~.V093~0.~582 1 1 ~ ~ ~ 6 PCT/C~/OO~I
1--
NoN-DE8EN8ITI8INa ANALOG8 OF GnR~
~ND OT~ER B~OLOG~CAL~Y ACTIVE LIaAND8
BACKGROUND OF THE INVENTION
Field of the In~a~ion
i
5The present invention is directed to novel
non-desensitizing snalogs of a biologically active
ligand containing at least one 6-membered aromatic ring
or 5-membered ring and a method for selecting non-
desensitizing analogs of a biologically active ligand
`' 10 containing at least one 6-membered aromatic ring or S-
membered ring. Additionally, the present invRntiOn is
directed to a novel method for treating a patient in a
non-desensitizing fashion with a non-desensitizing
analog of a biologically active ligand containing at
least one 6-membered aromatic ring or S-membered ring.
~oreover, the present invention is directed to a novel
method for ~alcing new and usef-ul non-desensitizing
analogs of G~ and opiate peptides as well as the
novel non-desensitizing compounds prepared thereby.
8tate o t~e ~rt
In living organisms, the interaction af a
biologically active ligand with a receptor to produce a
biological response is often accompanied by
desensitization of the receptor, such that a subseguent
interaction of the ligand with the receptor results in
attenuation of the subseguent biological response to
the ligand. Prior to the present invention, however,
very little was understood about the causes and effects
,' ~''', . ... . .
W093/0305X ~ ,i f, fi ~T/CA92/~281
surrounding the desensitization proce~ or how 6uch
detrimental desen~itization could be avoided. Thu6,
the primary focu~ up to thi~ point h~s been ~tte~pts to
develop agonistic and antagoni~tic analog6 of
biologically active ligands with l~ttle or no regArd to
their poten~ial desensitizing effect~.
For example, Gonadotropin-Relea6ing Hormone
(GnRH~ is one such biolog~cally actlve co~pound which
has been modified to produce both agoni~tic and
antagoni~tic analogs thereof. GnRH stimulates the
release of gonadotropins through interaction wlth
membrane-associated high affinity receptors on the
pituitary gonadotropes. Subsequently, these
gonadotropins act on the gonad~ to 6timulate the
synthesi~ of steroid 6ex hormonec. The pulsatile
release of GnRH, and thereby the release of
gonadotropins, controls the reproductive cycle in
domestic animals and humans.
Acute doses of GnRH agonists administered in
pulsatile fashion can increase the levels of
~uteinizing Hor~one (LH) and s~eroid 6ex hormones in
both animals and humans. Paradoxically, acute or
chronic doses of these agonists can 6uppress the levels
of LH and steroid hormones. Additionally, the effect
of multiple doses of GnRH agonists can be to suppress
estrogen formation in the female and suppress
testosterone formation in the male. Accordingly,
knowledge of the molecular process by which
desensitization occurs would be extremely useful in the
design of analogs of biologically active ligands in
which said receptor desensitization effects are
reduced.
.
~'
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~93/0~5X ~ 2 ~i ~; f) p( r/c~92/~o~,
Thus, it is an object of the pre~ent
invention to provide novel non~de~en6itizing ~nAlog~ of
~- ~ a biologically active ligand containing at lea~t one 6-
membered aromatic ring or 5-me~bered ring.
` 5 Yet another object of the present invention
- is to provide a novel method for selecting non-
; de6ensitizing analogs of a biologically active;lig nd
which cont~in at least one 6-membered aromatic ring or
5-membered ring.
still another object of the present invention
is to provide a novel method for treating a patient in
a non-desensitizing fashion with a non-desensitizing
~, analog of a biologically active ligand containing at
least one 6-membered arQmatic ring or 5-membered ring.
A further object of the present invention is
to provide a novel method for producing new and u~eful
analogs of Gnl~ and opiate peptides as well as the
novel analogs produced thereby.
~XaRY OF T9~ INVE~TION
,Jl 20 In accordance with the foregoing objectives,
~ there is provided a method for selecting non-
!~ ' desensitizing analogs of a biologically active ligand
¢ containing at least one 6-membered aromatic ring or 5-
membered ring. The method comprises selecting an
',25 analog of a biologically active ligand containing a 6-
; membered aromatic ring according to Formula I or
selecting a biologically active ligand containing a 5-
membered ring according to Formula II:
~5
~,
':~
~, ' ' .
W093/030~8 ~ r~ rr/~92/no~l
X X R2
S ~ R I R~ C C - ~ II
R~ 7 C
I
-N ~_ C~ R~ B R7
RP R9 N~ v~-
~ R9
In Formulafs I and II, above, X is selected
from H, R~, -ORI, h~lide, -CN, -CHO, C(halide)l, -alk-OH,
-alk-OR~, -aIk~ lk-CO~RI, -alk-SH, -alk-S~I,
-dlk-coNH2, -CO2H, -CO2RI, --CORI, --OCONH2, -OCH20H,
--OCH20RI,--OCORI,--N3,--N2,--NHCORI,--NO2,--NH2,--NHRI,
-NRI2, -Sf~H, -SO2RI, -SCORI, -NCS, -SCSRI, -SO2NH2,
-SO~nERI, -Sf~RI2, -SO4H, -PO3H, -PO~H2, -SH, -SRI, -N-N-RI,
'O, =NH, -N-RI, =N-OH, ~N-ORI, -CONH2, -CONHRI, -CONRI2,
-NHSO2RI, -SiRI3, tetrazole, imidazole, or 6ubstituted or
f 10 unsub~tituted phenyl, -alkylphenyl, -O-phenyl,
-f~-alkylphenyl, -O-benzyloxy-cnrbonyl. Additionally, R
is selected from alkyl of 1-7 carbon atoms, alkenyl or
alkynyl of 2-~7 carbon atoms or cycloalkyl of 3-7 carbon
: atoms, optionally halogenated at one or more hydrogen.
15 Likewise, R2, R3, ~, Rs, ~, R7, R~ and R9 are groups
selected from X or -OH. In the above, ~ is selected
from P, N, S, or C being in either the L- or
D-configuration and ~ can be deleted or extended by 1-2
carbon ~toms, substituted or unsubstituted. Moreover,
20 the nitrogen atom attached to ~ can be substituted or
unsubstituted C, S, O or P and the carbon atom attached
to ~ and oxygen can be substituted or unsubstituted N,
S, O, P or C. The aromatic ring of Formula I or the
ring of Formula II can contain 0-4 N, S or O atoms.
''.'~'
21.~ 6 pr/cAs2/oo2*l
Likewise, the aromatic ring cf Formul~ I or ring o~
Formula II can be fu~ed with a 6-membQred ~romat1a rlng
which can contain 0-4 N, S or 0 ~tom~ and which can be
substituted in the ~ame manner as the nromat~c r~ng o~
- S Formula I. Also included within Formulns I and II are
D-tyrosine ~nd D-histidine.
Al~o provided is a method of treating a
patient in a non-de6ensitizing fashisn with a non-
desensitizing analog of a biologically active ligand
containing at least one 6-membered aromatic ring or S-
~embered ring. The method comprises ~electing an
analog of a biologically active ligand containing a 6-
membered aromatic ring according to Formula I, a8
defined above, or selecting a biologically active
ligand containing a 5-membered ring according to
Formula II, ~s defined above. ~he next step in the
~ethod co~prises ~dministering to a patient in need
thereof a composition comprising a pharmaceutically
effective amount of the ~elected analog together with a
pharmaceutically acceptable carrier.
