisol (1 equivalent) for 1 hour at
O-C. It is washed with hexane then with ether before
being used for the following step. The mixture thus
constituted is placed under stirring overnight at room
temperature. After evaporation of the solvent, the
residue is solubilized in ethyl acetate and washed as in
a) before being characterized:
- oil: 2.17 mmoles; yield: 707~;
~ [a]D = 20- (C=1.2; MeOH);
- Rf: 0.23 (MeOH/CHCl3: 1/9);
- NMR performed in solution in CDCl3: in agreement.
Note: it is recalled that
TFA = trifluoroacetic acid, and
Bzl = benzyl.
- 15 l 33773t
c) SYnthesis of Boc-S(Bzl)-D(OBzl)-K(Z)-P-OH
(III):
According to the same sequence as in b), 1.47
mmoles of 80c-S-(Bzl)-Osu are added to 1.47 mmoles of
TFA.D(OBzl)-K(Z)-P-OH.
The product obtained has the following charact-
eristics:
- oil: 1.31 mmoles; yield: 89Yo;
- [a] D = -27- (C=1.0; MeOH);
- Rf: O. 29 (MeOH/CHCl3: 1/9);
- NMR carried out in solution in CDCl3 supplemented with
a small amount of MeOD to improve the solubility and the
resolution of the spectra: in agreement.
d) Svnthesis of AcS(Bzl)-D(OBzl)-K(Z)-P-OH (IV):
After elimination of the N-terminal Boc of pro-
duct (III), the new blocking group is added in a single
step. The final product, after washings, shows the
following characteristics when 1 mmole of TFA.S(Bzl)D-
(OBzl)K(Z)POH is used:
- oil: 0.77 mmoles; yield: 77%;
~ [~D = -22- (C=l.O; MeOH);
- Rf: O.12 (CHCl3/MeOH: 9/1); 0.54 (EtOActMeOH/CH3COOH;
16/3/1);
- NMR performed in solution in CDCl3 supplemented with
MeOD; in agreement.
e) Obtaininq of Ac-S-D-K-P-OH (V):
Product (IV) in solution in methanol is subject-
ed, after an addition of palladized carbon, to hydro-
genolysis. After completion of the reaction, the metha-
nol is evaporated and the residue is characterized.
This final product which is after lyophilization
in the form of a white solid, is characterized by the
same physico-chemical techniques as the natural peptide,
namely:
- analysis of amino acids;
- high performance liquid chromatography;
16 l 337731
- NMR in solution in D20, and
- FAB and FAB tMS-MS) mass spectrometry.
In all theses techniques, a good correspondence
is observed of the results obtained for the synthetic
product V with those obtained for the natural product.
In particular, the NMR spectra of the proton of
the natural and synthetic peptides are wholly superpos-
able as well as the mass spectra.
Analysis of this synthetic tetrapeptide by re-
verse phase HPLC on ODS-Hypersil C18 analytical column,
under isocratic conditions, with the mixture CH3CNtH20,
0.1% TFA [4.5t95.5], with a detection at 215nm, enables
the observation that the peak at 18 minutes, à 25-C
includes a slight shoulder which appears more distinctly
in the first part of the peak at 24 minutes, at 15-C.
This peak at 24 minutes, at 15-C can be split into a
peak of low intensity at 22 minutes, corresponding to
the preceding shoulder, and into a peak at 24 minutes.
The product corresponding to this peak possesses the
biological properties of the natural peptide.
It is subjected to the previously described
tests in relation with the natural inhibitor extracted
according to the invention.
The percentages of CFU-S in phase of DNA syn-
thesis obtained are collected in table II which follows.
17 l 33773~
TABLE II
Dose of synthetic Control ARA-C ARA-C +
tetrapeptidetmouse synthetic
tetrapeptide
___ __ _ ____ __ _ _ ____ ___ _ _ _ __
1. 100 ng 20 44 12
2. 100 ng 10 30
3. 100 ng - 55 17
104. 100 ng 0 26 6
5. 50 ng - 35 13
6. 25 ng 0 48 19
7. 100 ng 0 36 10
8. 100 ng 0 36 6
mean 5 38.7 10.5
Tests 1, 2 and 3 were carried ou with the tetra-
peptide obtained by a first synthesis, tests 4, 5, 6, 7
and 8 were performed with the tetrapeptide obtained by a
second synthesis.
The results obtained are to be compared with
those obtained with the natural product and which are
shown in table I, on one hand and in table III, on the
other hand.
The latter results were obtained with the
natural product extracted from two different batches A
and B of bone marrow.
TABLE III
Batch of bone dose/ Control ARA-C ARA-C +
marrow mouse natural
peptide
____ _ __ _ __ _ __ _ ____ __
A (5 exp.) 100 ng6.5+13.9 40.7+9.3 12.6+14.8
B (2 exp.) 100 ng11.9+16.3 47.4+9 2.99+13
mean 9.2 44.1 7.8
18 1 337731
Comparative study of tables I and III, on the
one hand, and II, on the other hand, enables the observ-
ation that the synthetic peptide has a quite similar
activity to that obtained with a natural peptide.
The results obtained are shown diagrammatically
in the single appended drawing.
