U.S. patent application number 10/695382 was filed with the patent office on 2006-08-24 for novel gnrh analogues with antitumour effects and pharmaceutical compositions thereof.
Invention is credited to Dezso Gaal, Adrienn Kalnay, Sandor Lovas, Imre Mezom, Melinda Mora, Richard F. Murphy, Istvan Palyi, Janos P. Pato, Janos Seprodi, Edit Z. Szabo, Henrietta Tanai, Istvan Teplan, Geza Toth, Gizella Turi, Zsolt Vadasz, Borbala Vincze.
Application Number | 20060189539 10/695382 |
Document ID | / |
Family ID | 29740482 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060189539 |
Kind Code |
A1 |
Lovas; Sandor ; et
al. |
August 24, 2006 |
Novel GnRH analogues with antitumour effects and pharmaceutical
compositions thereof
Abstract
The present invention provides peptides and pharmacologically
active compounds including gonadotropin-releasing hormone (GnRH)
analogues according to formulas of the invention, wherein the
compounds show an antitumour effect. The invention additionally
provides compositions including these peptides and compounds.
Inventors: |
Lovas; Sandor; (Omaha,
NE) ; Murphy; Richard F.; (Omaha, NE) ; Toth;
Geza; (Szeged, HU) ; Kalnay; Adrienn;
(Budapest, HU) ; Gaal; Dezso; (Budapest, HU)
; Palyi; Istvan; (Budapest, HU) ; Turi;
Gizella; (Budapest, HU) ; Vincze; Borbala;
(Budapest, HU) ; Mezom; Imre; (Budapest, HU)
; Tanai; Henrietta; (Budapest, HU) ; Vadasz;
Zsolt; (Budapest, HU) ; Teplan; Istvan;
(Budapest, HU) ; Seprodi; Janos; (Budapest,
HU) ; Szabo; Edit Z.; (Budapest, HU) ; Pato;
Janos P.; (Budapest, HU) ; Mora; Melinda;
(Budapest, HU) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581415
MINNEAPOLIS
MN
55458
US
|
Family ID: |
29740482 |
Appl. No.: |
10/695382 |
Filed: |
October 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09269954 |
Jul 27, 2000 |
6664369 |
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PCT/US95/10054 |
Aug 9, 1995 |
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10695382 |
Oct 28, 2003 |
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Current U.S.
Class: |
530/328 ;
514/10.1; 514/10.3; 514/19.3; 514/9.9; 530/326 |
Current CPC
Class: |
C07K 7/23 20130101 |
Class at
Publication: |
514/015 ;
530/326 |
International
Class: |
A61K 38/09 20060101
A61K038/09 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 8, 1994 |
HU |
94 02328 |
Oct 8, 1994 |
HU |
94 02329 |
Claims
1-17. (canceled)
18. A pharmacologically active compound of formula (I)
Y(W.sub.u,V.sub.z,X.sub.r,A.sub.k) (I) wherein Y represents the
molecular moiety of formula (Ia), ##STR5## wherein n is an integer
from 10 to 400; one of R.sub.1 and R.sub.2 represents hydrogen atom
whereas the other one represents a group of formula (B); ##STR6##
R.sub.3 represents a polymerization-initiating group; W represents
a hydroxyl group, optionally as a salt formed with an alkali metal
ion; V represents a C1-8 alkylamino group bonded through its amino
group or a valence bond; X is a "spacer" group being an amino acid
group or an oligopeptide group of at most six members wherein the
amino acid or oligopeptide group is coupled through its N-terminal
to the Y group and is optionally bearing a hydroxyl group or a
valence bond on its C-terminal, wherein the amino acids are Gly,
Ala, Leu, Ile, Val, Phe, Tyr, Ahx, Pro, Arg, or His; A is present
and represents a pharmacologically active polypeptide hormone group
containing an amino group and directly coupled therethrough to the
Y group when r is 0; or coupled to the C-terminal of the X group,
respectively, when r is larger than 0; r is an integer from 0 to
0.2 n; k is an integer being at most equal to r; z is an integer
from 0 to (n-r); and u is an integer from n to 2n-r-z, as well as
the salts and complexes of these compounds.
19. The pharmacologically active compound of formula (1) of claim
18, wherein R.sub.3 is a (CH.sub.3).sub.2CCN group.
20. The pharmacologically active compound of formula (I) of claim
19, wherein: A represents a native gonadotropin-releasing hormone
(GnRH) coupled through its amino group or a pharmacologically
active analogue thereof; and k, r, u, z, X, Y, V and W are as
defined in claim 18, as well as the salts and complexes of these
compounds.
21. The pharmacologically active compound of formula (I) of claim
20, wherein A represents
pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH.sub.2 (SEQ ID NO:2),
Ac-D-Trp.sup.1,3,
p-chlorophenyl-D-alanine.sup.2(D-Cpa.sup.2),D-LyS.sup.6,D-Ala.sup.10-gona-
dotropin-releasing hormone (GnRH)
Ac-D-Trp.sup.1,3,D-Cpa.sup.2,Lys.sup.5,[Asp(a-DEA)].sup.6,D-Ala.sup.10-Gl-
n.sup.8-GnRH, D-Phe.sup.2,D-Trp.sup.3,D-Lys.sup.6-GnRH,
Lys.sup.5,cyclo(Asp.sup.6-Lys.sup.8)-GnRH-III,
Lys.sup.4,[Lys(.epsilon.-Fmoc)].sup.8-GnRH-III, Lys.sup.4-GnRH-III,
D-Lys.sup.6-GnRH, Lys.sup.5,D-Trp.sup.6-GnRH coupled to X or Y
through the .epsilon.-amino group of their Lys side chains; and k,
r, u, z, X, Y, V and W are as defined in claim 18, as well as the
salts and complexes of these compounds.
22. The pharmacologically active compound of formula (I) of claim
18, wherein X represents an oligopeptide group consisting of four
members; and k, r, u, z, A, Y, V and W are as defined in claim 18,
as well as the salts and complexes of these compounds.
23. The pharmacologically active compound of formula (I) of claim
18, wherein X represents an oligopeptide group consisting of three
members; and k, r, u, z, A, Y, V and W are as defined in claim 18,
as well as the salts and complexes of these compounds.
24. The pharmacologically active compound of formula (I) of claim
18, wherein V is a C4-6 alkylamino group.
25. The pharmacologically active compound of formula (I) of claim
18, wherein r. is 0; and k, u, z, A, Y, V and W are as defined in
claim 18, as well as the salts and complexes of these
compounds.
26. A compound containing an activated ester group of formula (Ic),
Y[W.sub.u,V'.sub.z,(XOQ).sub.r] (Ic) wherein Y represents the
molecular moiety of formula (Ia), wherein n is an integer from 10
to 400; one of R.sub.1 and R.sub.2 represents hydrogen atom whereas
the other one represents a group of formula (B); R.sub.3 represents
a polymerization-initiating group; W represents a hydroxyl group,
optionally as a salt formed with an alkali metal ion; V' represents
a C1-8, alkylamino group bonded through its amino group; X
represents an amino acid group or an oligopeptide group of at most
six members coupled through its N-terminal to the Y group; OQ
represents an activated ester group on C-terminal of the X group; r
is an integer from 0 to 0.2 n; z is an integer from 0 to (n-r); and
u is an integer from n to (2n-r-z), as well as the salts of these
compounds.
27. Compounds of formula (Ic) as claimed in claim 26, wherein X
represents an oligopeptide group consisting of at most four
members, preferably -Gly-Phe-Leu-Gly-, -Gly-Phe-Gly-, -Phe-Leu-
Gly- or -Ahx-; OQ represents ONp group; and k, r, u, z, A, Y, V' as
well as W are as defined in claim 26, as well as the salts of these
compounds.
28. A pharmaceutical composition comprising a compound of formula
(I) of claim 18, wherein k. r, u, z, X, Y, V and W are as defined
in claim 18, or a pharmaceutically acceptable salt or complex
thereof in admixture with carriers and/or additives commonly used
in the pharmaceutical industry.
29. A tumour-inhibiting pharmaceutical composition comprising a
compound of formula (I) of claim 20 or a pharmaceutically
acceptable salt or complex thereof in admixture with carriers
and/or additives commonly used in the pharmaceutical industry.
30. A tumour-inhibiting and immunostimulatory pharmaceutical
composition comprising a compound of formula (I), wherein A
represents
Ac-D-Trp.sup.1,3,p-chlorophenyl-D-alanine.sup.2(D-Cpa.sup.2),Lys.sup.5,[A-
sp(a-DEA)].sup.6,D-Ala.sup.10-Gln.sup.8-GnRH, and k, r, z, u, X, Y,
V and W are as defined in claim 18, or a pharmaceutically
acceptable salt or complex thereof in admixture with carriers
and/or additives commonly used in the pharmaceutical industry.
31. A composition comprising a compound of claim 18 in combination
with a pharmaceutically acceptable carrier.
32. The pharmacologically active compound of claim 18, wherein n is
an integer from 20 to 200.
33. The pharmacologically active compound of claim 22, wherein X
represents -Gly-Phe-Leu-Gly- (SEQ ID NO:5).
34. The pharmacologically active compound of claim 23, wherein X
represents -Phe-Leu-Gly-.
35. The pharmacologically active compound of claim 23, wherein X
represents -Gly-Leu-Gly-.
36. The compound of claim 26, wherein n is an integer from 20 to
200.
37. The compound of claim 26, wherein R.sub.3 represents
(CH.sub.3).sub.2CCN.
38. The compound of claim 26, wherein W represents a hydroxyl grop
as a salt formed with a sodium ion.
39. The compound of claim 26, wherein V' represents a C4-6
alkylamino group.
40. The compound of claim 26, wherein the activated ester group is
selected from the group consisting of ONp, OPcp, Opfp, and
ONsu.
41. A tumour-inhibiting pharmaceutical composition comprising a
compound of formula (I) of claim 21 or a pharmaceutically
acceptable salt or complex thereof in admixture with carriers
and/or additives commonly used in the pharmaceutical industry.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to the fields of
biochemical endocrinology and anti-neoplastic pharmacology. More
specifically, the present invention relates to novel GnRH analogues
having antitumour effects and pharmaceutical compositions
thereof.
[0003] 2. Description of the Related Art
[0004] It is known that this factor of hypothalamic origin (a
peptide hormone built up of 10 amino acids) is responsible for the
secretion of luteinizing hormone (LH) and follicle-stimulating
hormone (FSH). A number of agonistic and antagonistic analogues of
GnRHs proved to be not only useful for the successful influencing
of processes of the reproduction biology but also suitable as
antitumour drugs.
[0005] GnRH analogues can exert their antitumour effect not only
through chemical castration but also in a selective way by directly
acting on the tumour cells. The presence of receptor(s)
specifically binding GnRH or GnRH analogues, respectively,: as well
as that of GnRH-mRNA were shown in cell cultures of cancers of
human mammary, prostate, ovary and pancreas. Furthermore, the in
vitro proliferation-inhibiting action of GnRH analogues was proven
on the same cell lines. The specific binding of tritium-labelled
D-Phe6-GnRH(1-9)-ethylamide (OVURELIN), a human GnRH superagonist,
to cells of MCF-7 and MDA-MB-231 human mammary carcinoma cell lines
was demonstrated in own experiments. These results confirm the
presence of receptor(s) specifically binding GnRH analogues, which
is a fundamental condition for development of a direct effect.
[0006] Similarly, D-Trp6-hGnRH (DECAPEPTYL), an agonistic analogue
brought into the therapeutical practice, proved to possess a
receptor on the MDA-MB-231 tumour cell line and a direct
growth-inhibiting effect on the human mammary tumour cell line
mentioned above.
[0007] Based on the effective concentration it can be supposed that
low-affinity binding site or sites may play an important role in
the development of direct antitumour effect. According to the
literature the direct antitumour action of GnRH analogues occurs
only at relatively high peptide concentrations (10.sup.-6-10.sup.-5
M). This pharmacological effect can be achieved only in the case
when the active molecule is present in the body not only in a high
concentration but also for a long time. For a long time, GnRH was
not believed to be a species-specific hormone; it has become known
as late as in the early 80's that the structure of gonadoliberin of
some fish and bird species, respectively, is different from that of
the mammals. In comparison to the mammalian GnRH, the structure of
fish- and bird-specific GnRH, respectively, differs in the amino
acid position(s) 7 and/or 8. In relation to the release of LH and
FSH, respectively, of mammals, the analogues of chicken GnRH or
salmon GnRH are not hyperactive and therefore, they do not
desensitize the gonadotropic cells of hypophysis in the
corresponding dose range. The composition of mammalian, e.g. human,
GnRH is as follows:
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.
[0008] In 1993, researchers isolated and synthesized the
Lamprey-III-GnRH decapeptide from lamprey (Petromyzon marinus)
(pGly-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2). This
Lamprey-III-GnRH (herein below GnRH-III) exerts a significant
tumour growth-inhibiting effect on human mammary tumour cell
lines.; Simultaneously, on investigating the endocrinological
effect of GnRH-III on rat hypophysis by using the superfusion
method it has been found that the LH-releasing effect of this
hormone is about thousand times weaker than that of the human GnRH.
In the course of in vivo investigations it has been found that,
during a prolonged treatment for three cycles, it did not inhibit
the ovulation of female rats even in high doses; therefore, it did
not induce desensitisation and chemical castration. In relation
thereto, a "flair up" tumour growth occurring at the beginning of
treatment did not appear on the tumour-bearing animals in contrast
to other known human hormone analogues acting through the same
mechanism of action. Thus, GnRH-III is a selective, highly active
antitumour compound.
[0009] During the therapeutical use of peptide hormones and their
synthetic analogues, a frequent demand is to retain the amount of
the pharmacologically active molecule at a high and steady level.
Thus, e.g. several agonistic and antagonistic synthetic analogues
of gonadoliberin (GnRH), a peptide hormone built up from ten amino
acids stimulating the release of luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) from the hypophysis, proved to
be useful not only for successfully influencing processes of
reproduction biology but also to provide the possibility of use as
antitumour drugs. Depending on the way of use, GnRH and its
analogues are capable to stimulate or inhibit the secretion of
gonadotropin. On carrying out a treatment repeatedly or
continuously for a prolonged time with GnRH analogues, a so-called
pharmacological gonadectomy is induced as a result of a
desensitizing effect exerted on the hypophysis by a pronounced
reduction of release of gonadotropin and steroids. Gonadectomy is a
required therapeutical intervention in the treatment of
steroid-dependent gonadal disease-entities. Simultaneously,
gonadectomy is reversible and does not psychically disturb the
patient. These analogues proved to be effective in the therapy of
prostate, mamma and endometrium carcinomas and even cancers of
pancreas and hypophysis on the basis of most recent data. Synthetic
GnRH antagonists are derivatives competitively inhibiting the
native hormone. In the case of potent antagonists the amino acids
in positions 1 to 3, 6 and 10 are usually exchanged for non-coded
amino acids, e.g. of D-configuration. The anti-ovulatory effect of
GnRH antagonists is well known. The GnRH analogues may exert their
antitumour action not only through chemical castration but also
through a direct effect on the tumour cells. Eidene et al. [J.
Clin. Endocrinol. Metab. 64, 423-432 (1987)] demonstrated the
presence of low-affinity binding sites in the cell cultures of
MCF-7 and MDA-MB-231 human mammary tumours; based on the direct
inhibitory effect of antagonistic analogues they concluded that
GnRH behaves as an autocrine regulatory factor in mammary carcinoma
cells. Results most recently published in the literature confirm
the expression of GnRH gene in mammary carcinoma cells, which
likely occurs during lactation and malignant transformation [Harris
et al.: Cancer Res. 51, 2577-2581 (1991)]. The presence in the
neoplastic tissue of GnRH-binding sites supports that GnRH has very
probably an immediate palliative effect in the therapy of patients.
suffering from mammary carcinoma. Human mammary carcinoma cell
lines or xenografts developed therefrom are useful in vitro and in
vivo model systems for demonstrating the direct action of GnRH.
GnRH agonists, when administered alone in substance form, are
proteolytically decomposed and rapidly eliminated; thus, a steady
and high GnRH analogue level, required for development of a direct
or indirect antitumour effect, cannot be achieved. The retention of
a high and steady level required for the inhibition of the tumour
can be solved by a very frequent administration (daily several
times) or by means of pharmaceutical compositions with prolonged
effect. Up to the present, the microcapsule or microgranular form
of GnRH analogues has been used for tumour inhibition in the
clinical practice (Buserelin retard.RTM. and Decapeptyl retard.RTM.
agonistic compositions).