Also provided ~re new, non-desensitizing
analogs of a biologically active ligand containing at
least one 6-membered aromatic ring according to Formula
. I or 5-membered ring according to Formula II, as
defined above, but excluding the following compounds:
(13 antagonist analogs obtained by
substituting D-amino acids at positions 1, 2 or 3, and
analogs which have position 2 deleted;
(2) the following agonist analogs based on
human GnRH:
vv~: - ' '; ~' ~ ' .... :.,. - '" ' '
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:~, ' ' ' ., '
.,
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21~ ) r~
-6-
(A) [D-Phe6~GnRH in which the nydroxyl
- gxoup of tyrosine i6 pro~cted with benzyl, acQtyl,
tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl,
2,4-dichlorobenzyl, benzyloxycarbonyl or
2-bromobenzyloxycarbonyl;
(B) tPhe~]GnRH, [Ala4Phe~]GnRH and
tPhe(N3)s]GnRH;
(C) tDes~he5-D-Ala~ProNHR9]GnRH in which
position 2 i8 Hi~, Tyr, Trp or Phe(4'-NH2), position 7
i~ Leu, Ile or Nle, and R is Et, Pr, CH2CH2OH or CHMe2;
(D) tDes~5Pro-B9]GnRH in which position
3 is Trp, Phe or 3tl-napthylalanine); X is His, Phe or
~ Phe(F5); position 6 i8 D-Ala(Y) wherein Y i8 naphthyl,
anthryl, fluorenyl, phenanthryl, biphenylyl,
benzhydryl, phenyl or cyclohexyl with 3 or more alkyl
groups, perhydronaphthyl, adamantyl,
. perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl,
heterocycle aryl, optionally N-nlkylated alkylanine or
cycloalkylamine, or NHCHt(CH2)~HC(~NR2)RI~CO wherein: n
is l-S; Rl is alkyl or NRR3, wherein R is H or alkyl and
R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl,
;~ ~lkyl-morpholino or (CH2).(RJ)2, wherein n is 1-5 ~nd R,
i8 alkyl; R2 is H or R3; and wherein C~NR~ cnn be a
ring; position 7 is Leu, Ile, MeLeu or Trp; position 8
is Arg, Gln, Tyr or Leu; B is selected from GlyNH2, NHE~
~ . wherein B2 is alkyl, cycloalkyl or haloalkyl, and
! NHCONHB3, wherein ~ is H or alkyl;
; (E) analogs containing a gamma (~) lactam in
the 6-7 position, in which position 1 is Pyr, N-acetyl,
N-Pyr-imino acid, or (C~7 cycloalkyl)acyl; positions 2
and 3 are aliphatic or aromatic amino acid; position 4
is Ser, Thr or Ala; position 5 is an aromatic amino
acid His, Trp or Phe; position 8 is an amino acid with
a basic sidechain; position 9 is an imino acid or
aliphatic amino acid; po~ition 10 is GlyNH2, AlaNH2,
NHEt, NHPr, NHCH2CH2OH; and
. ~
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~093/n~5~ l'(l/CA92/~ ~l
h~
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~ F) analogs in which po6ition 5 i~ Phe,
Tyr(Me), N-alkyl~Phe, N-alkyl-Tyr(Me) or N-alkyl-
Tyr(Et), wherein the nitrogen atom of at least one of
the amide bonds is Alkylated.
Also provided 18 a novel methc~ for
synthesizing new and u~e~ul ~nalogs of GnRH and opinte
peptides a6 well a8 the novel analc~6 produc~d thereby.
Also provided are the distinctly novel
non-de ensitizing ~nalogs having the For~ula III:
X~-X2~X3~X~~X5~X6~X7~~~xs~xlo III.
In Formula III, above, Xl is L- or D-Pyr, N-acyl-~oino
acid, ~-C-alkyl-Pyr, or N-~cyl-~-C-alkyl-~mino acid; X2
X3, ~, ~, X7 and X~ are independently selected frc~m
natural, synthetic, protected or ~-C-alkyl- amino
acids, or ~-X~ optionally contains a ~-lact~; ~ is an
imino acid or ~-C-alkyl-imino acid; X~0 is -N(H)R
wherein R is GlyNH2, azaGlyNH2, alkyl or alkenyl,
cycloalkyl, haloalkyl, hydroxyalkyl or aryl; and X5 is
selected from Formulas I or II, as defined above. The
compounds excluded from the compounds according to
- Formula~ I an~ II are likewise-excluded from the
compounds according to Formula III
: Also provided are novel agonist analogs
according to Formula III, wherein X~ is Pyr, N-acyl-
amino acid, ~-C-alkyl-Pyr, N-acyl-~-C-alkyl amino acid;
X2 and X3 are aromatic L-amino acids; X~, X~ and X~ are
independently natural, synthetic, protected or
~-C-alkyl- amino ac:ds; ~ is a natural, synthetic or
protected D-amino acid, Gly, ~-C-alkyl-amino acid; or
~-X~ optionally contains a ~-lactam; X9 is imino Acid or
~-C-alkyl-imino acid; XJO is -N(H)R wherein R is GlyNH2,
azaGlyNH2, alkyl, alkenyl, cycloalkyl, haloalkyl,
hydroxyalkyl or aryl; and Xs is according to For~ulas I
~' ' ' .;
~ ' ' .
,.
.'
: :
.
~VO 93J0305X l'(, l'/C,'A92/002~ 1
~ L .l '~
or II. The compounds excluded fro~ the compounds
according to Formulas I and II nre likewi~e excludQd
from thege compounds according to Formuln III.
DETA~LED D~8CRIP~ION OF ~R~ INV~NTION
Although biologically ~ctive lig~nds have
been the subject of numeroua ~tudies in which thore
have been a multitude of agonistic and antagonistic
analogs crea~ed, there has been no succe6sful atte~pt~
at developing criteria by which one can 6elect
agoni~tic and antagonistic analogs of biologically
active ligands which are non-desen~itizing in natuxe.
For thi~ rea~on, the discovery by the pre6ent inventors
is potentially very falr reaching in 6cope. That i8,
the pre~ent inventor~ have di6covered that by
~electively ~odifying a biologically active ligand
containing ~t least one 6-membered aromatic ring or
5-membered ring in the manner ~ccording to the pre6ent
invention, that it is po~sible to produce analog6 of a
biologically alctive ligand which are non-desensitizing
in nature. It is to be understood that the term ~non-
desensitizing" de~cribes n biQlogically ~ctive ligand
with at least partially reduced desensitizing
properties.
As used herein, the term "biologically active
ligand" refer~ to a molecule which binds to a
biologically active receptor molecule and which
directly or indirectly affects the activity of the
receptor molecule. The binding of such ligands to the
receptor (acceptor) molecule is accordingly a necessary
precondition for initiating, terminating, altering or
preventing the biological activity in the receptor
; molecule. Any ligand which effects the biological
activity of the receptor molecule is said to be a
2 ~ , '1 r(~r/~2/oo2~l
-9 -
biologically active ligand. The biologic~lly ~ct~ve
ligand can be a substrAte, an agoni~t, an ~ntagonlst,
an activntor, an inhibltor, etc. Example~ of
biologically active lig~nd~ according to the present
S .invention include GnRH, al60 known ns Luteinizing
Hormone-Releasing Hormone ~LH-RH), and Angiotensln ~II
or III), and the like. Additional examples of
biologically active ligands are well documented in the
art.
Moreover, the biologically active ligand can
be either peptidic or non-peptidic in nature. Such
ligands can be indigenou~ to the organism where the
biologically active receptor i~ found. When the ligand
is one which is naturally occurring in that organism,
then that ligand is referred to as a naturally
occurring biologically active ligand. On the other
hand, the biologically ~ctive ligands can be synthetic
molecules which are complementary to the biologically
active receptor and which ~ffect the biological
activity of the receptor. Thus, any molecule which is
complementnry to n biologically active receptor and
which affects the biologiGal ~ctivity of the receptor,
is a biologically active ligand.
~The term n ligand" according to the present
;25 invention refers to any organic compound for which a
receptor naturally exists or can be prepared. The term
"receptor" according to the present invention refers to
a molecule which binds the ligand.
When binding of the biologically active
ligand to the biologically active receptor and the
activation of the active site results in an alteration
-of the biological activity of the receptor, e.g.,
initiates, increases, decreases or terminates the
;
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`~VO 93/0305X ~ PCr/CA92/0~
--10--
biological activity of the receptor, the llgand 18 ~ld
to directly affect the activity of the receptor. On
the other hand, a biologically active ligand lndlrectly
affects the activity of the biologically actlve
receptor when the binding of the ligand to the receptor
results in an inability to activate the receptor.
Activation of the active site of the
naturally occurring biologically active ligand/receptor
complex is generally nccomplished by some sort of
chemical interaction between the ligand and the
r~ceptor. When the chemical interaction involve~ the
transfer of charge from one re6idue to another wherein
one of the residues is either a phenol or phenolate
re~idue, the ~nteraction is termed a charge-transfer
interaction. Such charge-transfer interactions are
believed to result in the alteration of the structure
of the ligand or ligand/receptor complex. Becau~e such
charge-transfer interactions can now be detected by the
techniques employed in the present invention, it is now
possible to incorporate such interactions into the
model created for the naturally occurring biologically
active ligand ~nd to create agonists and antagoni~ts
which have reduced desensitizing properties at the
complementary receptor.
Any biologically active ligand containing a
phenolic group, such as tyrosine, may utilize the
phenolic group to desensitize a complementary receptor.
Methods for determining biologically active phenolate
ligands using spectroscopic techniques have been
described in detail in U.S. Ser No. 07/458,926 filed
December 29, 1989, which is incorporated herein by
reference.
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W093/0305~ PCr/CA92/~2~l
2 1 1 ~~ ri ~i tj
: In one aspect of the method according to the present invention, the presence of a phenolate spec1e~
~ in a biologically active ligand i6 a~certained by
mea~uring the fluorescence lifetime due to 6aid
: ` 5 phenolate species in a suitnble environ~ent ~uch n6
propylene glycol. The pre~ence of long lifetime
phenolate (tyrosinate) fluorescence is diagno~tic of
the occurrence of the active phenolate ~pecies which
may cause receptor desensitization. The long
10 fluorescence lifetime is produced by intra~olecular
interactions within the biologically active ligand when
the ligand is dissolved in a receptor-si~ulating
solvent such a8 propylene glycol.
In ~nother method aspect, the lability of the
15 phenolic OH proton may be determined by NMR
~pectroscopy of the biologically acti~e ligand in a
~uit~ble ~olvent such aæ DMSO. The absence of a NMR
signal characteristic of the phenolic OH proton, at or
- ~bout 9.2 ppm, is also an indication of the presence of
20 n phenolate species which may be involved in a receptor
J desensitization process. Furthermore, and as described
in the pre~ent disclosure, modification or ~ubstitution
of the phenolic OH group of ~ biologically active
ligand provides a method for establishing the
~ 25 involvement of the phenolic OH group in a receptor
¦ desensitizing process, particularly when such
:~ modification results in a ligand with reduced receptor
~j desensitizing properties, as exemplified herein for
: peptides based on Angiotensin II and GnRH.
The term "agonist" according to the present
invention refers to a biologically active ligand which
binds to its complementary biologically active receptor
and activates the latter either to cause a biological
35 response in the receptor or to enhance pre-existing
:
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, , ~;
. ;',:
~1VO g3/03058 PC.~/C~92/0028 1
~12.'~
-12-
biological activity of the receptor. The ~gonist can
be the naturally occurring biologically active ligand
or it can be 2 synthetic molecule which can also
activate the receptor. For ex~mple, it i6 known in the
art that Angiotensin II acts a8 an agoni6t for it6
complementary receptor, the Angiotensin II receptor.
OthOE examples of ~goni6ts for the Angiotensin II
receptor include ts~r~] Angioten~in II and the like. A
common characteristic of all ligands in this invention
is that the ~harge-transfer interaction in the ligand
which is necessary to desensitize the biologically
active receptor i8 compromised. Thnt is to s~y that
the charge-transfer interaction i~ es6entially
inoperable in the ligand.
The term "antagonist~ refer~ to a
biologically active ligand which binds to its
complementary biologically active receptor and either
prevents the activation of the latter or deactivates
the latter 80 as to either prevent or diminish the
biological activity of the receptor. For example, it
i6 known in the art that the non-peptides 2-n-butyl-1-
4-c~rboxybenzyl]-4-chloroimidazole-5-acetic acid) and
(methyl 2-n-b~ltyl-1-[4-(2-carboxybenzamido)-benzyl]-4-
chloroimidazole-5-acetate) sodium salt act as
~ 25 antagonists of the Angiotensin II receptor. Other
j examples of art-recognized antagonists to other
biologically active receptors include propranolol for
the ~-adrenergic receptor, cimetidine for the
Histamine-H2 receptor and the like.
The term "Angiotensin II" refers to the
biologically active ligand which is an octapeptide
represented by the amino acid seguence of: -
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe. Similarly, the term
"Angiotensin III" refers to the biologically active
~93~0~58 ~'C~'/C~92/~28l
ri ,)
-13-
ligand which i6 a heptapeptide represented by the amino
acid sequence of: Arg-V~l-Tyr-Ile-Hi~-Pro-Phe.
, ~
It is to be understood that the disclo~ure of
the present invention employs the conventional
abbreviations for the v~riou~ common amino acid~ as
gener~lly accepted in the peptide art and ns
recommended by the IUPAC-IUB Commission on Biochemical
Nomencl~ture, Biochemistry II, 1726 (1972). Many other
terms used throughout the present di~closure are
believed to be readily understood in the art but a
definition of some of these terms is provided in Table
1 for convenience sake.
TABLE 1
DEFINITION OF VARIOU8 T~XM8
Term Definition
Aib 2-aminoi~obutyryl
Alk Cl-C~ alkyl or alkenyl group;
Bzl benzyl group
Cha L-cyclohexylalanyl
DCC N,N~Dicyclohexylcarbodiimide
DMF dimethylformamide
Et ethyl group
Halide F, Br, Cl, or I;
HBT l-hydroxybenzo~riazole
HPLC high-pressure liquid chromatography
Me ~ethyl group
Mtr 4-methoxy-2,3,6-Trimethylbenzenesulfonyl;
Pmc 2,2,5,7,8-pentamethylchromzn-6-sulfonyl;
Pr propyl
t-Boc t-Butoxycarbonyl
TFA trifluoroacetic acid
Tos tosyl group
Trt trityl qroup
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'~: ' `,
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~g3/03058 P-'l'/C~92/~ ~l
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As noted pxe~iously, the chemic~1 interaction
between the ligand and the receptor can cause th~
desenæitization of the receptor such that a subsequent
interaction of the ligand on the receptor results ln
attenuation of the subseguent biological response to
the ligand. One ~uch biologic~lly active liqand which
6erves to illu~trate the effects of desensitization of
a receptor i8 GnRH.
As mentioned earlier, GnRH is released from
the hypothalamus and binds to a receptor on the
pituitary gl~nd, causing the release of LN (Luteinizing
Hormone) and FSH (Follicle-Stimulating Hormone). GnRH
has been purified and identified in mammals to be a
decapeptide having the amino ~cid fiequence: Pyr-His-
Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2. The following are
other naturally occurring analogs of GnRH:
Salmon GnRH: Pyr-His-Trp-Ser-Tyr-Gly-~E~-Iç~-Pro-
~ Gly-NH2;
', Chicken GnRH-II: Pyr-His-Trp-Ser-His-Gly-~L~-~yE-Pro-
i 20 Gly- ~ ; and
Chicken GnRH-I: Pyr-His-Trp-Ser-Tyr-Gly-Leu-Çln-Pro-
Gly-NH2 .