[0010] According to another method to achieve a prolonged effect
the pharmacologically active molecule is chemically coupled to
molecules (e.g. polymers) slowly eliminating from the body.
Conjugates thus obtained slowly diffuse in biological systems and
alter the distribution in the body and absorption properties of the
active agent. Thus, targeted transport of the pharmacologically
active molecule and the reduction of undesired side effects can
also be attained.
[0011] Preferred carriers are the water-soluble native and
synthetic polymers, first of all homo- or copolymers, respectively,
of carboxylic acids as synthetic polymers. Styrene/maleic anhydride
copolymer has a molecular weight of about 1600 to 2000 D,
consisting of 7 to 8 styrene/maleic anhydride units wherein maleic
anhydride had partially been hydrolyzed or esterified,
respectively, was successfully used for improvement of the
pharmacological properties of neo carcinostatin. In this way a more
favourable distribution in the body together with a reduction of
toxicity of the active agent could be achieved [H. Maeda et al.: J.
Med. Chem. 28, 455-461 (1985)].
[0012] Polyanionic macromolecules with higher molecular weight may
also possess themselves biological activity (e.g. antitumour,
immunoadjuvant, interferon-inducing effects). However, their
molecular weight and the distribution of the molecular weight
proved to be crucial, i.e. their toxicity was increased by the
increase in the molecular weight [L. Gros: Encyclopedia of Polymer
Science and Technology, Vol. 2, pp. 243-267; as well as G. Butler:
J. Macromol. Sci. Chem. A 13, 351-368 (1979); and R. Ottenbrite:
ibid. A 22, 819-832. (1985)].
[0013] Thus, such polymers can be considered as carrier molecules
that can be prepared with a good reproducibility, small
polydispersity, which are not toxic and are eliminated at an
optimum rate from the body. It is also important that the
pharmacologically active molecules be capable to be coupled with a
good reproducibility to these polymers to obtain a product with an
appropriate solubility in water and the desired pharmacological
activity.
[0014] Up to the present, peptide hormones and within these GnRH or
GnRH analogues have not been coupled to known polycarboxylic acids
as carriers. Copolymers prepared from vinylpyrrolidone and maleic
anhydride are described in the Hungarian patent specification No.
194,286. These copolymers were prepared for cosmetical use; they
are very useful skin-cosmetics and can be employed both in
hydrophilic as well as lipophilic type emulsions. Depending on the
amount of the base added, the pH value of their aqueous solutions
can be varied within wide limits, they possess a high buffer
capacity and can be prepared with a narrow range of polydispersity
and with a good reproducibility. The vinylpyrrolidone and maleic
anhydride units are incorporated to the molecule in a 1:1 molar
ratio, with an alternating sequence.
[0015] Earlier, the toxicity, elimination and body distribution of
the N-vinyl-pyrrolidone/maleic acid copolymer (NVP-MA) had been
investigated in detail and the following statements were made [M.
Azori et al: Macromol. Chem. 187, 287-302 (1986)]. On the
intraperitoneal or intravenous, respectively, administration of the
polymer (with an average molecular weight of 20,000) in the form of
its sodium salt of pH 7.2, no death occurred up to 900 mg/kg of
body weight in the first case (i.p.) or up to 200 mg/kg of body
weight dose, respectively, in the second case (i.v.). This
indicates that NVP-MA is less toxic than many other polyanions
having a similar structure. [The intravenous (i.v.) LD.sub.50 value
of a divinyl ether/maleic acid copolymer with similar molecular
weight is 74 mg, that of polymaleic acid is 110 mg and that of
furan/maleic acid copolymer amounts to. 130 mg.] Body distribution
examinations carried out with a .sup.14C-labelled polymer (having
an average molecular weight of 8000) indicated that the polymer
cannot enter the -brain and spinal cord. The polymer is eliminated
mainly in the urine: 84% of the radioactivity introduced were
eliminated during 24 hours whereas after 56 hours altogether 95% of
the radioactivity introduced could be detected in the urine and
faeces. NVP-MA and its known derivatives till now have not been.
used in the therapy but on the basis of their favourable properties
it seems possible to introduce them into the body without toxic
effects.
[0016] The prior art is deficient in the lack of effective means of
inhibiting the a wide variety of neoplastic conditions. The present
invention fulfills this longstanding need and desire in the
art.
SUMMARY OF THE INVENTION
[0017] The invention relates to novel, pharmacologically active
compounds of general formula (I), their salts and complexes as well
as to a process for preparing same. In the general formula (I)
Y(Wu,Vz,Xr,Ak) (I) Y means the molecular moiety of general formula
(Ia), ##STR1## wherein n is an integer from 10 to 400, preferably
20 to 200; one of R.sub.1 and R.sub.2 stands for hydrogen whereas
the other one means a group of formula (B), ##STR2## R.sub.3 means
a polymeriation-initiating group, preferably (CH.sub.3).sub.2CCN
group; W means a hydroxyl group, optionally as a salt formed with
an alkali metal ion, preferably sodium ion; V represents a C1-8,
preferably C4-6, alkylamino group bonded through its amino group;
or a valence bond; [0018] X means an amino acid group or an
oligopeptide group of at most six members coupled through its
N-terminal to the Y group and optionally bearing a hydroxyl group
or a valence bond on its C-terminal, wherein the amino acids are
Gly, Ala, Leu, Ile, Val, Phe, Tyr, Ahx, Pro, Arg or His;
[0019] A represents a pharmacologically active polypeptide group
containing an amino group and directly coupled therethrough to the
Y group when r is 0; or coupled to the C-terminal of the X group,
respectively, when r is larger than 0;
r is an integer from 0 to 0.2 n;
k is an integer being at most equal to r;
z is an integer from 0 to (n-r); and
u is an integer from n to 2n-r-, as well as te salts and complexes
of these compouns.
[0020] The invention furthermore relates to the novel intermediates
of general formula (Ic), Y[W.sub.u, V'.sub.z,(XOQ).sub.r] (Ic)
wherein Y means the molecular moiety of general formula (Ia),
wherein n is an integer from 10 to 400, preferably 20 to 200; one
of R.sub.1 and R.sub.2 stands for hydrogen atom whereas the other
one means a group of formula (B); ##STR3## R 3 means a
polymerization-initiating group, preferably (CH.sub.3).sub.2 CCN
group; W means a hydroxyl group, optionally as a salt formed with
an alkali 10 metal ion, preferably sodium ion; V'stands for a C1-8,
preferably C4-6, alkylamino group bonded through its amino group; X
represents an amino acid group or an oligopeptide group of at most
six members coupled through its N-terminal to the Y group; OQ means
an activated ester group on C-terminal of the X group, preferably
ONp, OPcp, OPfp or ONSu group; r is an integer from 0 to 0.2 n; z
is an integer from 0 to (n-r); and u is an integer from n to
(2n-r-z), as well as the salts of these compounds.
[0021] Furthermore, te invention relates to tumour-inhibiting and
immunostimulatory pharmaceutical compositions comprising as active
ingredient a compound of general formula (I). The bioconjugates of
general formula (I) possess a selective tumour-inhibiting effect; a
part of the compounds of general formula (I) inhibit the growth of
both steroid-dependent and steroid-independent tumours,
particularly mammary carcinomas. The compounds of general formula
(I) show the effect of the pharmacologically active moiety in an
increased and prolonged degree.
[0022] The invention relates to novel peptides possessing
antitumour effect as well as their salts and esters. In addition to
antiestrogens, gonadotropin-releasing hormone (GnRH) analogues play
an important role in the treatment of hormone-dependent malignant
tumours. Within the malignant neoplasms, the scope of their use
extends to the cancers of prostate, breast (mammary), endometrium
and other hormone-dependent tumours. The present invention prepares
analogues of human GnRH (hGnRH) and Lamprey GnRH-III showing
antitumour effect in human tumour cell cultures. This aim was
solved by the preparation of peptides of general formula (IV) as
well as their pharmaceutically acceptable salts and esters.
[0023] The invention is based on the recognition that these
compounds exert a direct antitumour action against human tumour.
cells. Unexpectedly, compounds containing only natural L-amino
acids also show a direct antitumour effect. Namely, antitumour GnRH
analogues known at present contain at least one of non- natural
D-amino acids in the case of agonists and usually several
non-natural D-amino acids in the case of antagonists.
[0024] Furthermore, it has been recognized that for more amino acid
groups of antitumour GnRH analogues Lys groups may be substituted
without any decrease in the favourable antitumour effect of the
molecule. This is advantageous since, through the c-amino group of
Lys, the peptide can be connected to suitably selected larger
molecules containing an acylating group. In this case, the
macromolecules may be carrier molecules of the peptide and can
thereby promote the maintenance of a steady, high level of the GnRH
analogue in the body.
[0025] The invention relates to the peptides of general formula
(IV), X-R1-R2-R3-R4-R5-R6-R7-R8-Pro-R10-Y (IV)
[0026] wherein
[0027] X means hydrogen, acetyl group or propionyl group when
R.sup.1 is different from pGly; or an intramolecular acid amide
bond when R.sup.1 stands for pGlu;
[0028] R.sup.1 stands for pGlu, Glu, D-Trp, D-Cpa, D-Nal or
D-Phe;
[0029] R.sup.2 means His, D-Phe or D-Cpa;
[0030] R.sup.3 represents D-Cpa, D-Pal or L- or D-Trp optionally
protected on the indolyl moiety;
[0031] R.sup.4 stands for Ser; or Lys optionally protected on the
.epsilon.-amino group;
[0032] R.sup.5 means Tyr; or Lys optionally protected on the
.epsilon.-amino group; or His;
[0033] R.sup.6 stands for Asp, Glu, D-Lys and optionally
.epsilon.-amino methylated derivatives thereof; as well as D-Trp,
D-Phe, D-Leu, D-Ala, D-Cpa or D-Arg;
[0034] R.sup.7 represents Phe, Leu or N-Me-Leu; or L-Trp optionally
. . . protected on the indolyl moiety; :
[0035] R.sup.8 means Lys optionally protected on the
.epsilon.-amino group; Arg, Gln; or R.sup.6 and R.sup.8 together
can form an intramolecular ring through the .epsilon.-amino group
of Lys when R.sup.6 is Asp and R.sup.8 means Lys;
[0036] R.sup.10 stands for Gly, D-Ala or a valence bond; and
[0037] Y represents OH or NH.sub.2 group when R.sup.10 means Gly or
D-Ala; or an ethylamide group when R.sup.10 means a valence bond,
as well as the pharmaceutically acceptable salts and/or esters of
these compounds. The invention furthermore relates to
pharmaceutical compositions containing the peptide of general
formula (IV) and/or pharmaceutically acceptable salts and/or esters
thereof.
[0038] The abbreviations used in the description agree with the
-nomenclature accepted in the peptide chemistry and published in
J.
[0039] Biol. Chem. 241, 527 (1966); 247, 977 (1972); furthermore,
D-NaI stands for .beta.-(2-naphthyl)-D-alanine, D-Cpa means
p-chlorophenyl-D-alanine and D-Pal stands for
.beta.-(3-pyridyl)-D-alanine. When not noted otherwise, all amino
acids named in the description are in L-configuration.
[0040] In the peptides according to the invention the preferred
protective group of the indolyl moiety of Trp is For; the preferred
protective group of the e-amino group of Lys is Fmoc. The
pharmaceutically acceptable salts of the peptides of general
formula (IV) are acid-addition salts formed with pharmaceutically
acceptable organic or inorganic acids, e.g. acetates or
hydrochlorides.
[0041] The compounds of general formula (IV) can be prepared in
liquid phase by using methods known in the peptide chemistry (by
condensations carried out in the defined sequence of suitably
protected amino acids or fragments prepared therefrom) or in a
particularly preferable way--by using the solid-phase peptide
synthesis. A peptide obtained in the form of its salt can be
converted to an other salt in a known manner. If desired, the ester
groups of ester compounds obtained may be cleaved.
[0042] The peptide of general formula (IV) may be administered
mainly in the form of injectable solutions, infusions or intranasal
compositions. Being decomposed in the digestive system, they cannot
be administered orally in themselves but may be administered in any
other route. The injections may be given in intramuscular,
intravenous or subcutaneous route.
[0043] The active agents of general formula (IV) can be formulated
to pharmaceutical compositions by using known methods of the
pharmaceutical techniques. The active agent can be transformed also
to compositions with prolonged action (e.g. in the form of
microcapsules or microgranules) in the usual way. In addition to
the active agent, auxiliaries commonly used in the pharmaceutical
industry such as a liquid vehicle useful for injection purposes
(isotonic saline or phosphate buffer solution) may. be used. If
necessary, the compositions may contain stabilizers (e.g. ascorbic
acid), too.
[0044] The preparation of peptides according to the invention is
illustrated by the following Examples. The chemical purity and
identification of both intermediary and final products were
controlled by using thin-layer chromatography (TLC); those of the
final products were examined by means of HPLC chromatography, too.
The thin-layer chromatography values were determined on Kieselgel
sheets (DC Alufolien, Merck) by using the following solvent
mixtures: TABLE-US-00001 1. Ethyl acetate/pyridine/water/acetic
acid 15:20:6:11 2. Ethyl acetate/pyridine/water/acetic acid
30:20:6:11 3. Ethyl acetate/pyridine/water/acetic acid 60:20:6:11
4. Ethyl acetate/pyridine/water/acetic acid 120:20:6:11 5. Ethyl
acetate/pyridine/water/acetic acid 240:20:6:11 6. n-Butanol/acetic
acid/water 4:1:1 7. n-Butanol/acetic acid/water 4:1:2
[0045] The side chains of protected amino acids are protected by a
benzyl group in the case of Tyr and Ser; by a (benzyloxy)carbonyl
(Z) group or a 9-fluorenyl(methoxycarbonyl) (Fmoc) group for
preparing an intermediary peptide analogue in the case of Lys; by a
tosyl (Tos) group in the case of Arg and His; and by a cyclohexyl
(Chx) group in the case of carboxy groups of Asp and Glu.
[0046] The invention is illustrated in more detail by description
of the preparation process of the preferred analogues listed
hereinbelow:
[0047] 1. [Lys(.epsilon.-Fmoc)].sup.5-GnRH-III,
[0048] 2. Lys.sup.5-GnRH-III,
[0049] 3. Lys.sup.5,cyclo[Asp.sup.6-Lys.sup.8]-GnRH-III,
[0050] 4. Lys.sup.5,[Lys(.epsilon.-Fmoc)].sup.8-GnRH-III,
[0051] 5. Lys.sup.4,[Lys(.epsilon.-Fmoc)].sup.8-GnRH-III,
[0052] 6. Lys.sup.4-GnRH-III,
[0053] 7. [Lys(.epsilon.-Ac)].sup.4-GnRH-III,
[0054] 8. Glu.sup.6-GnRH-III,
[0055] 9. cyclo[Asp.sup.6-Lys.sup.8]-GnRH-III,
[0056] 10. D-Ala.sup.10-GnRH-III,
[0057] 11. HD-Trp.sup.1, [Lys(.epsilon.-Fmoc)].sup.8,
D-Ala.sup.10-GnRH-III,
[0058] 12. Ac-D-Trp.sup.1, D-Ala.sup.10-GnRH-III,
[0059] 13. H-D-Trp.sup.1, D-Ala.sup.10-GnRH-III,
[0060] 14. [Trp(For-Ind)].sup.3,7-GnRH-III,
[0061] 15. Phe.sup.7-GnRH-III,
[0062] 16. GnRh-III(1-9)-ethylamide,
[0063] 17. Lys.sup.5, D-Trp.sup.6-hGnRH,
[0064] 18. Lys.sup.4, D-Trp.sup.6-hGnRH,
[0065] 19. H-Glu.sup.1, D-Trp.sup.6-hGnRH,
[0066] 20. Lys.sup.5, D-Phe.sup.6-hGnRH(1-9)-ethylamide,
[0067] 21. Lys.sup.4, D-Phe.sup.6-hGnRH(1-9)-ethylamide,
[0068] 22. Lys.sup.5, D-Cpa.sup.6-hGnRH(1-9)-ethylamide.
[0069] On investigating the capacity factor of some analogues of
high importance by using HPLC the following results were obtained:
TABLE-US-00002 Analogue Methanol Example No. k' % 3 3.57 38 5 9.28
55 6 3.57 30 7 11.57 30 8 7.57 30 17 9.57 38 19 14.10 38 19 8.71
40
[0070] ISCO model 2350 pump 1 ml/min, ISCO V4 detector (215
nm).,
Column: BST, ODS Hypersil 5 m, 270.times.4 mm.