~ . .
How~ever, initial attempts at using GnRH to
induce ovulation were unsuccessful because of the
failure to rec~gnize the necessity of pulsatile
administration. Upon recognizing the therapeutic
potential of GnRH, biochemists began synthesizing both
agonistic and antagonistic analogs of GnRH.
.1 .
one approach to this goal has been the
replacement of an amino acid residue of GnRH with
-' another amino acid. Although in a few instances
decapeptides containing such a replacement have been
found to be more active than GnRH, due primarily to an
"
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W0~3/O~SX P(~/CA92/nn~l
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-15-
increase in re~i6tance to degr~dAtion by enzymes i~
vitro and in vivo, e.g., ~D-Aln6]-GnRH, A. Arimura, et
al., Endocrinology, 95, 1174 (1971) and [D-Leu~]-GnRH,
J. A. Vilchez-Martinez et al., Blochem. Blophys. Re~.
Commun, 59, 1226 (1974), for the most part the
replacement containing decapeptides have been less
active.
Thus, GnRH analogs fiynthesiz~d previou~ly
have been designed on the basis of resistance to
metabolic degradation, and have been particularly
concerned with the incorporation of a D-amino acid into
the GnRH peptide (as mentioned above), or with
N-methylation of one or more of the peptide bonds in
order to invoke intestinal stability to enzymes
(European Patent No. 0 328 090). The criteria for
selection of the analogs described herein is distinctly
different from previous work such as that of
EP-0,328,090 in that ~election is ba~ed on GnRH analogs
with receptor desens~tizing properties, achieved
2D principally by modifying the Tyr5 or His5 residue of
G~RH peptides iD the manner according to Formulas I and
II. -
. .
Contrary to the initial intent of theprevious workers, even when more active agonists of
GnRH were synthesized using the criteria for developing
agonists and antagonists with increased resistance to
degradation by enzymes, many were found to have
disadvantageous long-term effects. More specifically,
these analogs of GnRH stimulated the release of LH and
FSH initially, but continuous administration resulted
in desensitization of GnRH receptors on gonadotropes
and decreased gonadotropin secretion. Accordingly,
prolonged u6e of high concentrations of GnRH agonists
has caused reversible ovarian and uterine atrophy in
. ,
,
~'
~:
~ ~093/03058 P~T/CA92/00~l
21 ~2~ f-i
-16-
ani~als and has prevented implantation of ova ~nd
gestation. Likewise, high concentrations of previously
~ynthesi~ed GnRH agoni~ts in malQs has inhlbited
te~ticular synthe~is of te~to6terone.
; 5 However, through exten~ive rosQarch, the
present in~entors have di~covared a method for
6electing non-desen6itizing analog6 o~ a biologic~lly
active ligand containing at least one 6-me~bered
aromatic ring or 5-~embQred ring. ~he m-thod co~prise~
~electing an analog of a biologically active ligand
containing a 6-membered aromatic ring according to
Formula I or selecting an analog of a biologically
active ligand containing a 5-m~mbered ring according to
~ormula II:
X X R2
; ~ ~ R I R~ C C-----R3 II
R6 1 ~ \IcD
~ --N~ CO-- R6 ~----------R~
' R R -N f co - -
Rt R9
In Formulas I and II, above, X is selected
i from H, Rl, -ORI, halide, -CN, -CHO, C(halide)3, -alk-OH,
-alk-ORI, -alk-CO2H, -alk-CO2RI, -alk-SH, -alk-SRI,
-alk-CONH2, -CO2H, -C4RI, -CORI, -OCONH2, -OCH20H,
-OCH2ORI, -OCORI, -N3, -N2, -NHCORI, -NO2, -NH2, -NHRI,
-NRI2, -SO3~, -SO2RI, -SCORI, -NCS, -SCSRI, -SO2NH2,
-SO2NHRI, -SO2NRIz, -S04H, -PO3H, -PO4H2, -SH, -SRI, -N~N-RI,
=O, =NH, =N-RI, =N-OH, =N-ORI, -CONH2, -CONHRI, -CONRI2,
~ ,
~'VI) 93/0305X PC'r/CA92/002~ 1
,, .1 1 ,~ .i ~; ~,
-17-
-NHSO2RI, -SiRI3, tetrazole, imidAzole, or substltuted or
unsubstituted phenyl, -alkylphenyl, -O-phenyl,
-O-alkylphenyl. Addition~lly, Rl i~ selected from alkyl
of 1-7 carbon atom~, alkenyl or alkynyl of 2-7 cnrbon
S atoms or cycloalkyl of 3-7 carbon atoms, option~lly
halogenated at one or more hydrogen. Likewise, R2, R3,
~, Rs, R6, R7, R~ and R9 are groups ~elected from X or
-OH. In the above, ~ i8 selected from P, N, Sj or C
being in either the L- or D-configuration and B can be
deleted or extended by 1-2 carbon atoms, substituted or
unsubstituted. Moreover, the nitrogen atom attached to
can be ~ubstituted or un6ubst$tuted C, S, O or P and
the carbon ~tom attached to a and oxygen can be
6ubstituted or unsubstituted N, S, O, P or C. The
aroma~ic ring of Formula I or the ring of Formula II
can contain 0-4 N, S or O atoms. Likewise, the
aromatic ring of Formula I or ring of For~ula II can be
fused with a ~-me~bered aro~atic ring which can contain
0-4 N, S or O atoms nnd which can be substituted in the
s2me ~anner as the aromatic ring of Formula I. Also
' included within Formulas I and II are D-tyrosine and
D-Histidine.
..
! The analogs of a bioiogically active ligand
are selected 80 as to contain a ring modified according
to Formulas I or II and so that the resulting
biologically active ligand has reduced desensitizing
properties, i.e., the biologically active ligand i8
such that the subsequent interaction of the ligand with
the receptor does not substantially attenuate the
biological responce. The di~covery and use of analogs
according to Formulas I or II, and especially GnRH
analogs according to Formulas I or II, with reduced
desensitizing properties is new.
J
. ,': ' .
~ ."..
: ~
'. ~
~'093/0.~0~ 2/00~l
,~ I 1. ~ .i ~i ii
-18-
The an~log of a biologlcally ~ctive lig~nd
containing at least one 6-mQmberQd aromatic ring can be
~elected, for exnmple, from the peptides Angiot~nsin
(II or III) or GnRH. Thu6, one method according to the
5 pre~ent invention is the ~election of a non- -
de~ensitizing analog of tbe blologically active ligand
GnRH wherein the tyro~ine residue At the 5-position ha~
been modified according to Formula I.
More speciSically, some preferred analog6
6elected by the present method include GnRH analogs
represented by the formula III:
X~-X2-X3-X4-~-~-Xrx~~xs~xlo III.
In Formula III, above, X~-X~0 (positions 1-10) are
selected from the following: X~ i6 Pyr or N-acyl-amino
acid; X2 i5 an aromatic amino acid; X3 is an aromatic
amino acid, X4 i6 Ala, Thr, Ser, H6e, Ser(Me),
~-C-methyl-Ser(Me), Aib; Xs is Tyr(0-alkyl),
Phe(4'-halogen); Phe(4'-alkyl); }4 i6 Gly, Aib, or
D-amino acid (natural or 6ynthetic, including an amino
acid bearing a protecting group), ~-C-alkyl-amino acid;
X7 is Leu, Ile, Trp, Cha, ~-C-methyl-~mino acid, Val; X~
is Arg, Gln, Tyr, Leu; X9 i6 P~o, pipecolic acid,
nipecotic acid, azetidine-2-carboxylic acid,
dehydroproline, thioproline, hydroxyproline; and X~0 is
N(H)R wherein ]R i8 GlyNH2, azaGlyNH2, alkyl, haloalkyl,
hydroxyalkyl, cycloalkyl or aryl, wherein each alkyl or
aryl group has from 1 to 7 carbon atoms.
Another particularly preferred embodiment is
one in which the ~-carbon atom of at least one of the
amino acids is alkylated, in which ca6e position 5 may
be as described by Formulas I or Il or may be
~-C-methyl-histidine or ~-C-methyl-tyro6ine, or the
like. The modifications illustrated for the tyrosine
~V093/03058 .Jl~ ~r;~; fi PC r/cA92/~2#l
- ~ 9 -
residue of GnRH are also applicable to other Tyr-
containing peptides.
.
In a similar fashion, the role of ~yr in
receptor desensitization can ~160 b~ carried out by
Hi~tidine in certain peptides, sf which chicken GnRH-II
i~ an example. Therefore, modifications to the
imidazole group which remove the hydrogen bond donor
capability of imidazole, for exaDple alkylation at N~,
or N3, can be expected to dimini~h the desensitizing
~ 10 effects of certain ligands containing imidazole.
- More generally, selective ~odification or
sub~titution of the hydroxyl groups in a biologically
sctive ligand containing a 6-membered zlromatic ring or
S ne~bered ring according to Formulas I and II,
re~pectively, may produce zmalogs with reduced
desen~itizing effects. Such low-desensitizing analogs
may ~160 be used to attenuate the de6ensitization
evoked by a high-desensitizing ligand, as illu~trated
for GnRH in ~camples 4 nnd 5, infra.
Additionally, selection of a bioloqicz~lly
~ctive ligand having a phenolate group zlccording to
Formula I or a 5-membered ring according to Formula II
1 may likewise favorably alter the duration of zlction of
¦ the ligand. Thus, in the case of an zlgonistic ligand,
the duration of the biological response is increased.
For example, tTyr(Me)s]-GnRH has a longer duration of
action than GnRH. This increase in duration of action
and the non-desensitizing nature of the analog
; tTyr(Me)5]-GnRH iG illustrated in Example 2 and, infra.
The same method is al~o applicable to
antagonist analogs of a ligand. In the case of an
antagonist ligand, however, the duration of action is
. ~; `
~'~93/0305~ PCr/C~2/00
-20-
decreased. An example i6 seen with ~ngiotensin,
wherein methylation of the tyro~ine OH of the
angiotensin antagoni6t tSarl Ile~] ANG II to give tsar'
Tyr(Me)4 Ile~] ANG II result~ in a reduction of the
desensitization properties and duration of action. The
decrease in the duration of action and the non-
desensitizing nature of the above antagoni6tic analogs
is illustr~ted in Ex~mple 1, infra.
It i6 important to understand, however, that
the present invention i8 not limited to peptides such
as GnRH and Angiotensin II or III, but m~y be applied
to any desensitizing b~ologically active ligands having
a 6-membered aromatic rlng or 5-membered ring.
Accordingly, other example6 demonstrating the exten6ive
applicability of the ~election method according to the
present invention are as follows.
Additional examples of non-desensitizing
analogs of a biologically active ligand selected
according to the present invention include, but are not
limited to, the opioid ligands, wherein
de~ensitization, tolerance, dependence, and withdrawal
may be effectively reduced by modification of the
phenolic group or conjugated hydroxyl group of an
-opiate peptide (which have the N-terminal sequence Tyr-
Gly-Gly-Phe-Met/Leu such as enk~phalins, dynorphins and
endorphins) or non-peptide ligands. Thuc, methylation
of a phenolic hydroxyl group of morphine produces the
less active analog codeine. Furthermore, deletion of
both hydroxyl groups in morphine can result in the
agonistic mimetic metbadone which di6plays increased
potency and decreased withdrawal symptoms due to the
blockade of morphine dependence. Accordingly, it i6
understood in the present disclosure that GnRH is u~ed
as an example only, and that tbe ~tructural
~
~.,.~.
~'
~YO93/0305X PCT/CA~/0028l
,''ii.~,jfj,i
-21-
modific~tion6 to the 6-~ember aro~atlc rlng in GnPH c~n
likewise be applied to ~ny bloloqic~lly active ligand`
having a 6-membered aromatio ring or 5-memb4red ring
which can be modified according to Formula~ I or Il.
~8 wa6 mentioned, such biologically actlve
ligands are likewise not limlted to mQrely the peptidic
ligands but al~o include non-peptidic ligands.
Consequently, the present invention i6 u6eful in
selecting agonistic non-peptidic analogs of a
lo biologically active ligand containing at least ane 6-
membered aromatic ring according to Formula I or 5-
me~bered ring accord~ng to Formula II in which the
interrelnted properties of desensitization, tolerance,
dependence, withdrawal and addiction are reduced.
As used herein, the term "non-peptidic
ligands" ~8 ~pplied to the Formulas I and II refer to
the ring structures depicted in Formulas I and II
wherein the peptide backbone has been removed. For
example, representative example~ of non-desensitizing
analogs of a hiologically active lig~nd ~elected
according to the prefient invention include, but are not
limited to, certain steroid hormone6 and catechol~cine
hormones. Such modifications where applicable, may
also be applied to a biologically active ligand
containing a conjugated hydroxyl group, of which
-C=C-C-OH is an example.
To further illustrate the far-reaching
effects of the present invention, GnRH will once again
be used as a representative example to di6play the
numerous advantages which can be nchieved by the
practice of the present invention. GnRH analogs are
useful for increasing 6exual activity, fertility And
egg yield and inducing ovulation in animals, and are
~,
.,': .
, .. . .
~`
~'' .
~093/O~S8 PCr/CA~2/00~l
21~2ri~ fi
-22-
useful for increasing productivity in the farm1ng of
fifih, poultry and mammals, as well as for incre~elng
fertility in humans. GnRH analogs can also increase
lifetime gamete production, and permlt collection of
gametes from pre- and poQt-natal, juvenile, prepubertal
and mature mammals for natural and artificial
fertilization. GnRH analog6 are also effective in the
treatment of gonadotropin-related disease6 suah a8
prostatic cancer, breast cancer, endometriosis and
~0 fibroid shrinkage.
However, as was mentioned, agonistic and
super agonistic analogs of GnRH often reguire
continuous or rapid pulsatile dosing ~n order to avoid
the potent desensitizing effects associated with these
peptides. The development of GnRH agonist analogs with
reduced receptor desen~itizing properties is
advantageous because such analogs obviate the need for
continuous or rapid pulsatile dosing methods and may be
administered in a single dose or witb considerably
lower dose frequency. The advantage of eliminating the
need for pulsatile dosing likewi~e extends to all
~ analogs of a biologically active ligand according to
.~ the present invention.
i~ .
~; The use of tbe present metbod of selecting a
-non-desensitizing analog of a biologically active
ligand quite surprisingly possesses tbe additional
advantage of not requiring tbe continued use of solely
the non-desensitizing analog but allows for the use of
"normallyH desensitizing analogs witbout tbe expected
desensitizing effects upon tbe biologically active
receptor. For example, a non-desensitizing GnRH analog
may be administered alone or in combination with
another normally desensitizing GnRH agonist since the
desensitizing properties of the latter are suppressed
~, ,'' ' '' ~'
S~
W093/0~s8 , r~/CA92/~28l
-23-
by the pre6ence of, or pretreatment with, the ~or~er.