[0071] Eluent: MeOH/0.1 M NaH2PO4 (pH=2.22). k ' = T R - t O t 0
##EQU1##
[0072] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention given for
the purpose of disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The abbreviations used in the description agree with the
nomenclature accepted in the peptide chemistry and published in J.
Biol. Chem. 241, 527 (1966); and ibid 247, 977 (1972); furthermore,
D-NaI stands for a-(2-naphthyl)-D-5-alanine, D-Cpa means
p-chlorophenyl-D-alanine and D-Pal stands for
a-(3-pyridyl)-D-alanine. When not noted otherwise, each of the
amino acids mentioned in the description is in L-configuration.
[0074] The new compounds of general formula (I) can be prepared by
coupling pharmacologically active polypeptides, preferably GnRH
analogues, to molecules of general formula (Ia) in the following
way: a) pharmacologically active compounds containing a free amino
group are coupled to N-vinylpyrrolidone/maleic anhydride copolymer
of general formula (Ib) ##STR4## known per se but not used in the
therapy up to the present, wherein R.sub.1, R.sub.2 and R.sub.3 are
as defined for the general formula (Ia); b) a compound of general
formula (Ib) is reacted with an activated ester of general formula
(III) H--X--OQ (II) of an amino acid or an oligopeptide, then the
pharmacologically active polypeptides are coupled to the carrier
compound of general formula (Ic) obtained by means of the activated
ester groups. The compounds obtained can be transformed to salts of
a pH value of 7.2; preferably to sodium salts.
[0075] The pharmacologically active molecule (A) appearing in the
general formula (I) is preferably an agonistic or antagonistic GnRH
analogue. The X group of compounds of general formula (I) means an
amino acid group or an oligopeptide group of at most six members
built up from native or non-natural amino acids. These can be
prepared by using processes known in the peptide chemistry.
[0076] The invention is based on the following recognitions:
[0077] 1. In comparison to the corresponding pharmacologically
active peptide hormones, chiefly GnRH analogues, in relation to the
original effect of the peptide hormone molecules, the compounds of
general formula (I) show an increased action prolonged in time.
[0078] 2. Compounds of general formula (I) containing a spacer
group as X, i.e. wherein r is different from 0, show an even more
favourable effect than those compounds containing no X group.
Simultaneously, no toxic side effects are shown by the compounds
containing X group. or those without X group; and these compounds
retain all effects of the pharmacologically active polypeptide
molecule A.
[0079] 3. Compounds of general formula (I) and their salts, wherein
A represents a GnRH analogue of formula (IIa) coupled through the
amino group of the side chain of the Lys group of the peptide
hormone, Y, W, V, X, r, k, z and u are as defined for the general
formula (I), are tumour-inhibiting compounds retaining and even
exceeding the known pharmacological effects of the analogue (IIa),
i.e. Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH.sub.2;MI-1544:
Ac-D-Trp.sup.1,3,D-Cpa.sup.2,D-Lys.sup.6,D-Ala.sup.10; thus, they
inhibit the growth of both steroid-dependent and
steroid-independent tumours. In addition, they induce a reversible
chemical castration enhancing the antitumour action in the case of
steroid-dependent tumours.
[0080] 4. Compounds of general formula (I), wherein A stands for a
GnRH antagonist analogue of formula (IIb), i.e., MI-1892:
Ac-D-Trp.sup.1,3,D-Cpa.sup.2,Lys.sup.5,[Asp(`-DEA)]6,D-Ala.sup.10-(Gln.su-
p.8-GnRH) coupled through the n-amino group of lysine in position
5, and Y, W, V, X, r, k, z as well as u are as defined for the
general formula (I), are selective tumour-inhibiting agents
according to their antagonistic character; thus, they inhibit the
growth of both steroid-dependent and steroid-independent tumours,
actually to a degree surpassing the effect of the compound of
formula (IIb). Both the antagonist of formula (IIb) and its
derivative of general formula (I) possess an irreversible chemical
castration effect which, together with the selective direct
tumour-inhibiting effect, is preferred from the view-point of
effectivity of tumour inhibition. It has unexpectedly been observed
that the antagonist of formula (IIb) possesses also an effect
stimulating the immune system, and this effect is retained by the
derivative of general formula (I), too. This recognition is
surprising, since the therapeuticuse of tumour-inhibiting GnRH
antagonists known up to now has been impeded just by their
immunosuppressive action.
[0081] 5. Compounds of general formula (I), wherein A stands for
the lamprey GnRH-III coupled through the n-amino group of the
lysine moiety in position 8, whereas Y, W, V, X, r, k, z and u are
as defined for the general formula (I), are selective
tumour-inhibiting agents under in vivo conditions and therefore,
they inhibit the growth of both, steroid-dependent and
steroid-independent tumours in spite of the fact that the GnRH-III
alone shows only a negligible in vivo effect. Conjugates of general
formula (I) of GnRH-III, exceeding the effects of sustained-release
hGnRH compositions, resulted in a tumour-free state of the
experimental animals in a treatment period, where only a decrease
in the tumour growth was observed by using known compositions. The
most preferable compounds of general formula (I) according to the
invention were prepared by conjugating the following GnRH analogues
of formulae (IIa) to (IIi): human GnRH (hereinafter GnRH):
[0082] Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2; chicken
GnRH (hereinafter Gln.sup.8-GnRH):
[0083] Glp-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH.sub.2; lamprey
GnRH-III (hereinafter GnRH-III):
[0084] Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH.sub.2;MI-1544:
Ac-D-Trp.sup.1,3,D-Cpa.sup.2,D-Lys.sup.6,D-Ala.sup.10-GnRH
antagonist analogue of formula (IIa) of human GnRH;
[0085] MI-1892:
Ac-D-Trp.sup.1,3,D-Cpa.sup.2,Lys.sup.5,[Asp(`-DEA)]6,D-Ala.sup.10-(Gln.su-
p.8-GnRH); antagonist analogue of formula (IIb) of chicken
GnRH;
[0086] SJ-1004: D-Phe2,D-Trp3,D-Lys6-GnRH; antagonist analogue of
formula (IId) of human GnRH;
[0087] TH-614: Lys.sup.5,cyclo(Asp.sup.6-Lys.sup.8)-GnRH-III;
analogue of formula (IIe) of lamprey GnRH-III;
[0088] HB-694: Lys.sup.4,[Lys(n-Fmoc)]8-GnRH-III; analogue of
formula (IIf) of lamprey GnRH-III;
[0089] TH-602: Lamprey GnRH-III; see above the formula; GnRH of
formula (IIc);
[0090] HB-685; Lys4-GnRH-III; analogue of formula (IIg) of lamprey
GnRH-III;
[0091] TH-609: D-Lys6-GnRH; analogue of formula (IIh) of human
GnRH;
[0092] TH-615: Lys5,D-Trp6-GnRH; analogue of formula (IIi) of human
GnRH.
[0093] The GnRH analogues were coupled either directly or through
an X group, i.e. spacer moiety, to the polymer designated as P. For
naming the spacers, the one-letter marking of the amino acids are
used, e.g. H-GFLG-OH: H-Gly-Phe-Leu-Gly-OH (peptide in the form of
its sodium salt); P: polyvinylpyrrolidone/maleic acid copolymer
sodium salt, i.e. a salt of formula (Id), wherein one of R.sub.1
and R.sub.2 means hydrogen whereas the other one is a group (B), n
is 66, transformed to the sodium salt by n equivalents of
NaHCO3.
[0094] The biological study of the following substances was carried
out: P-GLG-1892: compound of general formula (I), wherein A=(IIb)
group coupled to the Y=(Ia) group through X=-GLG-moiety; n=66,
r=0.1 n, k=0.3 r, z=0, u=1.9 n and W=OH or ONa, respectively,
pH=7,2;
[0095] P-GFLG-1544: compound of general formula (I), wherein
A=(IIa) group coupled to the Y=(Ia) group through the
X=-GFLG-moiety; n=66, r=0.1 n, k=0.3 r, z=0, u=1.9 n and W=OH or
ONa, respectively, pH=7.2;
[0096] P-1892: compound of general formula (I), wherein A=(IIb)
group, n=66, r=0, k=0.024 n, z=0, u=1.9 n, W=OH or ONa,
respectively, pH=7.2;
[0097] P-GFLG-1892: compound of general formula (I), wherein
A=(IIb) group coupled to the Y=(Ia) group through an
X=-GFLG-moiety; n=66, r=0.1 n, k=0,3 r, z=0, u=1.9 n and W=OH or
ONa, respectively, pH=7.2;
[0098] P-GFLG-609: compound of general formula (I), wherein A=(IIh)
group, coupled to the Y=(Ia) group through an 20 X=-GFLG-moiety;
n=66, r=0.1 n, k=0.3 r; z=0; u=1.9 n and W=OH or ONa, respectively,
pH=7.2; P-GFLG-1004: compound of general formula (I), wherein
A=(IId) group, coupled to the Y=(Ia) group through an 25
X=-GFLG-moiety; n=66, r=0.1 n, k=0.3 r; z=0; u=1.9 n and W=OH or
ONa, respectively, pH=7.2;
[0099] P-GFLG-614: compound of general formula (I), wherein A=(IIe)
group, coupled to the Y=(Ia) group through an X=-GFLG- moiety;
n=66, r=0.1 n, k=0.3 r; z=0; u=1.9 n and W=OH or ONa, respectively,
pH=7.2;
[0100] P-GFLG-685: compound of general formula (I), wherein A=(IIg)
group, coupled to the Y=(Ia) group through an X=-GFLG- moiety;
n=66, r=0.1 n, k=0.3 r; z=0; u=1.9 n and W=OH or ONa, respectively,
pH=7.2;
[0101] P-GFLG-602: compound of general formula (I), wherein
A=(GnRH-III) group, coupled to the Y=(Ia) group through an
X=-GFLG-moiety; n=66, r=0.1 n, k=0.3 r; z=0; u=1.9 n and W=OH or
ONa, respectively, pH=7.2;
[0102] P-GFLG-615: compound of general formula (I), wherein A=(IIi)
group, coupled to the Y=(Ia) group through an X=-GFLG- moiety;
n=66, r=0.1 n, k=0.3 r; z=0; u=1.9 n and W=OH or ONa, respectively,
pH=7.2;
[0103] P-FLG-892: compound of general formula (I), wherein A=(IIb)
group, coupled to the Y=(Ia) group through an X=-GFLG- moiety;
n=66, r=0.1 n, k=0.3 r; z=0; u=1.9 n and W=OH or ONa, respectively,
pH=7.2;
[0104] P-Ahx-1892: compound of general formula (I), wherein A=(IIb)
group, coupled to the Y=(Ia) group through an X=-Ahx-moiety; n=66,
r=0.1 n, k=0.3 r; z=0; u=1.9 n and W=OH or ONa, respectively,
pH=7.2.
[0105] The biological tests were carried out by using the materials
and methods following hereinafter. Certain human mammary carcinoma
cell lines or xenografts prepared therefrom are useful for the
biological testing of the conjugates.
[0106] Human cell lines used in in vitro experiments. MCF-7 human
mammary tumour cell line was stabilized in 1973 by Soule et al.
from the pleural fluid of mammary carcinoma patients. The cells
grow in monolayer and are epithelial in their character. MDA-MB-231
human mammary carcinoma cell line was isolated and stabilized in
1974 by Cailleau et al. similarly from pleural fluid. These cells
also grow in monolayer. PC3 human prostate carcinoma cell line was
stabilized 20 in 1979 in cell culture by Kaighn et al.; the cells
are of simple epithelium type and form compact colonies in
clonogenic assays. Ishikawa cell line originated from
adenocarcinoma of human endometrium [Nishida et al.: Obstet.
Gynecol. Jpn, 37, 25 1103-1111 (1985)], has epithelial character
and contains steroid as well as GnRH receptors. Human tumour cell
lines are maintained in plastic flasks (Greiner) in
Dulbecco-6modified Eagle-MEM (DMEM GIBCO) nutritive medium. The
medium used herein contained 10% of fetal calf serum (FCS).
[0107] The MCF-7 cell line is estradiol-receptor (ER) positive and
GnRH receptor-positive. Therefore, it is suitable to study the
receptor-mediated direct effect of GnRH; being an in vivo model
system, it is useful for investigating both the direct and indirect
effects of GnRH. Being an ER-negative and GnRH receptor-positive in
vitro and in vivo model. system, MDA-MB-231 cell line is useful to
study the direct effect of GnRH. Based on literature data, PC3
human prostate carcinoma cell line contains receptors specifically
binding GnRH, whereby the essential condition for the direct effect
is fulfilled. Ishikawa cell line is originated from human
endometrium adenocarcinoma, contains ER and PgR proteins and is
GnRH receptor-positive. Thus, it is useful for investigating the
direct effect of GnRH. Buserelin, chemically
[D-Ser(tBu)]6,desGly10-hGnRH(1-9)EA, is a tumour-inhibiting agonist
being used in a retard form in clinical practice; conclusively, the
substance itself but not its retard form was employed in the in
vitro experiments.
[0108] The preparation of the known polymeric carrier and the
investigation of compounds of general formula (I) as well as of the
spacers of general formula X were carried out as follows.
[0109] NVP-MA samples used herein were prepared according to the
literature [Boreddy et al.: Polymer 25, 115-120 (1983)]. Activated
esters of oligopeptides employed as spacers were preferably
nitrophenyl esters obtained by using classic methods of peptide
chemistry. The coupling of peptides to polymers can be accomplished
in solvents, such as dimethylsulfoxide (DMSO), dimethylformamide
(DMF) or dimethylacetamide. DMF was used as solvent.
[0110] Nitrophenyl esters of peptides were characterized by amino
acid analysis. The retention factors referred to were determined on
Kieselgel 60 F254 thin layer by using the following developing
systems:
1. pyridine: acetic acid:water:ethyl acetate 20:6:12:62;
2. chloroform:methanol:water 10:5:1.
[0111] High pressure liquid chromatography (HPLC) examinations were
carried out on a C-18 reverse-phase column at a flow rate of 0.5.,
ml/min by using the following gradients:
Eluent A: S % of acetonitrile plus 95% of triethylamine--phosphate
buffer of pH 2.25;
[0112] Eluent B: 80% of acetonitrile plus 20% of
triethylamine-phosphate buffer of pH 2.25; 100% of eluent A up to
the 5th minute; to 60% of eluent A up to the 10th minute; to 35% of
eluent A up to the 30th 25 minute; to 0% of eluent A up to the 35th
minute; 0% of eluent A up to the 40th minute; and to 100% of eluent
A up to the 45th minute. The detection was performed at 280 nm with
an UV detector. The conjugates were purified by ultrafiltration on
AMICON PM10 membrane.
[0113] The results advantages of the new compounds can be
summarized as follows. Based on the in vitro examinations it can be
stated that the proliferation- and colony formation-inhibiting
effects of the conjugates P-GFLG-1544 and P-GFLG-1892--containing
the GFLG tetrapeptide spacer group - highly exceed the antitumour
effect of both the GnRH analogue substances (i.e. MI-1544 or
MI-1892) as well as that of the spacer-containing carrier
(P-GFLG-OH) and even that of the compound P-1892. The conjugates
(i.e. compounds built up from the carrier and GnRH analogue)
resulted in colony formation-inhibition of 100% above a
concentration of 20 .mu.M. Such a high grade of inhibition could
till now be achieved only by the use of cytostatics. Based on the
results both the polymeric starting substance and the
spacer-containing polymer as a polyanion in themselves also exert a
direct antitumour effect, and, when coupled covalently to GnRH
antagonists, they enhance the direct antitumour action of the
antagonists.
[0114] According to the in vivo studies the conjugates P-GFLG-1544
and P-GFLG-1892 as well as the carrier P-GFLG-OH proved to be
non-toxic. The conjugates P-GFLG-1544 and P-GFLG-1892 resulted in a
tumour volume diminution of 30 to 35% in the second week and 37 to
49% in the fourth week of treatment on the ER-positive, GnRH
receptor-positive MCF-7human mammary carcinoma xenograft; whereas a
tumour volume diminution of 37 to 42% could be observed on the
ER-negative, GnRH receptor-positive MDA-MB-231 human-mammary
carcinoma xenograft. This result is considered to be significant
since the degree of tumour inhibition observed by us on human
carcinoma xenograft is nearly identical with that of the
sustained-release composition of SB-75, a GnRH antagonist
[Szepeshzi et al.: Breast Cancer Res. Treat. 21, 181-192 (1992)],
which had been: achieved on the MXT mouse mammary carcinoma which
is more. sensitive.