Stated in another wny, by treating a pntient with ~
non-desensitizing GnRH analog ~ccording to the present
invention, it is po66ible to sub~equently or
simultaneou61y treat the p~tient with an normally
desen6itizing analog of GnRH becau~e tho pretreatment
with, or pre~encQ of, the non-desen~itizing GnRH analog
~ccording to the present invention act6 to eliminate or
suppres6 the de6en6itizing effect of the normally
de6ensitizing GnRH analog. A "normally" desen6itizing
analog is an analog of GnRH which has not been modified
in a manner which reduce6 the de6ensitizing properties
as illu~trated in the present invention. The use of a
non-desQnsitizing GnRH analog in combin~tion with
normally de6ensitizing GnRH or 6ynthetic analogs
thereof i8 a preferred method a6pect of the present
invention.
A "biologically active receptor" according to
the present invention is a molecule, having a specific
binding 6ite for its complementary ligand, and includes
classical hormon~l receptors, binding and/or transport
proteins, enzyme~, antibodies and the like. One
embodiment of a biologically active receptor includes
membrane-bouncl proteins which control certain cellular
proces6es in which themselves Are regulated by the
binding, or lack of binding of a complementary
naturally occurring biologically active ligand.
Because such membrane bound biologically active
receptors are bound to membrane, it i6 believed that
the conformation of the biologically active ligand
necessary to activate such receptors are lipid induced.
On the other hand, there are other biologically active
- receptor6 which are not membrane bound. In 6uch cases,
cuch receptors may not require a lipid induced
conformation of the biologically active ligand and, in
~' ' `' .
i~.''
~' .
~!O 93/0305X I'CI /~,A92/01)2~ 1
--24--
fact, may require an aqueous induced conformation of
the complementary biologically active lig~nd in order
to activate such receptors.
Examples of biologically active receptors
have been well documented in the art. Specific
examples include insulin receptor (wherein the
complementary ligand i~ insulin) and the Angioten6in II
receptor (wherein the complementary ligand i6
Angiotensin II~, and the like.
A6 de6cribed above, the non-desen6it$zing
analogs of a biologically active ligand ~elected
according to the present invention have a multitude of
uses in hu~ans and animals, either of which are
considered "patients~ as that term is employed in the
present disclosure or claims. Additional exa~ples of
desired use6 of non-de~ensitizing GnRH analogs of a
biologically active lig~nd selected according to the
present invention include, for example, the ability to
more readily ~ynchronize estrus in live6tock, e.g.,
cattle, sheep or 6wine, either in order to be able to
~ mate all the female6 in a given group with a male of
i the desixed genetic quality, or so as to be able to
¦ perform artificial insemination OD a ~aximum number of
females, both within a comparatively short period of
time. It will be appreciated that 6uch a method is of
particular importance for breeders of race horse6 and
show animals, where the fee is paid for the services of
an exceptional male animal sften amount to considerable
sums of money.
Thus, in accordance with the multitude of
potential uses for non-desensitizing analogs of
biologically active ligands, the present invention i5
also directed to a method of treating a patient in a
~ ' ' ' ~, ' ''"''' ;' ' . '' ,
, ~ ''' ' '' , , "`-,
~. .. . . ..
W0 93/0305X ~ rj r~ . j P(~l /CA92/0028 1
non-desensitizing fashion with a non-de~Qnsitizlng
analog of a biologically active ligand containing at
least one 6-membered aromatic ring or 5-~e~bered ring.
The method compri6es first selecting ~n nnalog Or a
biologically active lig~nd cont~ining a 6-membered
aromatic ring according to Formula I or selectlng an
analog of ~ biologically ~cti~e ligand containing a
5-me~bered ring According to Formula II:
X X R2
R~ ~,,R2 C~C
~5 ~ R3 I R~ C C ~ II
\~/
R~-----~ R~ C
----N_ F~-- B~ --R7
Rt RP --N Co----
RJ R9
In Formula~ I and II, above, X is selected
from H, R~, -ORI, halide, -CN, -CHO, C(balide)3, -alk-OH,
-alk-ORI, -alk-C ~ , -alk-C~R~, -alk-SH, -alk-SR~,
-alk-CONH2, -CO2H, - ~ Rl, -CORI, -OCONH2, -OCH20H,
-OCH20RI, -OCORI, -N3, -N2, -NHCORI, -NO2, -NH2, -NHRI,
-NRI2 ~ -SO3H, -SCiRI, -SCORI, -NCS, -SCSRI, -SO~NH2 ~
-SO~nERI, -SO2NRI2, -SO4H, -PO3H, -PO4H2, -SH, -SRI, -N~N-RI,
=0, =NH, -N-RI, =N-OH, ~N-ORI, -CONH2, -CONHRI, -CONRI2,
-NHS~RI, -SiRI3, tetrazole, imidazole, or substituted or
unsubstituted phenyl, -alkylphenyl, -O-phenyl,
-O-aIkylphenyl, -O-benzyloxy-carbonyl. Additionally, R
is selected from alkyl of 1-7 carbon atoms, alkenyl or
. alkynyl of 2-7 carbon atoms or cycloalkyl of 3-7 carbon
atoms, optionally halogenated at one or more hydrogen.
Li~ewi~e R2 R3 R4, R5, R6, R7, R3 and R9 are groups
;
~ .
" ~ .
, . ~ .
.
WO 93/0305X ~ I'CI'/C'~92/00~#1
-26-
selected from X or -OH. In the above, ~ i8 ~elected
from P, N, S, or C being in either the L- or
D-configuration and B can be deleted or extended by 1-2
carbon atoms, sub6tituted or unfiubstituted. Moreover,
S the nitrogen atom attnc~ed to ~ can be ~ubstituted or
~nsubætituted C, S, O or P and the carbon atom attached
to ~ and oxygen can be ~ub~tituted or un~ub~tituted N,
S, O, P or C. The ~romatic ring of Formula I or the
ring of Formula II can contain 0-4 N, 8 or O ato~s.
Likewise, the aromatic ring of Formula I or ring of
Formula II can be fu6ed with a 6-membered aromatic ring
which can also contain 0-4 N, S or O atom6 which can be
substituted in the ~ame manner as the aromatic ring of
Formula I. Al60 included within Formulas I ~nd II,
above, are D-tyro6ine and D-histidine.
Step two of the method compri6es
administering to a patient in need thereof the ~elected
analog of a biologically active ligand a6 a compo6ition
compri6ing (i) a pharmaceutically effective amount of
the selected analog and (ii) a pharmaceutically
acceptable carrier for the sQlected analog. While it
is possible for the particular active ingredient to be
administered as the raw chemical, it i~ preferable, in
view of the potency thereof, to present it a8
pharmaceutical formulation containing an acceptable
carrier.
In a preferred method, at least one non-
desensitizing an~log of GnRH i6 given in combination
with at least one normally desensitizing GnRH or
synthetic analog thereof. The non-desen6itizing
analog(s) of GnRH may be given before a normally
desenstizing GnRH analog, simultaneously therewith, or
both.
,
~J~O 93/0305~ 1'( '1/( `A92/1~2S~ 1
i~ :1 1. ,' ,,, ~,
-27-
For each of t~e utilities mentioned nbov¢,
the amount of the analog of the biologic~lly ~ctlv~
ligand will, of course, vary both with the p~rtlcular
active ingredient and with the route of adminifitratlon.
In general, however, for e~ch of t~e~e utilities the
dosage for na~al or parental ad~inistration will be in
the rAnge of about 0.005 to 200~g per kilogr~m body
weight of the patient, preferably between about 0.01
and 100 ~g/kg. For ornl or vaginal administr4tion the
dosage would generally be in the range of about 0.005
to lOOO~gJkg, preferably about 0.05 to 20~g/kg. It i6
under tood that all dos~ges are calculated with
reference to the base peptide. These dose~ apply to
both a non-desensitizing and normally desensitizing
analog of GnRH when ~dministered in one of the
combination protocols described above.
It should be recognized that by "non-
desensitizing fashion", it is understood that the
present method treats a patient in a manner such that
the patient does not need to be treated with the type
of pulsatile dosing that was heretofore required in
order to successfully ~chieve the hormonal changes in
the body of ani~als and humans. ~ather, the analogs,
alone or in combination with normally desensitizing
analogs, according to the present invention obviate the
need for continuous or rapid pulsatile dosing methods
and thus may be administered in a single dose or with
considerably lower dose frequency.
The formulations of the present invention
comprise an active ingredient or combination thereof,
as described above, together with one or more
pharmaceutically acceptable carriers as well as other
optional therapeutic ingredients. The carriers must be
pharmaceutically "acceptable" in the sense of being
.
~. ' ''
~. ''
~"'~ ' ' .
~093tO~SX ~ ,it;lj P~ A92/oo~l
-28-
compatible with the other ingredient~ of the
formulation and not deleterious to thQ reciplent
thereof. Desirably, the formulatione ~hould not
include oxidizing agents and other substances with
S which peptides, or non-peptides, Are known to be
incompatible.
The foruul~tions include those suitable for
oral, rectal, nasal, topical, vaginal or parenteral
(including subcutaneous, intr~muscul~r, and
intravenous) administration, although the more suitable
route in any given case will depend upon the sel~cted
active ingredient.
A6 another preferred route of adcinistration,
an active ingredient(s) may be presented as a depot
formulation having a slow-release characteristic
suiting it for implantation in the body of the
recipient, for example, sub-cutaneously,
intramuscularly, intraperitoneally or intravaginally.
For example, injections may be given in depot form with
a slow rele~se agent such as an emulsion, sesa~e oil, a
10% ~queous polyvinylpyrolido~e, or in biodegradable
microspheres ~uch ~s polylactic/glycolic acid.
Alternatively, administration may be given with the aid
of a mini osmopump, located internally or externally.
In animals such as fish, administration may be
optionally given with a dopamine antagonist such as
domperidone.
The formulations may conveniently be
presented in unit dosage form and may be prepared by
any of the methods well known in the art of pharmacy.
Typical methods include the step of bringing into
association the active ingredient with the carrier
which optionally contains one or more accessory
~X ~ :
~'.. :..... - ~ ,~, . ....
.'
. ...
~ .
:~
.
W~93/03~5X ,~ 92/~2~l
-29-
ingredient~. In gener~l, the formulatlon~ a~e prepared
by uniformly and intimately bringlng into ~seoci~tion
the active ingredient with liquid carrierG or finely
divided solid carriers or both, and then, if neces~ry,
~haping the product into the desired formulation.
Pormulations of the present invention
suitable for oral administrntion may be presented as
discrete units such as capsule , cnchets or tablets
each containing a predetermined amount of the active
ingredient; as a powder or granules; or as a solution
or a suspension in an aqueous liquid or a non-aqueous
liquid; or a5 an oil-water liquid emulsion or a water-
oil liquid emulsion. The active ingredient ~ay also be
presented as a bolus, electuary or pa6te.
A tablet ~ay be made by compres6ing or
~olding, optionally with one or morç accessory
ingredients. Compressed tablets may be prepared by
compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or grnnules,
optionally mixed with a binder, lubricant, inert
dilution, lubricating, surface active or disper6ing
agent. Molded tablet6 ~ay be made by molding in a
suitable machine a mixture of the powder compound
moistened with an inert liquid diluent.
For~ulations for rectal administration may be
presented as a suppository with the usual carriers such
as coco-butter, while a suitable formulation for nasal
administration is nasal drops comprising an active
ingredient in an aqueous or oily solution.
Formulations suitable for oral administration
include lozenges comprising the active ingredient in a
flavored basis, usually sucrose or acacia or
~'' "
~'
W093/03058 2 11 ~ rj !~ r~ Ag2/~l
-30-
tragacanth; and pa6tilles comprising the activ~
ingredient in an inert basiG such as gelatln, glycerlne
or sucrose and acacia.
Formulations suitable for vaginal
administration may be presented as pessaries, creams,
pastes, or sprny formulations containing in addition to
the active ingredients such carriers as are known in
the art to be appropriate.
Formulations suitable for parentnl
~dministration conveniently comprise sterile aqueous
solutions of the active ingredient, which solutions are
preferably isotonic with the blood of the recipient.
Such formulations may be conveniently prep~red by
dissolving a solid active ingredient in water to
produce an agueous solut~on, and rendering said
solution sterile and isotonic with the blood of the
recipient.
Also provided herein are new, non-
desensitizing analogs of biologically active ligands
containing at least one 6-m~mhered aromatic ring or 5-
membered ring. The non-desensitizing analogs of a
biologically active ligand contain at least one 6-
membered aromatic ring according to Formula I or
5-membered ring according to Formula II:
X X RZ
3 I~ C o~ 5 II
l i
- N C0-- R4-~ R7
R~ R~ -N~ 0--
, ,, ' ,~
WO93/0305R ,)i l~'JrjJj;; r(~r/cA92/~28
-31-
In Formulas I ~nd II, ~bove, X le ~e~ected
from H, Rl, -ORI, hnlide, -CN, -CHO, C~h~1ido)3, -~lk-OH,
-alk-OR~, -Alk-CO2H, -a1k-CO2R~, -alk-SH, -alk-8R~,
-alk-CONH2, -CO2M, -CO2RI, -CORI, -OCONH2, -OCH20H,
-OCH2ORI, -oCORI, -N3, -N2, -NHCORI, -NO2, -NH2 ~ -NHRI,
-NRI2, -SO~H, -SO2RI, -SCORI, -NCS, -SCSRI, -SO2NH2,
-SO2NHRI, -SO2NRI2~ -S04H, -PO3H, -PO4H2, -SH, -SRI, -N-N-RI,
-O, =NH, -N-RI, ~N-OH, -N-ORI, -CONH" -CONHRI, -CONRI2,
-N~SO2RI, -SiRI3, tetrazole, i~idazole, or substituted or
unsubstituted phenyl, -alkylphenyl, -O-phenyl,
-O-alkylphenyl, -O-benzyloxy-carbonyl. Additionally, R
is selected from alkyl of 1-7 carbon atoms, ~lkenyl or
alkynyl of 2-7 carbon atoms or cycloalkyl of 3-7 carbon
atoms, optionally halogenated at one or more hydrogen.
Likewise, R2, R3, R4, R5, R6, R7, R~ and R9 are groups
selected from X or -OH. In the above, ~ is selected
from P, N, S, or C being in either the L- or
D-configuration and B can be deleted or extended by 1-2
carbon atoms, 6ubstituted or unsubstituted. Moreover,
the nitrogen atom nttached to ~ can be substituted or
unsubstituted C, S, O or P and the carbon atom attached
to ~ and oxygen can be 6ub6tituted or unsub6tituted N,
S, O, P or C. The ~ro~atic ring of Formula I or the
ring of Formula II can contain 0-4 N, S or O atom6.
Likewise, the aromatic ring of Formula I or ring of
Formula II can be fused with a 6-membered aromatic ring
which can also contain 0-4 N, S or O atoms which can be
substituted in the same manner as the aromatic ring of
Formula I. Also included within the Formulas I and II,
above, are D-tyrosine and D-histidine.