[0115] The substance MI-1892 and. its conjugate P-GFLG.-1892
possess a direct, selective tumour-inhibiting effect. Namely, in
contrast to substance -MI-1544 and its conjugate P-GFLG-1544, they
exert a minimum chemical castration effect. Based on this fact it
can be supposed that they act on a broader spectrum of mammary
carcinomas and therefore, on hormone- (estrogen-)-independent
tumours, too.
[0116] When investigating the effect of the compounds on the
cellular *1 5 and humoral immune system it was stated that MI-1892
as selective chicken GnRH antagonist as well as the conjugate
P-GFLG-1892 containing the antagonist and the spacer-containing
P-GFLG, a novel polyanionic macromolecule, increased the activity
of T-lymphocytes against bovine red blood cell (BRBC) antigen (by
using the method of rosette formation). On the basis of
examinations of the antibody production of B-lymphocytes, MI-1892
and P-GFLG-OH as well as the conjugate P-GFLG-1892 containing
MI-1892 moiety strengthened the humoral immune response, too.
[0117] The immunosuppressive side effect of cytostatics used in
tumour therapy is commonly known. This effect can be compensated to
a certain grade by various immunostimulants (endotoxin,
levamisole). Polypeptides with much lower molecular weights (about
40,000 to 80,000) are also capable of protecting against
immunosuppressive effect [Gal et al.: J. Biol. Resp. Modif. 5,
148-159 (1986)). The molecular weight of the novel polyanionic
P-GFLG-OH macromolecule tested by us is about 10,000, that of
MI-1892 is about 2000, i.e. relatively low; therefore, their
immunostimulatory activity was unexpected. The importance of these
results is reflected by the fact that MI-1892 administered to mice
in a dose of 50 .mu.g/mouse or 100 .mu.g/mouse, respectively,
increased the humoral immune response. to more than twofold or
threefold, respectively; the increase was twofold in the case of
P-GFLG-OH. The conjugate P-GFLG-1892 also possesses an
immunostimulatory effect. The investigation of the cellular immune
response showed that MI-1892 given in a dose of 100 .mu.g/mouse
increased the response to nearly twofold; this increase was twofold
in the case of P-GFLG-OH and P-GFLG-1892.
[0118] The novel polycarboxylic acid derivatives according to the
invention are very preferable carriers for peptide hormones since
they can be prepared with a good reproducibility and with a low
polydispersity in the aimed range of molecular weights and they are
water-soluble.
[0119] Known polycarboxylic acid derivatives also possess a weak
tumour-inhibiting action and can similarly favourably be used as
carrier compounds. By coupling pharmacologically active compounds
such as GnRH analogues to the polymers of the invention, novel
compounds are obtained which exert an increased therapeutic
efficiency in comparison to that of their structural moieties.
[0120] The new conjugate P-GFLG-1544 is a compound with favourable
therapeutic effect for the following reasons: It has a selective
tumour-inhibiting effect proved by the direct inhibition of cell
proliferation. Thus, it inhibits not only the growth of
steroid-dependent but also that of steroid-independent tumours as
demonstrated by investigations carried out on MDA-MB-231 mammary
tumour cells. Due to the antagonistic effect, the coupled MI-1544
possesses a reversible castration effect. This effect is proved by
the change of uterus weight measured during in vivo treatments as
well as the decrease in the cytosolic progesterone receptor (cPgR)
level of uterus. A very important advantage of this effect is that
the hormone status is re-established by suspending the treatment.
This is opposed to the chirurgical intervention and irradiation,
which are irreversible and refused by many patients The advantage
of castration effect is that in the case of steroid-dependent
tumours the selective direct and castration effects are summarized
to result in a stronger inhibition of the tumours.
[0121] The castration effect is illustrated by the following data.
After a treatment for four weeks the uterus weights of animals
bearing MCF-7 xenografts were decreased by 42% (control=0.1980 q
0.0120 g; P-GFLG-1544=0.1149+0.0110 g); after a treatment for
twelve weeks the uterus weights of animals bearing MDA-MB-231
xenografts were decreased by 50% (control=0.0263+0.0028 g,
P-GFLG-1544=0.0133 q 0.0018 g). The weight of uterus was not
decreased but slightly increased by P-GFLG-OHalone (0.0337 q 0.0034
g, 128%). The change of progesterone level was proved by the
significant decrease (of 54%) of the uterus cPgR level of animals
bearing MCF-7 xenografts (control=517 q 45 femtomol/mg of protein,
P-GFLG-1544=241 q 28 femtomol/mg of protein).
[0122] In opposition to several known GnRH antagonists, P-GPLG-1544
does not possess immunosuppressive effect. Summing up: the
conjugate P-GFLG-1544 not only retained but also substantially
exceeded both the selective tumour-inhibiting as well as the
castration effects of the MI-1544, an antagonist analogue of GnRH,
mainly in respect of tumour inhibition. No adverse side effects
have been observed during the investigations on the conjugate. Due
to its prolonged effect this novel antagonist compound can be
utilized for in vivo tumour inhibition. The new conjugate
P-GFLG-1892 is a favourable compound with therapeutic effect for
the following reasons: It has a selective tumour-inhibiting effect.
Therefore, it inhibits the growth of both steroid-dependent and
steroid-independent tumours, which was proved by investigations on
MDA-MB-231 cells.
[0123] The GnRH antagonist MI-1892 and the conjugate containing the
antagonist possess a weaker castration effect in comparison to
MI-1544 and its conjugate. The castration effect of the conjugate
was demonstrated by the change in uterus weight measured during in
vivo treatments as well as by the decrease in the cytosolic
progesterone receptor (cPgR) level of uterus. After a treatment for
four weeks the uterus weight of animals bearing xenograft was
decreased by 39% (control 0.1980 q 0.0120 g, P-GFLG-1892=0.1215 q
0.0130 g). After a treatment for twelve weeks the uterus weight of
animals bearing MDA-MB-231 was decreased by 20% (control=0.0263 q
0.0028 g, P-GFLG-1892=0.0215 q 0.0017 g). The weight of uterus was
not decreased but slightly increased by P-GFLG-OH alone (0.0337 q
0.0034 g, 128%). The change in the progesterone level was proved by
the 23% decrease in the uterus cPgR level of animals bearing MCF-7
xenograft (control =517 q 45 femtomol/mg of protein,
P-GFLG-1892=376 q 33 femtomol/mg of protein).
[0124] In opposition to several known GnRH antagonists, it has no
immuno-suppressive effect. It is a new recognition that this novel
GnRH antagonist analogue and the conjugate containing this analogue
possess immunostimulatory effect as proved by the humoral and
cellular immune response investigations.
[0125] On comparison to healthy individuals, the functioning of the
protective mechanism (immunostatus) of patients suffering from
tumour is more unfavourable. Since the protective (immune)
mechanism is enhanced by the compound, its tumour-inhibiting action
can more strongly become valid. The novel conjugate P-GFLG-GnRH-III
is a compound possessing favourable therapeutic action for the
following reasons: It has a selective tumour-inhibiting effect.
Therefore, it inhibits the growth of both the steroid-dependent and
steroid-independent tumours which was proved by investigations on
MDA-MB-231 cells. It has no castration effect since the cycle of
female rats is not influenced even by a high dose of -the coupled
GnRH-III during three cycles observed. This effect may particularly
be preferred on young patients suffering from mammary tumour, where
a castration would cause psychic disturbances. In a way different
from other conjugates, P-GFLG-GnRH-III gradually induces an
inhibition of continually increasing grade during the long-lasting
treatment for 7 weeks; and at the end of treatment, tumour-free
animals are observed whereas in the case of other conjugates the
degree of inhibition increases only up to the fifth week of
treatment, then a stagnation in the grade of inhibition can be
observed.
[0126] The pharmaceutical compositions containing the compound of
general formula (I) according to the invention may be prepared by
transforming the compound of general formula (I) or a
pharmaceutically acceptable salt or complex thereof to a
composition with carriers and/or additives commonly used in the
pharmaceutical industry by using known operations of the
pharmaceutical techniques.
[0127] The pharmaceutical composition for therapeutic use may
contain any filling material and carrier used in the therapy (e.g.
calcium carbonate, talc); solvent (such as water, an aqueous
solution containing ethanol and/or polyalcohol, e.g. polyethylene
glycol and/or glycerol and the like); salts (e.g. sodium chloride
for adjusting the physiological osmotic pressure; or e.g. chlorides
of iron, cobalt, zinc or copper and the like for supplementing
trace elements); solubilizing additives, e.g. complex-forming
agents (cyclodextrins, crown ethers, native proteins, saponins and
the like); compounds diminishing the relative permittivity of the
solvent such as ethanol, polyols (polyethylene glycol or glycerol);
tablet-disintegrating agents; complex-forming agents commonly used
in sustained-release compositions (e.g. water-insoluble
cyclodextrin derivatives, native and artificial polymers, crown
ethers and the like); pH-adjusting compounds such as mineral and
organic buffers; taste-improving agents (beet-sugar, fructose and
dextrose, saccharins, in verted sugar and. the like); antioxidants
(e.g. vitamin C); as well as other active ingredients promoting the
effectuation of the action of active agents of general formula (I).
The pharmaceutical compositions may be oral such as tablets, pills,
drages, hard or soft capsules, microcapsules, solutions, emulsions
or suspensions; or parenteral, e.g. injectable solutions, slow and
rapid infusions; as well as pharmaceutical compositions useful for
rectal administration such as suppositories; furthermore creams or
jellies. There also exists the possibility of incorporating to
liposomes the pharmaceutical compositions developed for the above
uses. The bioconjugates of general formula (I) can be utilized also
in aerosol compositions targeted at the absorption through the skin
surface or the lungs, respectively. For the preparation of tablets,
drages or hard gel capsules, e.g. calcium carbonate, talc, fats,
waxes or polyalcohols, having an appropriate density, are useful
carriers.
[0128] For the preparation of solutions and syrups, e.g. water,
polyalcohols (e.g. polyethylene glycol or glycerol), beet-sugar or
dextrose may be used as carriers. Parenteral compositions may
contain water, alcohol, polyalcohols or vegetable oils as carriers.
Carriers of suppositories may be e.g. oils, waxes, fats or
polyalcohols having a suitable density. The bioconjugates of
general formula (I) are useful for therapeutic utilization in
combination with artificial and native active agents, too. The
bioconjugates of general formula (I) are effective in a dose range
of 0.01 to 100 .mu.g/kg in subcutaneous, intramuscular or
intravenous injections. The dose to be used in the practice is
dependent on the type of disease as well as on the state and age of
the patient and it should be determined by the physician.
[0129] The invention is further illustrated by the following
non-limiting Examples, wherein the term "active substance" relates
to the GnRH analogue moiety of the conjugates. The following
examples are given for the purpose of illustrating various
embodiments of the invention and are not meant to limit the present
invention in any fashion.
EXAMPLE 1
Preparation of poly(N-vinylpyrrolidone-co-maleic
acid)-Gly-Phe-Leu-Gly nitrophenyl ester
[0130] After dissolving poly(N-vinylpyrrolidone-co-maleic
anhydride) [average molecular weight: 10,000, IR: 1840 cm-1, 1790
cm-1 (anhydride), 1660 cm-1 (NCO of pyrrolidone)] in anhydrous
dimethylformamide 10 mol % of H-Gly-Phe-Leu-Gly nitrophenyl ester
trifluoroacetate (Rf1: 0.50) and an equivalent amount of
triethylamine were added. After 3 hours the product was
precipitated by adding diethyl ether, filtered and thoroughly
washed with ether. The presence of nitrophenyl ester content in the
product obtained was proved by IR spectroscopy (1840 cm-1, 1790
cm-1 anhydride, 1740 cm-1 COOH, 1660 cm-1 NCO, 1540 cm-1, 1350 cm-1
NO2) and determined by its ultraviolet absorption measured at 400
nm in sodium 10 hydrogen carbonate solution (molar extinction
coefficient: 1660). By ultrafiltration of an aliquot part it was
controlled that the product was free from unbound peptide
nitrophenyl ester. After dissolving the polymer in absolute DMF a
calculated amount (2 equivalents) of water was added for
hydrolyzing the anhydride groups. The termination of hydrolysis was
determined by the IR spectrum of a sample (IR: 1740 cm-1 COOH, 1540
cm-1, 1350 cm-1 NO2).
EXAMPLE 2
Preparation of poly(N-vinylpyrrolidone-co-maleic acid)-Phe-Leu-Gly
nitrophenyl ester
[0131] The product was prepared and characterized as described in
Example 1 by coupling H-Phe-Leu-Gly nitrophenyl ester hydrochloride
(Rf1: 0.59) (IR: 1740 cm-1 COOH, 1540 cm-1, 1350 cm-1 NO2).
EXAMPLE 3
Preparation of poly(N-vinylpyrrolidone-co-maleic acid)-Gly-Leu-Gly
nitrophenyl ester
[0132] The product was prepared and characterized as described in
Example 1 by coupling H-Gly-Leu-Gly nitrophenyl ester
trifluoroacetate (Rf2: 0.53). (IR: 1740 cm-1 COOH, 1540 cm-1; 1350
cm-1 NO2).
EXAMPLE 4
Preparation of poly(N-vinylpyrrolidone-co-maleic acid)-Ahx
nitrophenyl ester
[0133] The product was prepared and characterized as described in
Example 1 by coupling H-Ahx nitrophenyl ester acetate (Rf1: 0.7).
(IR: 1740 cm-1 COOH, 1540 cm-1, 1350 cm-1 NO2).
EXAMPLE 5
[0134] Preparation of
Ac-D-Trp.sup.1,3,D-Cpa.sup.2,{Lys[n-poly(N-vinyl
pyrrolidone-co-maleic acid)-Gly-Phe-Leu-Gly-]}5, [a-Asp(DEA)]6,
D-Ala10-GnRH (abbreviated: P-GFLG-1892) After adding 1 equivalent
of MI-1892 (calculated for the nitrophenyl ester groups) to the
solution prepared according to Example 1, the pH of the solution
was adjusted between 7 and 8 by adding triethylamine. After 24
hours the reaction mixture was diluted to its 20-fold volume by
adding water and an 5% NaHCO3 solution was added for the complete
hydrolysis of the unreacted nitrophenyl ester groups. Low molecular
materials were removed by ultrafiltration and the high molecular
fraction was lyophilized. The purity of the product was controlled
by HPLC (Rt: 34.48 min, the retention time of MI-1892 is 20.61 min)
and the active ingredient content was determined by UV
spectrophotometry in the range of 200 to 450 nm (the specific
extinction of MI-1892 is 10,400 at 280 nm). Simultaneously, it was
stated that the product did not contain any free or unbound
nitrophenol (between 380 and 420 nm).
EXAMPLE 6
Preparation of
Ac-D-Trp1,3,D-Cpa2,{D-Lys[n-poly-(N-vinylpyrrolidone-co-maleic
acid)-Gly-Phe-Leu-Gly]}6.
[0135] D-Ala10-GnRH (abbreviated: P-GFLG-1544) Example 5 was
followed, except that MI-1544 was used as GnRH analogue. The purity
of the product was controlled by HPLC [retention time (Rt): 29.96
min, that Rt of MI-1544 is 22.93 min], its active ingredient
content was determined by 10 UV spectrophotometry in the 200 to 450
nm range. The molar specific extinction of MI-1544 is 11,000 at 280
nm.
EXAMPLE 7
Preparation of {D-Lys[n-poly(N-vinylpyrrolidone-co-maleic
acid)-Gly-Phe-Leu-Gly]}6,-GnRH (abbreviated: P-GFLG-609)
[0136] Example 5 was followed, except that D-Lys6-GnRH was used as
GnRH analogue. The purity of the product was controlled by HPLC and
the active ingredient content was determined by UV 20
spectrophotometry in the 200 to 450 nm range.
EXAMPLE 8
Preparation of
D-Phe2,D-Trp3,{D-Lys[n-poly(N-vinylpyrrolidone-co-maleic
acid)-Gly-Phe-Leu-Gly-]}6.-GNRH (abb.: P-GFLG-1004)
[0137] Example 5 was followed, except that SJ-1004 was used as GnRH
analogue. The purity of the product was controlled by HPLC (Rt:
20.7 min, that of SJ-1004 is 17.3 min) and the active ingredient
content was determined by UV spectrophotometry in the 200 to 450 nm
range. The molar specific extinction of SJ-1004 is 6500 at 280
nm.
EXAMPLE 9
Preparation of {Lys[n-poly(N-vinylpyrrolidone-co-maleic
acid)-Gly-Phe-Leu-Gly-]}5,cyclo(Asp6-Lys8)-GnRH-III (abbr.:
P-GFLG-614)
[0138] Example 5 was followed, except that TH-614 was used as GnRH
analogue. The purity of the product was controlled by HPLC (Rt: of
TH-614 is 16.4 min) and the active ingredient content was
determined by UV spectrophotometry in the 200 to 450 nm range. The
molar specific extinction of TH-614 is 8800 at 280 nm.
EXAMPLE 10
Preparation of {Lys[n-poly(N-vinylpyrrolidone-co-maleic
acid)-Gly-Phe-Leu-Gly-]}4,-GnRH-III (abbreviated: P-GFLG-685)
[0139] To the polymer solution prepared according to Example 1 0.5
equivalent (calculated for the nitrophenyl ester groups) of HB-694
hormone analogue containing a free amino group was added. The pH
value of the reaction mixture was adjusted to 10 by using
(diisopropyl)-ethylamine. After a few hours piperidine was
portionwise added to remove the protective group from the
[Lys(n-Fmoc)]8 derivative. After stirring for a few additional
hours, the reaction mixture diluted with water was ultrafiltered
and the ultrafiltration was repeated after dilution of the
supernatant with sodium hydrogen carbonate solution. The
thus-purified product was lyophilized, its purity was controlled by
HPLC and the active ingredient content was determined by UV
spectrophotometry in the 200 to 450 nm 5 range. The molar specific
extinction of HB-685 is 9800 at 280 nm.
EXAMPLE 11
Preparation of {Lys[n-poly(N-vinylpyrrolidone-co-maleic
acid)-Gly-Phe-Leu-Gly-]}8,-GnRH-III (abbr.: P-GFLG-602)
[0140] Example 5 was followed, except that TH-602 was used as GnRH
analogue. The purity of the product was controlled by HPLC (Rt:
14.8 min, that of TH-602 is 13.4 min) and the active ingredient
content was determined by UV spectrophotometry in the 200 to 450 nm
range. The molar specific extinction of TH-602 is 9800 at 280
nm.
EXAMPLE 12
Preparation of Ac-D-Trp.sup.1,3,D-Cpa.sup.2,{Lys[n-poly(N-vinyl
pyrrolidone-co-maleic acid)-Phe-Leu-Gly-]}.sup.5,a-Asp
(DEA)].sup.6, D-Ala10-GnRH (abbreviated: P-FLG-1892)
[0141] Example 5 was followed, except that the product prepared
according to Example 2 was used as carrier and MI-1892 was employed
as GnRH analogue. The purity of the product was controlled by HPLC
(Rt: 30.77 min) and the active agent content was determined by
spectrophotometry in the 200 to 450 nm range.
EXAMPLE 13
Preparation of
Ac-D-Trp.sup.1,3,D-Cpa.sup.2,{Lys[n-poly(N-vinylpyrrolidone-co-maleic
acid)-Gly-Leu-Gly-]}.sup.5,[a-Asp(DEA)].sup.6,D-Ala.sup.10-GnRH
(abbreviated: P-GLG-1892)
[0142] Example 12 was followed, except that the product prepared in
Example 3 was used as carrier and MI-1892 was employed as GnRH
analogue. The purity of the product was controlled by HPLC (Rt:
25.39 min) and the active ingredient content was determined by UV
spectrophotometry in the 200 to 450 nm range.
EXAMPLE 14
Preparation of
Ac-D-Trp.sup.1,3,D-Cpa2,{Lys[n-poly(N-vinylpyrrolidone-co-maleic
acid)-Ahx]}5,[a-Asp(DEA)]6,D-Ala10-GnRH (abbreviated:
P-Ahx-1892)
[0143] Example 12 was followed, except that the product prepared in
Example 4 was used as carrier and MI-1892 was employed as GnRH
analogue. The purity of the product was controlled by HPLC (Rt:
19.98 min) and the active ingredient content was determined by UV
spectrophotometry in the 200 to 450 nm range.
EXAMPLE 15
Preparation of Ac-D-Trp1,3,D-Cpa2,{Lys[n-poly(N-vinyl
pyrrolidone-co-maleic acid)-]}5,[a-Asp(DEA)]6,D-Ala10-GnRH
(abbreviated: P-1892)
[0144] After dissolving poly(N-vinylpyrrolidone-co-maleic an
hydride) in DMF, calculated amounts of MI-1892 and triethylamine
were added. After standing for 12 hours the product was
precipitated by diethyl ether, filtered, then dissolved in sodium
hydrogen carbonate solution of 5% and purified as well as analyzed
as described in the preceding Examples (Rt: 19.59 min).
EXAMPLE 16
Preparation of {Lys[n-poly(N-vinylpyrrolidone-co-maleic
acid)-Gly-Phe-Leu-Gly-]}5,D-Trp6-GnRH (abbreviated: A-GFLG-615)
[0145] Example 5 was followed, except that TH-615 was used as GnRH
analogue (see Example 9). d: 10300.
EXAMPLE 17
Preparation of Gly-Phe-Leu-Gly-GnRH analogues coupled to the
poly(N-vinyl-pyrrolidone-co-maleic acid)-hexylamide derivative
[0146] A calculated amount of hexylamine was added to the solution
of poly(N-vinylpyrrolidone-co-maleic anhydride) in dimethyl
formamide under stirring. After 2 hours the product was
precipitated by adding diethyl ether, then filtered and washed with
ether until it became amine-free (negative ninhydrin test, IR: 293
cm-1 CH2). The solid was again dissolved in DMF and
H-Gly-Phe-Leu-Gly nitrophenyl ester hydrochloride was added. The pH
value of the reaction mixture 20 was set to 7-8 with triethylamine
and the precipitation was repeated after 3 hours. The coupling of
the GnRH analogue and purification of the products were carried out
as described in Example 5. The purity of the product was controlled
by HPLC and the active agent content was determined by UV 25
spectrophotometry in the 200 to 450 nm range.
EXAMPLE 18
Investigation on the Dose-Survival Relation
[0147] The investigation gives a precise information about the
cell-damaging effect of the tested substance. In the case of
cytostatics the cell-damaging effect is not cell-specific. GnRH
analogues possess a phase-specific action, they block but do not
kill the cells in the G0/G1 phase. One part of the arrested cells
again go into the cycle, the other part may perish (apoptosis). The
colonies formed from the arrested cells do not reach the countable
colony size at the time of evaluation (the count of colonies may be
identical but the sizes of colonies are different). In the case of
hormone analogues, a specific inhibition of 10 to 30% can be
achieved on the cells possessing a receptor for the hormone in
question. The investigations were carried out on MCF-7 and
MDA-MB-231 human mammary carcinoma cell lines, PC3 human prostate
15 carcinoma cell line and Ishikawa human endometrium carcinoma
cell line. Threehundred cells were set into Petri dishes of 3.5 cm
in diameter each. The treatment was carried out once, 24 hours
following the explantation, then the colonies formed were counted
after 9 to 12 days.
[0148] After dissolving the substances in the nutrient medium, the
cultures were treated with 1 to 50 .mu.M of MI-1544 human GnRH
antagonist; MI-1892 chicken GnRH antagonist; SJ-1004 human GnRH
antagonist analogues and synthetic lamprey GnRH-III substance as
well as with Lys4-GnRH-III and Lys5-cyclo (Asp6,Lys8)-GnRH-III
lamprey GnRH-III analogues; furthermore P-GFLG-1544, P-GFLG-1892,
P-GFLG-GnRH-III, P-GFLG-Lys4-GnRH-III,
P-GFLG-Lys5-cyclo(Asp6,Lys8)-GnRH-III conjugates or P-FLG-1892 and
P-GLG-1892 conjugates, respectively, corresponding to the same
amounts of the active substances.
[0149] MI-1892 chicken GnRH antagonist substance (administered in a
concentration of 50 fm) inhibited the colony formation of both
mammary carcinoma cell cultures (MCF-7, MDA-MB-231) to a nearly
identical degree, namely by 15 to 20% and 35 to 38%, respectively.
When administering in a concentration of 50 .mu.M, the MI-1544
hGnRH antagonist showed an inhibition of 45% of the colony
formation on the MCF-7 cell line and 20% on the MDA-MB-231 cell
line.
[0150] Both conjugates P-GFLG-1544 and P-GFLG-1892 exerted an
inhibition of 80 to 85% on the colony formation in a concentration
of 10 .mu.M and 100% inhibition in a concentration of 50 .mu.M of
the active substance, respectively, (Table 1). TABLE-US-00003 TABLE
I Inhibition of colony formation by GnRH analogue/macromolecule
conjugates (P-GFLG-GnRH analogues) on human tumour cell lines Exam.
No.(comp.) Cell line Dose(.mu.M) % Inhibition 6. P-GFLG-1544 MCF-7
10 85 50 100 MDA-MB-231 10 80 50 100 Ishikawa 50 100 PC3 10 100 50
100 5. P-GFLG-1892 MCF-7 10 60 50 93 MDA-MB-231 10 92 30 100
Ishikawa 10 72 30 100 PC3 10 86 8. P-GFLG-1004 MCF-7 10 75 50 95
MDA-MB-231 10 62 50 92 Ishikawa 10 3 50 46 PC3 30 100 50 100 11.
P-GFLG- MCF-7 50 9 GnRH-III Ishikawa 50 15 PC3 50 16 10. P-GFLG-
Lys4-GnRH-III MCF-7 50 68 MDA-MB-231 50 75 Ishikawa 50 25 PC3 30 94
9. P-GFLG-Lys5- MCF-7 50 78 cyclo(Asp6, MDA-MB-231 50 82 Lys8)-
Ishikawa 50 19 GnRH-III PC3 50 100
[0151] The tripeptide spacer-containing conjugates P-FLG-1892 and
P-GLG-1892 exerted a somewhat weaker, namely 70 to 80%, inhibition
on the survival of the cells in an active substance concentration
of 50 .mu.M. P-GFLG-1544 and P-GFLG-1892 were the most strong 20
inhibitors of colony formation of PC3 prostate and Ishikawa
endometrium carcinoma cell lines, too: by using a concentration of
30 to 50 .mu.M, an inhibition of 100% was observed both in the PC3
and the Ishikawa cell cultures (Table 1). Both conjugates highly
surpassed the inhibition of 25 colony formation of MI-1544 GnRH
antagonist and MI-1892 chicken GnRH antagonist analogue active
substances, which has been found to be 5% and 10%, respectively, on
Ishikawa cell line and 20% or 5%, respectively, on the PC3 cell
line by 37 using a concentration of 50 .mu.M.
[0152] When administered in a concentration of 50 .mu.M, SJ-1004 as
a weak GnRH antagonist analogue substance inhibited the degree of
colony formation by 31% on the MCF-7 cell line and 5 by 26% on the
MDA-MB-231 cell line whereas it did not show any inhibition on the
PC3 cell line (0%) or on Ishikawa cell line (5%), respectively. In
contrast to this, when given in an active substance concentration
of 50 .mu.M, the P-GFLG-1004 conjugate resulted in 10 an inhibition
of 92 to 95% on mammary carcinoma cell lines; 100% on PC3 prostate
carcinoma cell culture; and 46% on Ishikawa cell line (Table 1). In
opposition to the own or human and chicken GnRH analogues, the
synthetic lamprey GnRH-III resulted in the most significant
inhibition of colony formation as the active substance in itself
but not as a conjugate on the MCF-7, MDA-MB-231 and PC3 cell lines.
At a concentration of 50 .mu.m, GnRH-III inhibited the colony
formation by 65% (on MCF-7), 69% (on MDA-MB-231) and 21% (on PC3),
respectively, whereas 20 the P-GFLG-GnRH-III conjugate did not
result in an inhibition of higher than 16% on any cell line (Table
1).
[0153] In the cases of Lys4-GnRH-III and
Lys5-cyclo(Asp6,Lys8)-GnRH-III lamprey GnRH-III analogues, the
inhibiting effects of the conjugates, namely, P-GFLG-Lys4-GnRH-III
and P-GFLG-25 Lys5-cyclo(Asp6,Lys8)-GnRH-111 proved again to be
more significant (Table 1). In the form of active substance,
Lys4-GnRH-III exerted on the colony formation an inhibition of 40%
on the MCF-7 cell line, 35% in the MDA-MB-231 cell culture 38 and
21%, respectively, on Ishikawa .cells. It did not show any
inhibition in the case of PC3 cells.
[0154] The P-GFLG-Lys4-GnRH-III conjugate showed on the colony
formation an inhibition of 68% on MCF-7 cell line, 75% on 5
MDA-M8-231 cell culture and 25% on Ishikawa cells; interestingly,
it inhibited by 94% the colony formation on the PC3 cell line
(Table 1).
[0155] The Lys5-cyclo(Asp6,Lys8)-GnRH-III lamprey analogue exerted
on the colony formation an inhibition of 44% on MCF-7 cell line and
13% on Ishikawa cell line. As a conjugate,
P-GFLG-Lys5-cyclo(Asp6,Lys8)-GnRH-III inhibited the colony
formation in mammary carcinoma cell cultures much more strongly (78
to 82%) than the active substance. On Ishikawa cell line, the
inhibiting effect of the conjugate (19%) was nearly the same as
that of the active substance (13%). The conjugate blocked the
colony formation by 100% on the PC3 cell line.
EXAMPLE 19
In Vitro Inhibition of Proliferation of the Human Mammary Carcinoma
Cell Line
[0156] The procedure of treatment was as follows. After
trypsinization the cells, 400,000 cells of MCF-7, MDA-MB-231 human
mammary carcinoma, PC3 human prostate carcinoma or Ishikawa human
endometrium tumour each were passed into Petri dishes of 10 cm in
diameter. Starting from the day following transfer, the cells were
treated with the substances to be tested in solution prepared with
the nutrient medium during the exponential growth phase. The
cultures were treated with 1 to 50 .mu.M (calculated for the total
volume of the culture) of MI-1544 GnRH antagonist, MI-1892 chicken
GnRH antagonist or SJ-1004 GnRH antagonist analogues as well as
synthetic lamprey GnRH-III hormone in every two days and the cell
count was determined on the 7th day. Simultaneously with the above
examinations, the cells were once treated with the conjugates
P-GFLG-1544, P-GFLG-1892, P-GFLG-1004 as well as P-GFLG-GnRH-III in
amounts corresponding to 1 to 50 .mu.M of active substance on the
day, following transfer, then the cell count was determined on the
7th day after explantation. The GnRH peptide hormone substances
used in a concentration of 30 .mu.M resulted in the most
significant inhibition of cell proliferation. The inhibitory effect
was not enhanced by increasing the concentration.
[0157] In the case of the MCF-7 estradiol receptor (ER)-positive
human cell line (10% FCS), a significant difference was observed
between the direct antitumour effect of the weak GnRH antagonist
SJ-1004 and the GnRH antagonist MI-1544 or chicken GnRH antagonist
MI-1892, respectively. SJ-1004 elicited only a decrease of 17% in
the cell count whereas both antagonists (30 .mu.M) resulted in a 34
to 36% inhibition in the case of a treatment carried out every
other day for five days.
[0158] When used in the same dose of the active substance,
P-GFLG-1544 conjugate induced an inhibition of 58%. It may be
supposed that the antitumour effect of MI-1544 antagonist is
additively strengthened by the copolymer covalently bound thereto
(Table II).
[0159] When administered in a concentration of 30 .mu.M of the
active substance, the P-GFLG-[1892 conjugate containing MI-1892
showed 45% inhibition on the cell proliferation. Considering that
one single treatment was performed with the conjugate, the polymer
improved the inhibitory effect of the antagonist in this case, too
(Table II). TABLE-US-00004 TABLE II Inhibition of cell division by
GnRH analogue/macromolecule conjugates on various human tumour cell
lines Exam. No.(compd.) Cell line Dose (.mu.m) % Inhibition 6.
P-GFLG-1544 MCF-7 30 58 MDA-MB-231 30 45 Ishikawa 30 95 PC3 30 68.5
5. P-GFLG-1892 MCF-7 30 45 MDA-MB-231 30 42 Ishikawa 30 91 PC3 30
51 8. P-GFLG-1004 MCF-7 30 63 MDA-MB-231 30 68 Ishikawa 30 20 PC3
30 35 11. P-GFLG-GnRH-III MCF-7 30 10 MDA-MB-231 30 11 13.
P-GLG-MI-1892 MCF-7 30 33 MDA-MB-231 30 35 12. P-FLG-MI-1892 MCF-7
30 33 MDA-MB-231 30 35 MCF-7: mammary tumour cell line of human
origin; MDA-MB-231: mammary tumour cell line of human origin; PC3:
prostate tumour cell line of human origin; Ishikawa: endometrium
tumour cell line of human origin.
[0160] P-FLG-1892 and P-GLG-1892 conjugates containing a tripeptide
spacer group and given in an active substance content 10 of 30
.mu.M resulted in a direct antitumour effect of only 30 to 35%
(Table 2). In the case of MDA-MB-231 ER-negative cells (10% FCS),
the treatment in every two days with SJ-1004 resulted in a decrease
of 23% of the cell count, whereas the treatment with 15 MI-1544 and
MI-1892 (30 .mu.M) decreased the cell count by 35% to 36%.
[0161] When administered in an active substance concentration of 30
.mu.M, P-GFLG-1544 conjugate resulted in 45% and P-GFLG-1892 in 42%
inhibition of the proliferation (Table II). By 20 using P-FLG-1892
or P-GLG-1892 a lower, namely 33 to 35%, inhibition was seen (Table
II).
[0162] On the PC3 prostate and Ishikawa endometrium carcinoma cell
lines, the proliferation-inhibiting effect of the effective
GnRH-analogue substances (MI-1544 and MI-1892) was 25 significantly
increased by. the copolymer covalently coupled thereto. When used
in a concentration of 30 .mu.M, after two treatments on days 2 and
4, MI-1544 inhibited the proliferation of Ishikawa cell line by 8%
and that of the PC3 cell line by 24%; whereas P-GFLG-1544 once
administered resulted in a direct inhibition of 95% of the
proliferation on Ishikawa cell line and 68.5% on the PC3 cell
line.
[0163] After two treatments, the MI-1892 chicken GnRH antagonist
active substance resulted in a 14% and 33% inhibition on Ishikawa
or PC3 cell cultures, respectively. After a single administration
the P-GFLG-1892 conjugate exerted a direct proliferation-inhibiting
effect of 51% on the PC3 cell line and a more significant
inhibition of 91%, respectively, on Ishikawa cell line (Table
II).
[0164] SJ-1004 as a weak GnRH antagonist showed a weak antitumour
effect. After two treatments (days 2 and 4), an inhibition of only
17% was observed on the MCF-7 cell line 15 whereas this inhibition
was found to be 23% on the MDA-MB-231 cell line, 20% on Ishikawa
cell line and 10% on PC3 cell line, respectively. When administered
once in the conjugate form, P-GFLG-1004 resulted in a nearly
identical significant inhibition (63 to 68%) on both mammary
carcinoma cell lines 20 (Table II).
[0165] The P-GFLG-1004 conjugate exerted a weaker direct
proliferation-inhibiting effect than P-GFLG-1554 or P-GFLG-1892;
this was found to be 35% on PC3 and 20% on Ishikawa cell line,
respectively. In the conjugates of the GnRH-III synthetic lamprey
GnRH or P-GFLG-GnRH-III, the investigation on the proliferation-
inhibiting effect was carried out only on the MCF-7 and MDA-MB-231
cell lines. In agreement with investigations on the inhibition of
colony formation, this was the single GnRH hormone out of the
agonistic. and antagonistic derivatives tested, which showed as,
the active substance alone a more significant inhibition of colony
formation or proliferation; respectively, in comparison to the
results achieved with the P-GFLG-GnRH-III conjugate. By using the
active substance, two treatments (days 2 and 4) resulted in a
direct inhibition of 40 to 39% whereas an inhibition of 10 to 11%
was only observed after one treatment (on day 2) with
P-GFLG-GnRH-III (Table II).
[0166] As shown in Table II, the proliferation-inhibiting effect is
in agreement with the results of inhibition of the colony formation
within a certain error. Based on our observations, a strict
correlation exists also between the inhibition of colony formation
and the in vivo effect of the substances, too.
EXAMPLE 20
The In Vivo Toxicological Study of the P-GFLG-1892 and P-GFLG-1544
Conjugates
[0167] These experiments were carried out on 21 control and 20
treated female CBA/Ca mice each. The treatments were as follows: 7
animals as controls without treatment; 7 animals once administered
subcutaneously (s.c.) with the 10-fold of the effective dose
(active substance concentration: 400 .mu.g/animal); 7 animals once
administered intraperitoneally 25 (i.p.) with the 10-fold of the
effective dose (active substance concentration: 400 .mu.g/animal).
None of the substances proved to be toxic. On the 5th day following
the i.p. treatment and on the 7th day following the s.c. treatment,
the body weights of the animals were increased by 10% whereas the
weights of the control animals did not change.
EXAMPLE 21
Investigation on the In Vivo Antitumour Effect of P-GFLG-1892 and
P-GFLG-1544 Conjugates on Immunosuppressed Mice Bearing Human MCF-7
or MDA-MB-231 Xenograft
[0168] When inoculated to immunosuppressed CBA/Ca mice, the 10
MCF-7 and MDA-MB-231 cells had formed a tumour which could
successfully be transplanted. The immunosuppression of mice was
carried out by using the method of Steel et al. (1978).
Immunosuppression and Xeno-Transplantation
[0169] CBA/Ca female mice of about 6 weeks old were thymectomized
and then, after one week, they were irradiated with 60Co (whole
body irradiation; the lethal dose is 9.5 Gray). Within 24 hours 3
to 5.times.10.sup.5 myelocytes were injected to the animals.
Beneath the skin of the immunosuppressed mice 2.times.10.sup.7
tumour cells or, in the case of later passages, about 2 mm.sup.3 of
tumour cells or in the case of later, additional passages about 2
mm.sup.3 of tumour pieces were implanted. After
xeno-transplantation the mice bearing MCF-7 xenograft were treated
weakly once with the mixture of 50 .mu.g of estradiol valerate and
30 .mu.g of Norgestomet (Intervet International B.V.). The
treatment with steroid hormone was stopped by at least one week
before beginning the investigation. (The take usually occurred
during 24 days.)
[0170] In the case of MCF-7 tumours (xenografts) the examinations
were carried out with passages being in the phase of the 37th and
38th, respectively, transplantations. Based on the observations,
the growth of tumours was accelerated after transplantation of
several years and showed a two-fold growth rate in comparison to
the state after the 4th or 5th passage. (In the case of control
animals the tumour after 4-5 passages grew from 0.3 g to 3-4 g
whereas the weights of tumours after the 37th or 38th
transplantation increased from 0.3 g to 8 g during 6 weeks). It
should be noted that likely, a clonal selection occurred during the
passages; namely, clones growing more rapidly prevailed the
slower-growing populations (clonal selection).
[0171] In the case of mice bearing MCF-7 xenograft, the treatment
was begun in the 4th week following xeno-transplantation. This
examination was carried out on 50 animals (with a tumour volume of
255 to 319 mm.sup.3; (d12.times.d2.times.3.14):6].The treatments
were carried out as follows. Daily twice MI-1892 (2.times.25 .mu.g
in every 12th hour) s.c. given to 8 animals each; daily 50 .mu.g or
in every 3rd day 150 .mu.g of P-GFLG-1892 or P-GFLG-1544 conjugate
(calculated for the active substance) to 8 animals or 6 animals
each, respectively; P-GFLG-OH given daily in an s.c. dose
corresponding to 50 .mu.g of active substance to 6 animals each;
and 150 .mu.l of physiological saline daily administered s.c. to 8
control animals each. At the end of the 4th week of treatment the
daily two treatments with the MI-1892 antagonist substrate and the
daily single treatment with P-GFLG-1-892 and P-GFLG-1544 conjugates
resulted in a decrease of 20 to 30% in the tumour volume in
comparison to the tumour volume of controls of the same age. Thus,
the treatments diminished the rate of tumour growth in such a
manner that the substance administered daily twice and the
conjugate administered daily once led to about the same result.
(The former treatment caused a descrease of 20%, the latter one a
decrease of 30%, respectively, in the tumour growth.) The rate of
tumour growth was not influenced by P-GFLG-1892 or P-GFLG-1544
given in every 3rd day or by daily given P-GFLG-OH.
[0172] Recent investigations have begun on 28 animals bearing MCF-7
xenograft .and on 28 animals bearing MDA-MB-231 xenograft. On
animals bearing MCF-7 tumour the treatment was commenced in the 5th
week following xeno-transplantation; the tumour volume was 205 to
225 mm.sup.3. On animals bearing MDA-MB-231 tumour, the treatment
was started in the 4th week after xeno-transplantation with a
tumour volume of 140 to 150 mm.sup.3. The treatments were carried
out in such a manner that daily 75 .mu.g of P-GFLG-1892 conjugate
s.c. (calculated for the active substance) or daily 50 .mu.g,
respectively, of P-GFLG-1544 conjugate s.c. (calculated for the
active substance) or daily 50 .mu.g, respectively, of P-GFLG-OH
carrier s.c. (calculated for the active substance) were used. The
control groups were daily treated with physiological saline s.c.
(Each treated group consisted of 7 animals.)
[0173] It should be emphasized at repeated investigations carried
out on mice bearing MCF-7 xenograft that both conjugates
P-GFLG-1892 and P-GFLG-1544 resulted in a 30 to 35% inhibition of
tumour growth even in the 2nd week of treatment (Table 3). By
exceeding the results of former investigations in the 4th week, the
decrease caused by P-GFLG-1892 was 37% (the tumour volume was 2.428
cm3), the decrease caused by P-GFLG-1544 was 49% (the tumour volume
was 1.806 cm3) in the tumour volumes in comparison to controls of
the same age. In the 6th week, the inhibitory effects of both
conjugates were found to be essentially equal; even a tumour-free
animal was found in the case of P-GFLG-1544. A 43 to 49% inhibition
of tumour growth was achieved (Table III). TABLE-US-00005 TABLE III
Effect of the tumour growth on MCF-7 xenograft Example No.
Treatment Tumour volume 5 (compound) weeks as control % Inhibition
% MI-1892 4 78.5 22.5 2 .times. 25 .mu.g 6 79 21 5. P-GFLG-1892 2
70 30 1 .times. 50 .mu.g 6 57 43 6. P-GFLG-1544 2 70 30 1 .times.
50 .mu.g 6 51 49 Xenograft: transplantation of human tumour into
immunosuppressed mice
[0174] TABLE-US-00006 TABLE IV Effect on the tumour growth on
MDA-MB-231 xenograft Tumour volume (cm3) as control (%) weeks
Example No.(compd) 0 1 2 3 4 5 6 7 6. P-GFLG-1544: 0.15 0.21 0.21
0.24 0.28 0.33 0.36 -- 1 .times. 50 .mu.g 100 83 66 66 67 75 63 --
(%) 5. P-GFLG-1892: 0.15 0.21 0.25 0.27 0.34 0.33 0.33 -- 1 .times.
75 .mu.g 100 84 79 74 81 75 58 -- (%) Control: 0.15 0.25 0.32 0.37
0.41 0.45 0.57 -- 11. P-GFLG-GnRH-III: 0.17 0.25 0.33 0.40 0.41
0.49 0.40 0.31 1 .times. 100 .mu.g: 104 107 80 72 64 53 32 24 (%)
GnRH-III: 0.19 0.26 0.36 0.47 0.55 0.81 1.04 1.28 (%) 115 113 86 85
85 87 85 101 6. P-GFLG-1544: 0.17 0.24 0.3 0.36 0.35 0.46 0.58 0.75
1 .times. 100 .mu.g: 101 105 72 65 53 49 47 59 (%) Control: 0.17
0.23 0.42 0.56 0.65 0.93 1.23 1.27 Xenograft: transplantation of
human tumour into immunosuppressed mice. The animals were daily
treated in s.c. route.
[0175] TABLE-US-00007 TABLE V Effect on the tumor growth on
MDA-MB-231 xenograft Example No. Treatment Tumour volume 5
(compound) weeks as control % Inhibition % MI-1892 4 75.5 24.5 2
.times. 25 .mu.g 6 78 22 5. P-GFLG-1892 2 73.5 26.5 1 .times. 50
.mu.g 6 56 44 6. P-GFLG-1544 2 76.5 23.5 1 .times. 50 .mu.g 6 60 40
6. P-GFLG-1544 2 71 29 1 .times. 100 .mu.g 6 47 53 11.
P-GFLG-GnRH-III 2 80 20 1 .times. 100 .mu.g 4 64 36 6 33 67
GnRH-III 2 86 14 4 85 15 6 85 15 Xenograft: transplantation of
human tumour into immunosuppressed mice. The animals were daily
treated in s.c. route.
[0176] TABLE-US-00008 Volumes and weights of tumours after 6-week
treatment Volume (cm.sup.3) Weight (g) Treated with P-GFLG-1892
4.327 + 0.379 4.0 + 0.9 Treated with P-GFLG-1544 3.502 + 0.321 4.8
+ 0.8 Control 6.868 + 0.573 7.9 + 0.8
[0177] In the case of mice bearing MDA-MB-231 tumour, a 21 to 34%
decrease in the tumour volume was observed even from the 2nd week
in comparison to the control group of the same 20 age (Table IV).
The growth of the tumours (estradiol receptor-negative,
estradiol-independent tumours) was not inhibited by chemical
castration; therefore, the effect observed was unequivocally the
result of a direct antitumour action. The treatment was terminated
in the 6th week; nearly the same inhibition of 37 to 42% was
observed after the treatment with two conjugates (P-GFLG-1892 and
P-GFLG-1544, respectively,) (Table III). A tumour-free animal was
found at the end of the 6th week in the groups treated with the
conjugates.
[0178] A more recent study was started on 28 animals bearing.
MDA-MB-231 xenograft. The treatment was begun in the 4th or 5th
week following xenotransplantation. The tumour volume was 0.165 to
0.194 cm3. The treatments were carried out daily with 100 .mu.g of
active substances (calculated to GnRH-III, P-GFLG-GnRH-III or
P-GFLG-1544, respectively) administered in s.c. route. (For
treatment of the control group, see the preceding Examples). Each
group consisted of 7 animals. During the treatments the 10 tumour
volumes were weakly measured for 9 weeks. The results are
summarized in Table III.
[0179] The GnRH-III active substances in themselves did not inhibit
the rate of tumour growth. The increase in the dose of P-GFLG-1544
GnRH antagonist conjugate (50 .mu.g in the preceding series, 100
.mu.g in this series of experiments) did not raise the inhibitory
effect of the conjugate on tumour growth. It is considered to be a
very important result that 2 animals from 7 ones treated with
P-GFLG-GnRH-III and 1 animal from 7 ones treated with P-GFLG-1544
became tumour-free in 20 the 6th week of treatment. TABLE-US-00009
Volumes and weights of tumours after 9-week treatment Volume
(cm.sup.3) Weight (g) P-GFLG-GnRH-III 0.613 + 0.307 0.45 + 0.4
GnRH-III 2.017 + 0.585 1.84 + 0.52 P-GFLG-1544 1.618 + 0.518 0.94 +
0.52 Control 2.249 + 0.787 1.505 + 0.29
[0180] An excellent 73 to 78% inhibition, not observed till now,
was obtained with the P-GFLG-GnRH-III conjugate at the end of the
9th week. No significant difference was found between the body
weights of treated and control animals, respectively, either during
the treatment or at the termination of treatment.
EXAMPLE 25
Investigation on the In Vivo Antitumour Effect of P-GFLG-1892 and
P-GFLG-1544 on Mice Bearing MXT Mouse Mammary Tumour
[0181] When inoculated to CBA/Ca mice, the MXT mouse mammary 10
tumour cells developed a tumour. The take of tumour occurred in
100%; thus, the. treatments could be started before the development
of tumours, on the day following xenotransplantation. A further
importance of the model lies therein that, in opposition to the
immune system of mice bearing human xenograft, the immune system of
mice bearing MXT mouse mammary tumor was not artificially
suppressed; therefore, this model was equally useful to investigate
the antitumour effect as well as the action exerted on the immune
system. The MXT mouse mammary carcinoma contains ER and PgR
proteins; furthermore, it contains also GnRH receptors; therefore,
this in vivo tumor model is suitable for studying both the direct
and indirect effects. The treatment of mice bearing MXT mammary
tumour was commenced on the day following transplantation. The
investigation was accomplished on 35 animals in such a way that 75
.mu.g of MI-1892, MI-1544, P-GFLG-1892 and P-GFLG-1544,
respectively (calculated for the active substances), were s.c.
injected daily once to each mouse while the tumour volume was
systematically measured from the 9th day of treatment. The results
are shown in Table VI. TABLE-US-00010 TABLE VI Effect on the tumour
growth on MDA-MB-231 xenograft Tumour volume (cm3) as control (%)
Example No. days (compound) 9 11 13 17 18 20 24 26 P-GFLG-1544:
0.38 0.74 1.26 2.43 3.91 4.74 7.34 9.03 (%) 55 41 39 46 52 51 58 65
P-GFLG-1892: 0.29 0.66 0.91 1.86 3.1 3.61 6.07 8.91 (%) 40 36 28 36
42 38 48 64 Control: 0.7 1.8 3.22 5.11 7.42 9.26 12.66 13.82
MI-1544: 0.44 1.03 1.74 2.63 3.94 5.02 6.08 8.42 (%) 63 57 54 51 53
54 48 60 MI-1892: 0.61 1.05 1.59 3.17 4.8 5.73 6.32 11.30 (%) 86 58
49 62 64 61 50 82
[0182] The animals were daily treated in s.c. route starting from
the day following transplantation of MXT mouse mammary tumour. The
treatment was performed as follows: P-GFLG-1544: 75
.mu.g/day/animal related to MI-1544 active substance; P-GFLG-1892:
75 .mu.g/day/animal related to MI-1892 active substance; MI-1544:
75 .mu.g/day/animal; MI-1892: 75 .mu.g/day/animal.
[0183] The percentages of inhibition are summarized in Table VII.
The strongest inhibition of tumour growth was observed at the end
of the 2nd week of treatment. The percentages of inhibition
measured at the end of 2nd and 3rd weeks are shown in Table VII.
The results show that, in this model, the P-GFLG-1892 conjugate is
much more effective than MI-1892 active substance in itself. No
difference exists between the inhibitory effect of MI-1544 and that
of P-GFLG-1544. TABLE-US-00011 TABLE VII Effect on the tumour
growth on MXT mouse mammary tumour Example No. Treatment Tumour
volume 5 (compound) weeks as control % Inhibition % MI-1892 13 49
51 1 .times. 75 .mu.g 20 61 39 MI-1544 13 54 46 1 .times. 75 .mu.g
20 54 46 5. P-GFLG-1892 13 28 72 1 .times. 75 .mu.g 20 38 62 6.
P-GFLG-1544 13 39 61 1 .times. 75 .mu.g 20 51 49 The animals were
daily treated in s.c. route starting from the day following
transplantation of the MXT mouse tumour.
EXAMPLE 26
Preparation of [Lys(.epsilon.Fmoc)]5-GnRH-III
[0184] The peptide is prepared on a benzhydrylamine resin (of 0.65
milliequivalent/g capacity) by using an automatic peptide
synthetizer. The protected amino acid Boc-glycine is used in an
excess of three equivalents calculated for the capacity of the
resin; DIC as condensing agent and HOBt as catalyst are employed in
amounts equivalent to the protected amino acid. The coupling of
Boc-Gly-OH to the resin lasts for 12 hours. Thereafter, the
completion of coupling to the resin is controlled by means of
ninhydrin reaction of the resin/protected amino acid compound. The
coupling to the resin of Boc-Gly-OH is usually complete in the
first coupling; if in some cases the ninhydrin reaction gives a
positive result (indicating that the amino groups of the
benzhydrylamine resin are not fully substituted), the coupling to
the resin can be made complete by using the symmetric anhydride
method. (Based on the weight increase, the capacity of the resin
amounts to 75-80% of the capacity value given by the manufacturing
company.) After cleaving and neutralizing the Boc-Gly-BHA resin in
the usual way, the peptide synthesis is carried out stepwise
according to the following scheme: TABLE-US-00012 Minutes 1.
washing 3 times with dichloromethane 2 2. cleavage once with a
mixture of 1:2 ratio by 2 volume of TFA and dichloromethane 3.
cleavage once with a mixture of 1:2 ratio by volume of TFA and
dichloromethane 4. washing 3 times with dichloromethane 2 5.
washing 3 times with ethanol 2 6. washing 3 times with chloroform 2
7. neutralization twice with a mixture of 1:9 3 ratio by volume of
triethylamine and chloroform 8. washing twice with chloroform 2 9.
washing 3 times with dichloromethane 2 10. addition of Boc-amino
acid 11. coupling once with diisopropylcarbodiimide 120-300 13.
washing with ethanol
[0185] On cleaving the Boc protective group, a mixture of 0.5 10%
by, weight of indole with 0.2% by volume of thioanisole or a
mixture of 2% by volume of anisole with 0.2% by weight of
L-methionine is. employed for preventing side reactions. Protected
amino acids are usually coupled by employing the carbodiimide
method, but BOP reagent is used for bulky amino acids (with high
steric demand) (e.g. Leu, Trp, Cpa).
[0186] In the case of a positive ninhydrin reaction, the coupling
is carried out with symmetric anhydride after carbodiimide
coupling; or with BOP reagent after BOP-reagent coupling. In the
course of the synthesis, a dimethylformamide (DMF) solution-.
containing a three-fold excess of Boc-amino acid, molar ratio of
DIC coupling agent and HOBt catalyst calculated for 0.5 mmol of BHA
resin are used in an order according to the sequence. In the
present Example, Boc-L-Lys(.epsilon.-Fmoc)-OH protected amino acid
is used as amino acid in position 5.
[0187] The protective groups of the side chains and the peptide are
removed from the resin by liquid HF in such a way that 0.25 mmole
of peptide--BHA resin is maintained in 20-25 ml of HF in the
presence of 2.5 ml of 10% by weight of p-cresol containing anisole
and 100 mg of dithiothreitol at 0.degree. C. for 1 hour. After
removing HF under reduced pressure and treating the residue with
absolute diethyl ether, the peptide is dissolved from the solid
residue in a 15-33% by volume acetic acid solution. In the present
case, the hydrofluoride salt of the peptide is purified by gel
filtration on Sephadex G-25 column in 33% by weight acetic acid to
obtain 360 mg of crude product with a chemical purity of 85%;
Rf2=0.5. Subsequently, the peptide is purified by using
medium-pressure liquid chromatography (MPLC) on C18 reverse-phase
silica gel column with gradient solutions.
EXAMPLE 27
Preparation of Lys.sup.5-GnRH-III
[0188] After dissolving 240 mg of crude intermediary peptide
derivative in 10 ml of DMF-water mixture of 1:1 ratio, 2 ml of
piperidine are added while stirring and cooling with ice-water.
After 2.5 hours the mixture is evaporated and the residue dissolved
in 33% acetic acid and purified on a Sephadex G-25 column. The
fractions are investigated by means of TLC and the main product is
collected. The crude product is purified by using MPLC
procedure.
[0189] The conditions of the column included: Prepex C-18
(25-40.mu., Phenomenex, USA) Column: 400 mm (in length).times.25 mm
(in diameter) Eluent A: 70% by vol. of 0.05 M ammonium acetate
solution (pH 4.00) and 30% by vol. of methanol B: 50% by vol. of
0.05 M ammonium acetate solution (pH 4.00) and 50% by vol. of
methanol.
[0190] The elution is carried out by gradient elution with an
eluent composed of solutions A and B. The pure main fraction is
collected, then made free from salts and purified by carrying out
gradient elution on the above column. Eluent: A: 10% acetic acid
400 ml B: a 80:20 mixture of A and isopropanol 400 ml. The pure
fractions are collected and the residue is lyophilized to give 77
mg of the aimed product; Rf1=0.40, Rf2=0.15.
EXAMPLE 28
Preparation of Lys5,cyclo[Asp6-Lys8]-GnRH-III
[0191] a) The crude intermediary peptide derivative (120 mg)
described in Example 26 is transformed to its hydrochloride salt by
dissolving the peptide 1 in 6 ml of water and adding 2 ml of 0.1 N
hydrochloric acid solution. After evaporation of the solution to
dryness under reduced pressure, 102 mg of hydrochloride salt are
obtained; R.sub.f2=0.5.
[0192] b) Cyclization: A solution containing 29 mg of peptide
hydrochloride of step 3/a) in 25 ml of DMF is cooled to 0.degree.
C. and 200 .mu.l of 1% sodium hydrogen carbonate solution are
added. Simultaneously, 10 mg of BOP reagent and 10 mg of
1-hydroxybenzotriazole are dissolved in 5 ml of DMF and added
slowly, portionwise to the above aqueous solution. Subsequently,
from a stock solution containing 170 .mu.l of diisopropyl
ethylamine (DIEA) and 5 ml of DMF, 200 .mu.l are added to the
reaction mixture which is then stirred at room temperature
overnight. The intermediary peptide formed (Rf2=0.6) is subjected
to the next transformation without isolation. c) Removal of the
Fmoc group. After evaporating the reaction mixture 3/b) under
reduced pressure, the residue is triturated with ethyl acetate and
filtered. The solid is dissolved in 5 ml of DMF, then a mixture of
5 ml of DMF with 200 .mu.l of piperidine is added and the reaction
mixture is evaporated under reduced pressure. The residue is
dissolved in 33% by volume acetic acid and purified on a Sephadex
G-25 column. The fractions containing the main product are further
purified by using MPLC method.
[0193] Eluent A: 70% by vol. of 0.05 M ammonium acetate solution
and 30% by vol. of methanol B: 30% by vol. of 0.05 M ammonium,
acetate solution and 70% by vol. of methanol. The pure fractions
are lyophilized twice to obtain 11 mg of the aimed product;
R.sub.f2=0.35 Rf7=0.187.
EXAMPLE 29
Preparation of Lys.sup.5[Lys(.epsilon.-Fmoc)].sup.8-GnRH-III
[0194] The method described in Example 26 is followed with the
difference that Boc-L-Lys(.epsilon.-Fmoc)-OH is used instead of
Boc-L-Lys(.epsilon.-Z)-OH in position 8 of the GnRH-III sequence
whereas in position 5 Boc-L-Lys(.epsilon.-Z)-OH is employed instead
of Boc-His(Tos)-OH as protected amino derivative. The purification
is accomplished by using the method of Example 26. A crude product
is obtained in a yield of 335 g (with a chemical purity of about
80%); R.sub.f2=0.55.
EXAMPLE 30
Preparation of Lys.sup.4[Lys(.epsilon.-Fmoc)].sup.8-GnRH-III
[0195] By using 1 mmol of BHA resin Example 26 is followed with the
difference that Boc-Lys(.epsilon.-Fmoc)-OH is used as protected
amino acid in position 8 and Boc-Lys(.epsilon.-Z)-OH in position 4
of the peptide. After being purified on Sephadex G-25 column, the
crude peptide is obtained in a yield of 1.04 g, with a purity of
80%; R.sub.f2=0.29.
[0196] For purification, a solution containing 550 mg of crude
product in 20% by volume acetic acid was applied onto a MPLC column
according to Example 27 and eluted first with 200 ml of 20% by
volume acetic adid (isocratic elution) and then purified by using
gradient elution using 400 ml each of the following solutions:
Solution A: 20% by volume acetic acid. Solution B: a 3:1 mixture of
solution A and isopropanol. The fractions are collected and
lyophilized to result in a yield of 217 mg of the aimed product;
R.sub.f2=0.45, R.sub.f7=200.18.
EXAMPLE 31
Preparation of Lys4-GnRH-III
[0197] To a solution of 200 mg of the crude peptide obtained in
Example 31 in 10 ml of DMF, 15 ml of DMF containing 10% of
piperidine are added under cooling with ice-water. One hour later
the reaction mixture is evaporated and the residue is purified by
using MPLC and gradient elution with 25% acetic acid up to a 2:1
mixture of 25% acetic acid and methanol. Thus, 68 mg of a pure
product are obtained which proved to be uniform on the basis of TLC
and HPLC analysis; R.sub.f1=0.5, R.sub.f2=0.67, R.sub.f3=0.05.
EXAMPLE 32
Preparation of [Lys( -Ac)]4-GnRH-III
[0198] To a solution containing 200 mg of crude peptide obtained in
Example 31 in 30 ml of DMF, DIEA and 130 mg of imidazole are added,
then the mixture of 5 ml of dichloromethane (DCM) and 200 .mu.l of
acetic acid anhydride are dropped thereto under stirring and
cooling. After one hour the reaction mixture is evaporated under
reduced pressure. The residue is triturated with diethyl ether, the
ethereal supernatant is decanted and the residue is dissolved in 15
ml of DMF. Then 2 ml of DMF containing 200 .mu.l of piperidine are
added and one hour later it is evaporated. The residue is dissolved
in 20% acetic acid and purified by using MPLC method. The elution
is carried out by using gradient elution (composed of 20% acetic
acid and a 6:4 mixture of 10% acetic acid and methanol) to give 70
mg of a pure product which proved to be uniform on the basis of TLC
and HPLC analysis; R.sub.f2=0.067, R.sub.f7=0.087.
EXAMPLE 33
Preparation of Glu.sup.6-GnRH-III
[0199] Example 26 is followed with the difference that
Boc-Glu-25-(OChx)-OH is used as protected amino acid in position 6
of the GnRH-III sequence. The purification is accomplished
according to Example 2 by using gradient elution in ammonium
acetate buffer solution. Characteristic values of the product:
R.sub.f=18 0.19, R.sub.f7=0.086.
EXAMPLE 34
Preparation of cyclo[Asp.sup.6-Lys.sup.8]-GnRH-III
[0200] After preparing hydrochloride salt according to Example 28
by using the lamprey GnRH-III decapeptide as starting substance,
the cyclization is carried out according to Example 28. The product
is purified according to Example 27.
EXAMPLE 35
Preparation of D-Ala.sup.10-GnRH-III
[0201] Example 26 is followed with the difference that, instead of
Boc-Gly-OH, Boc-D-Ala-OH protected amino acid is coupled as first
amino acid to the benzhydrylamine resin. The purification is
accomplished according to Example 27.
EXAMPLE 36
Preparation of
H-D-Trp.sup.1,[Lys(.epsilon.-Fmoc)].sup.8,D-Ala.sup.10-GnRH-III
[0202] Example 26 is followed with the difference that in the
GnRH-III sequence Boc-D-alanine is coupled as first amino acid: in
the case of lysine of position 8 Boc-Lys(.epsilon.-Fmoc)-OH
protected amino acid and to the N-terminal of the decapeptide
Boc-D-Trp-OH protected amino acid are coupled. The purification is
accomplished according to Example 26 on Sephadex G-25 column. The
intermediary derivative is purified by using gradient elution
according to Example 26.
EXAMPLE 37
Preparation of Ac-D-Trp.sup.1,D-Ala.sup.10-GnRH-III
[0203] From the protected peptide-BHA resin prepared according to
Example 36 the Boc group is removed according to Example 26, then
the peptide-BHA resin having a free amino terminal is acetylated
with a mixture containing acetic acid anhydride and imidazole.
Thereafter, the aimed peptide is cleaved from the resin by liquid
HF according to Example 1, the protecting group is split off
according to Example 32, and the product is purified according to
Example 27.
EXAMPLE 38
Preparation of H-D-Trp1,D-Ala.sup.10-GnRH-III
[0204] The Fmoc protective group is removed according to Example 32
from the protected intermediary peptide prepared according to
Example 36. Then the purification of the crude product is carried
out by following Example 27.
EXAMPLE 39
Preparation of [Trp(For-Ind)].sup.3,7-GnRH-III
[0205] Example 26 was followed with the difference that in position
8 of the GnRH-III sequence Boc-Lys(.epsilon.-Z)-OH, in positions 3
and 7 Boc-Trp(For-Ind)-H protected amino acid derivative are used.
The purifications are carried out by following Example 26.
EXAMPLE 40
Preparation of Phe.sup.7-GnRH-III
[0206] Example 26 was followed with the difference that
Boc-phenylalanine is used in position 7 of the GnRH-III sequence.
The purification is carried out by following Example 26.
EXAMPLE 41
Preparation of GnRH-III(1-9)-ethylamide
[0207] According to the GnRH-III sequence the protected amino acid
is coupled by starting from 1.46 mmol of Boc-Pro-Merrifield resin
by following Example 26. The peptide protected on its side chains
is cleaved from the resin by stirring it with a 20% by volume
ethylamine solution in DMF at 10.degree. C. for 48 hours.
R.sub.f2=0.5.
[0208] After evaporating DMF under reduced pressure, the residue is
triturated with diethyl ether. The protective groups of the side
chain are removed from the crude protected peptide by using liquid
hydrogen fluoride according to Example 26. The crude peptide 16 is
purified first on a Sephadex G-25 column followed by medium
pressure liquid chromatography to give 215 mg of the aimed product,
R.sub.f2=0.31.
EXAMPLE 42
Preparation of Lys.sup.5,D-Trp.sup.6-hGnRH
[0209] Starting from 0.5 mmol of BHA resin and following the method
of Example 26, as protected amino acids Boc-D-Trp-OH is used for
the synthesis instead of Boc-Gly in position 6 and
Boc-L-Lys(.epsilon.-Z)-OH instead of Boc-L-Tyr(Bzl)-OH in position
5 according to the sequence of amino acids of human GnRH. The
purification is carried out according to Example 1 by using
Sephadex G-25. The gradient elution is performed with 400 ml each
of the following solutions: A: 70% by vol. of 0.05 M ammonium
acetate solution (pH 4.00) and 30% by vol. of methanol 25 B: 30% by
vol. of 0.05 M ammonium acetate solution (pH 4.00) and 70% by vol.
of methanol. The solution obtained is lyophilized 3 times for
removing the ammonium acetate to give 52 mg of the aimed 21
product; R.sub.f2=0.34, Rf7=0.21.
EXAMPLE 43
Preparation of Lys.sup.4,D-Trp.sup.6-hGnRH
[0210] Example 26 is followed according. to the amino acid sequence
of hGnRH with the difference that in position 6 Boc-D-Trp-OH is
used, and in position 4 Boc-L-Lys(.epsilon.-Z)-OH protected amino
acid derivative is used instead. of Boc-L-Ser(Bzl)-OH. The
purification of the product is performed also according to Example
42 to obtain 60 mg of pure product.
EXAMPLE 44
Preparation of H-Glu.sup.1,D-Trp.sup.6-hGnRH
[0211] Example 26 is followed according to the amino acid sequence
of hGnRH with the difference that in position 6 Boc-D-Trp-OH is
used instead of Boc-glycine whereas in position 1
Boc-L-Glu(OChx)-OH protected amino acid derivative is used instead
of pyroglutamic acid. The product is purified according to Example
42, except that after the gradient elution with ammonium acetate a
step of making free from salts is inserted, wherein the gradient
elution is carried out on MPLC column by using 300 ml each of the
following solutions: Solution A: 20% by volume acetic acid;
Solution B: a 3:1 mixture of solution A and isopropanol. The pure
fractions are collected and lyophilized to give 53 mg of the aimed
product; R.sub.f1=0.29; T.sub.f17=0.19.
EXAMPLE 45
Preparation of Lys.sup.5,D-Phe.sup.6-hGnRH(1-9)-ethylamide 22
[0212] According to Example 41 Boc-Pro-Merrifield resin is used as
starting material but the synthesis is carried out according to the
sequence of human GnRH, except that in position 6 Boc-P-Phe-OH and
in position 5 Boc-Lys(.epsilon.-Z)-OH are used as protected amino
acids. The peptide is cleaved from the resin by stirring in
ethylamine at 0.degree. C. for 8 hours, then stirring at room
temperature overnight while ethylamine evaporates. Thereafter, the
protected nonapeptide ethylamide is dissolved from the resin by
washing with methanol and DMF. The solution obtained is evaporated
and the residue is triturated with ether until it solidifies. The
product obtained is treated with liquid HF according to Example 26,
then the product is purified by HPLC method according to Example
27.
EXAMPLE 46
Preparation of Lys.sup.4,D-Phe.sup.6-hGnRH(1-9)ethylamide
[0213] Example 45 is followed with the difference that
Boc-Lys(.epsilon.-Z)-OH is used as protected amino acid in position
4.
EXAMPLE 47
Preparation of Lys.sup.5,D-Cpa.sup.6-GnRH(1-9)ethylamide
[0214] Example 45 is followed with the difference that instead of
Boc-D-Phe-OH Boc-D-Cpa-OH is used as protected amino acid in
position 6.
Human Cell Lines Used in the In Vitro Experiments
[0215] MCF-7 human mammary carcinoma cell line was stabilized in
1973 by Soule et al. from the pleural fluid of a patient suffering
from mammary carcinoma. MDA-MB-231 human mammary carcinoma cell
line was isolated and stabilized by Cailleau et al. in 1974
similarly from pleural fluid. Both cell lines grow in monolayer.
MCF-7 and MDA-MB-231 human mammary carcinoma cell lines are
maintained in plastic flasks (Greiner), in Dulbecco-modified
Eagle-MEM (DMEM, GIBCO) liquid nutrient medium containing 10% of
fetal calf serum. MCF-7 cell line is estradiol receptor
(ER)-positive containing proteins specifically binding GnRH. Thus,
it represents, a useful model system for investigating the direct
effect of GnRH mediated by the GnRH receptor. Being ER-negative and
GnRH receptor-positive, MDA-MB-231 cell line is similarly suitable
to study the direct effect of GnRH. PC3 human prostate carcinoma
cell line was stabilized in cell culture by Kaighn et al. in 1979.
The cells are of epithelial type and form compact colonies in
clonogenic assays. The Ishikawa cell line originates from human
endometrial adenocarcinoma [M. Nishida et al.: Acta Obstet.
Gynecol. Jpn. 37, 1103-1111 (1985)], it is epithelial in its
character and contains steroid-as well as GnRH receptors.
EXAMPLE 48
Dose-Survival with GnRH Analogues on MCF-7, MDA-MB-231 Human
Mammary Carcinoma, PC3 Prostate Cancer and Ishikawa Endometrium
Tumor Cell Cultures
[0216] The examination gives accurate information about the
cell-damaging effect of the substance tested as a function of
varying doses. The treatment was carried out once; the colonies
formed from the surviving cells were counted after 8-12 days. When
using hormones, the substances were in general not toxic and
possessed the great advantage of being cell- or receptor-specific,
respectively. GnRH analogues were phase-specific, they inhibited
but did not destroy the cells in GO/G1 phase. A part of the
arrested cells again entered the cycle, an other part of them might
be destroyed (apoptosis). The colonies formed from arrested cells
did not achieve the countable colony size in the day of counting
the colonies (the colony number may be identical, the colony size
cannot be identical). In the case of a dose-response investigation,
colonies containing less than 15 cells were not counted. 300 cells
were put into each Petri dishes of 3.5 cm in diameter. The
treatment was once carried out 24 hours after setting, then the
colonies formed were counted after 8-12 days. The cells were
treated once after 24 hours with 1-50 .mu.M of various GnRH
analogues of Examples above or with 1-50 .mu.M of GnRH-III,
respectively. The percentages of inhibition obtained on MCF-7,
MDA-MB-231, PC3 and Ishikawa cells, respectively, together with the
dose of the active agent used are shown in Table VIII.
[0217] Based on these investigations, when used in a dose of 50
.mu.M, the known synthetic GnRH-III peptide resulted in a 45% and
49% inhibition, respectively, of colony formation of MCF-7 and
MDA-MB-231 cell cultures, respectively. Such a significant
inhibition has not been observed till now by using any GnRH agonist
(Ovurelin, Buserelin) or even GnRH antagonist (MI-1544). GnRH-III
did not inhibit the Ishikawa cell line within the dose range used
and it resulted only in a 21% inhibition of colony formation of the
PC3 prostate cell culture. The effect of GnRH-III peptide was
exceeded by the compound of Example 17 which caused an inhibition
of 65% on MCF-7 and 63% on MDA-MB-231 (see Table VIII). Beside the
compound of Example 42, the peptides of Examples 31 and 32 proved
to be most effective (35-42%) on the MDA-MB-231 mammary carcinoma
cell culture (Table VIII). On PC3 prostate tumour cell culture, the
compound of Example 8 administered in a dose of 50 .mu.M induced
the strongest inhibition (31%) whereas compounds 6 and 7 did not
influence and compound 17 weakly inhibited the colony-forming
ability of the PC3 cell line within the dose range used (Table
VIII). When employed in 50 .mu.M dose on the Ishikawa endometrium
tumour cell culture, the following results were achieved: 13%
inhibition of colony formation by compound 3; 21% by compound 6;
14% by compound 7; 2% by compound 8; 15 % by compound 17; and 24%
by compound 14.
[0218] In these studies the D-Trp6-hGnRH analogue (Sigma Co.)
commonly used in the tumour therapy was employed as control. It
appears from the data of Table VIII that, among the analogues
tested, the analogues of Examples 26 and 40 showed a weak if any
effect. Analogues 14 and 19 approximated the activity of the
control. Other analogues (3, 6, 7 and 8) exceeded the inhibitory
effect of control; analogue 17 resulted in a twice stronger
inhibition.
EXAMPLE 49
Inhibition of the Cell Division of Human Tumour Cell Lines
[0219] After being treated with trypsin, 300,000 each of MCF-7,
MDA-MB-231, PC3 or Ishikawa cells, respectively, were passed into
Petri dishes of 10 cm in diameter. Starting from the day following
the setting, the cells were treated with 1-50 .mu.M of the GnRH
analogue in the exponential growth phase. During the experiment
lasting for 5 days, the cells were treated in every two days with
the peptide hormone dissolved in the nutrient medium, then the cell
count was determined on the sixth day.
[0220] When used in 30 .mu.M dose, GnRH-III exerted an inhibition
of 35-40% both on MCF-7 as well as MDA-MB-231 cells. Based on these
investigations, Ovurelin or Buserelin as hGnRH agonists did not
induce a significant change in the cell count on the MCF-7
ER-positive cell line (5-15% inhibition) after 6 days' treatment;
whereas they resulted in a 25-30% inhibition of cell proliferation
on the DMA-MB-231 ER-negative cell line.
[0221] When used in a dose of 30 .mu.M, the anti-proliferation
effect of GnRH-III agreed with the direct antitumour effect of
known GnRH antagonists (mammary GnRH derivatives containing several
D- amino acids, MI-1892 and MI-1544) on both mammary tumour cell
lines (Table IX). The proliferation-inhibiting effect of both
substances 6 and 7 was nearly identical (27-31%) with the direct
proliferation-inhibiting effect of GnRH antagonists containing
several D-amino acids (MI-1544, MI-1892, SJ-1004) (Table IX).
[0222] As shown in Table IX, the proliferation-inhibiting effect of
the substances, except compound 15, agreed with the results of
inhibition of colony formation within certain limits. A close
correlation exists between the inhibition of colony formation and
the in vivo effectivity of the substances, too. The compositions of
known peptides used as reference substances are as follows.
[0223] MI-1892:
[0224]
Ac-D-Trp.sup.1,3,D-Cpa.sup.2,Lys.sup.5,[.beta.-Asp(DEA)].sup.6,D-A-
la.sup.10-hGnRH
[0225] MI-1544:
[0226] Ac-D-Trp.sup.1,3,D-Cpa.sup.2,D-Lys.sup.6, D-Ala.sup.10-hGnRH
[0227] [M. Kovcs et al.: Peptides 10, 925-931 (1989)]
[0228] SJ-1004:
[0229] D-Phe.sup.2, D-Trp.sup.3, D-Lys.sup.6-hGnRH
[0230] GnRH-III:
[0231] pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2
[0232] Decapeptyl:
[0233] pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2
TABLE-US-00013 TABLE VIII Inhibition of the colony formation on
various human tumor cell lines Compound Example No. Cell line Dose
% Inhibition 2. Lys.sup.5-GnRH-III MCF-7 50 .mu.M 0 MDA-MB-231 50
.mu.M 0 3. Lys.sup.5-cyclo- MCF-7 50 .mu.M 44 (Asp.sup.6,
Lys.sup.8)- 50 .mu.M 13 GnRH-III 6. Lys.sup.4-GnRH-III MCF-7 10
.mu.M 15 50 .mu.M 40 MDA-MB-231 10 .mu.M 18 50 .mu.M 35 Ishikawa 50
.mu.M 21 PC3 50 .mu.M 0 7. [Lys(.epsilon.-Ac)].sup.4 MCF-7 10 .mu.M
40 GnRH-III 50 .mu.M 52 MDA-MB-231 10 .mu.M 31 50 .mu.M 42 Ishikawa
50 .mu.M 14 PC3 50 .mu.M 0 8. Glu6-GnRH-III MCF-7 50 .mu.M 44
MDA-MB-231 50 .mu.M 25 Ishikawa 50 .mu.M 2 PC3 50 .mu.M 31 17.
Lys5-DTrp6- MCF-7 10 .mu.M 39 hGnRH 50 .mu.M 65 MDA-MB-231 10 .mu.M
30 50 .mu.M 63 Ishikawa 50 .mu.M 17 PC3 50 .mu.M 18 14.
[Trp(For-Ind)].sup.3,7- MCF-7 50 .mu.M 31 GnRH-III Ishikawa 50
.mu.M 24 15. Phe7- MCF-7 50 .mu.M 26 GnRH-III MDA-MB-231 50 .mu.M
21 19. H-Glu1, D-Trp6- MCF-7 10 .mu.M 16.8 hGnRH 50 .mu.M 32.7
D-Trp6-hGnRH MCF-7 10 .mu.M 17.1 (Decapeptyl) 50 .mu.M 36.7
MDA-MB-231 10 .mu.M 15.7 50 .mu.M 38.7
[0234] TABLE-US-00014 TABLE IX GnRH analogue Inhibition of cell
proliferation in human tumor cell lines Compound Example No. Cell
line Dose % Inhibition MI-1544 MCF-7 (2x) 30 .mu.M 36 MDA-MB-231
(2x) 30 .mu.M 34 Ishikawa (2x) 30 .mu.M 8 PC3 (2x) 30 .mu.M 24
MI-1892 MCF-7 (2x) 30 .mu.M 35 MDA-MB-231 (2x) 30 .mu.M 35 Ishikawa
(2x) 30 .mu.M 14 PC3 (2x) 30 .mu.M 33 SJ-1004 MCF-7 (2x) 30 .mu.M
17 MDA-MB-231 (2x) 30 .mu.M 23 Ishikawa (2x) 30 .mu.M 8 PC3 (2x) 30
.mu.M 10 6. Lys.sup.4-GnRH-III MCF-7 (2x) 30 .mu.M 27 MDA-MB-231
(2x) 30 .mu.M 28 7. [Lys(.epsilon.-Ac)]4- MCF-7 (2x) 30 .mu.M 31
GnRH-III 8. Glu.sup.6-GnRH-III MCF-7 (2x) 30 .mu.M 18 15.
Phe7-GnRH-III MCF-7 (2x) 30 .mu.M 0 GnRH-III MCF-7 (2x) 30 .mu.M 40
MDA-MB-231 (2x) 30 .mu.M 39 PC3 (2x) 30 .mu.M 10 Ishikawa (2x) 30
.mu.M 8
[0235] Any patents or publications mentioned - in this
specification are indicative of the levels of those skilled in the
art to which the invention pertains. These patents and publications
are herein incorporated by reference to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0236] One skilled in the art will readily appreciate that the
present. invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The present examples along with the methods, procedures,
treatments, molecules, and specific compounds described herein are
presently. representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention as
defined by the scope of the claims.
Sequence CWU 1
1
6 1 10 PRT Homo sapiens MOD_RES (1)..(1) PYRROLIDONE CARBOXYLIC
ACID 1 Xaa His Trp Ser Tyr Gly Leu Arg Pro Xaa 1 5 10 2 10 PRT
Petromyzon marinus MOD_RES (1)..(1) PYRROLIDONE CARBOXYLIC ACID 2
Xaa His Trp Ser His Asp Trp Lys Pro Xaa 1 5 10 3 10 PRT Artificial
GnRH analogue 3 Xaa His Trp Ser His Asp Trp Lys Pro Xaa 1 5 10 4 10
PRT Artificial GnRH analogue 4 Xaa His Trp Ser Tyr Gly Leu Arg Pro
Xaa 1 5 10 5 4 PRT Artificial Spacer moiety 5 Xaa Phe Leu Xaa 1 6
10 PRT Artificial GnRH analogue 6 Xaa His Trp Ser Tyr Xaa Leu Arg
Pro Xaa 1 5 10
* * * * *