Excluded from the compounds according to this
aspect of the present invention are those antagonist
analogs of mammalian GnRH obtained by substituting
~ ~ '
` ~;~93tO3058 2 L i ~ j ~ fJ I'CI/CA92/00281
--32--
D-amino acids at positions 1, 2 or 3, and analogs whlch
have position 2 deleted. Also excluded are the
following agonist analog6 based on mammalian GnRH:
(A) ~D-Phea]GnRH ~n which the hydroxyl
group of tyrosine is protected with benzyl, acetyl,
tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl,
2,4-dichlorobenzyl, benzyloxycarbonyl or
2-bromobenzyloxycarbonyl;
(B) lPhe5]GnRH, tAla~Phe51GnRH and
10 tPhe(N3)5~GnRH;
(C) tDesl~hes-D-Ala~roNHR9~GnRH in which
position 2 is Hi~, Tyr, Trp or Phe(4'-NH2), position 7
is Leu, Ile or Nle, and R i6 Et, Pr, CH2CH20H or CHMe2;
(D) tDes~qKsPro-B93GnRH in which position
3 is Trp, Phe or 3(1-napthylal~nine); X is His, Phe or
Phe(F5); position 6 is D-Ala(Y) wherein Y is naphthyl,
~nthryl, fluorenyl, phenanthryl, biphenylyl,
benzhydryl, phenyl or cyclohexyl with 3 or more alkyl
groups, perhydronaphthyl, adamantyl,
perhydrobenzhydryl, phenyl, cyclohexyl, dicyclohexyl,
heterocycle aryl, optionally N-alkylated alkylamine or
cycloalkylamine, or NHCHt(CH2)~NHC(eN~0 RI~CO wherein: n
is 1-5; Rl is ~lkyl or NRR3 whe~ein R i8 H or aIkyl and
R3 is H, alkyl, fluoroalkyl, cycloalkyl, phenyl, benzyl,
alkyl-morpholino or (CH2).(~)2 wherein n is 1-5 and RJ is
alkyl; R2 is H or R3; or wherein C=NR~R~ can be a ring;
position 7 is Leu, Ile, MeLeu or Trp; position 8 is
Arg, Gln, Tyr or Leu; and B is selected from GlyNH2,
NHB2 wherein 82 is alkyl, cycloalkyl or haloalkyl, and
NHCONH~ wherein ~ i~ H or alkyl;
(E) analogs containing a ~-lactam in the
6-7 position, in which position 1 is Pyr, N-acetyl,
N-Pyr-imino acid, or (C~7 cycloalkyl)acyl; positions 2
and 3 are aliphatic or aromatic amino acid; position 4
is Ser, Thr or Ala; posit:ion 5 is aromatic amino acid
His, Trp or Phe; position B is an amino acid with a
~'" ', ,.., ~' , '~"'', '"
~; .,.~ : .' ,.,
W093/0~58 PCr/CA92/~ ~l
2 1 1 2 5 6 G
-33-
basic sidechain; position 9 i6 imino ~cid or aliphatic
amino acid; position 10 ~8 GlyNH2, Al~NH2, NHEt, NHPr,
NHCH2CH20H; nnd
(F) analog~ in which po~ition 5 i~ Phe,
Tyr~Ne), N-alkyl-Phe, N-alkyl-Tyr(Phe) or N-alkyl-
Tyr(Et~, wherein the nitrogen atom of at least one of
the amide bonds is alkylated.
Some preferred novel analogs selected by the
present invention include GnRH analogs having the
Formula III:
Xl~X2~X3~X~~X~~X6~X7~x~~xs-xlo III.
In Formula III, above, X~-X~0 (positions 1-10) are
~elected from the following: X~ is Pyr or N-acyl-amino
acid; X2 is an aromatic amino acid; X3 is an aromatic
amino acid, ~ is Ala, Thr, Ser, Hse, Ser(Me),
~-C-methyl-Ser(Me) or Aib; X5 is Tyr(0-alkyl),
Phe(4'-halogen) or Phe(4'-alkyl); X6 is Gly, Aib, or
D-amino acid (natural or synthetic, including an amino
,~ acid bearing a protecting group) or ~-C-alkyl-~mino
acid; X~ is Leu, Xle, Trp, Cha, Val or
~-C-methyl-a~ino acid; X~ is Arg, Gln, Tyr or Leu; Xg is
Pro, pipecolic acid, nipecotic acid, azetidine-2-
carboxylic acid, dehydroproline, thioproline or
hydroxyproline; and XlO is N(H)R wherein R is GlyNH2,
25 : azaGlyNH2, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl or
aryl, wherein each alkyl or aryl group has from 1 to 7
carbon atoms.
Another preferred embodiment is one in which
the ~-carbon atom of at least one of the amino acids is
alkylated, in which case position 5 may be as described
by For~ulas I or II or may be ~-C-methyl-histidine or
~-C-methyl~tyrosine. GnRH analogs and other analogs of
biologically active ligands containing ~-C-alkyl-amino
acids are new. A particularly preferred embodiment
,,
~09?~/030~ P~'r/C~92/0021~1
2 I l ~ r) !; ~;
includes GnRH analogs in wh~ch at lea6t one o~
po~itions 4, 5, 6 and 7 contaln an ~-C-alkyl-amlno
acid. Such modifications are advantageous for
promoting and stabilizing ~urns occurring in thi~
region of the peptide backbone, and for lim~ting
proteolysis of the peptide.
M~ny of the analogs of a b~ologically ~ctive
ligand according to the pre~ent invention can be
synthesized by the methods well-known to one skilled in
the art. For example, compounds can be synthesized by
the solid phase method by one or more str~tegies Xnown
to one skilled in the nrt (J. Stewart and J. Young,
Solid Phase Peptide Svnthesis, 2nd Ed. (1984), Pierce
Chemical Co.). In one such example, chloromethylated
polystyrene or benzhydrylamine resin can be used with
N-t-butyloxycarbonyl protected amino acids and
sidechain protection such as His(Tos), Ser(Bzl) and
Arg(Tos). The peptide-resin bond may be cleaved and
protecting groups removed by treatment with ~nhydrous
HF, or the peptide-resin bond may be cleaved and
a~inoalkylated with alkylamine and the protecting
groups subse~ently removed with HF. The peptide may
then be purified by reversed-phase HPLC.
Purification of peptides can be accomplished,
for example, with the use of a Varian HPLC system
equipped with a Vista 401 micro-processor controller.
Separations can be achieved, for example, on a Bio-Rad
~i-Pore 313 reverse-phase preparative column (25.0 x
2.15 cm) at 25C with a stepped linear gradient of
acetonitrile in 0.1~ CF3CO~ at a flow rate of 7.5
ml/min. Automated repetitive injections of peptides (5
x 5 mg) can then be made from a nitrogen pressurized
Rheodyne injector with a 2.0 ml sample loop. One-fifth
~";,
:
93/03~ 3 , i` ~ l/<A92/~2~l
-35-
of the total sa~ple may then be injected durlng each
run by lowering the flow r~te to 4.0 ml/min for a
O.l-min Hinject" period. One cycle could thus con~i~t
of the following event6: o - lo min, 7.5 ml/min, 90%
H20/l0% of 1% ~queous CF3C02H; lo - 11 min, 4.0 ml/min;
11 - 11.1 ~in, "in~ectn; 11.1 - 13 min, 7.5 ml/mln, 70%
H20/20% CR3CN/lOS of 1% CF3C~; 13 - 30 m~n, 45% ~0/45%
CH3CN/10% of 1% CF3CO~; 30 - 42 min, so% ~HlCN/io% of 1%
CF3CO2H; 42-50 min, 100% H20.
In a novel solid phase peptide ~ynthes~s
method according to the present invention, synthesis i~
carried out at room temperature, the resin is a novel
chloro-(orthochloro)-trityl-resin to which the FMOC-
lS protected, terminal amino acid is attached in the
presence of diethylpropylamine (1.1 equivalents) in
methylene chloride for 1 hour.
Traditionally, two solid phase peptide
synthesis strategies using Boc-amino..acids and FMOC-
amino acids, respectively, have been used. In the Bocstrategy, the peptide-resin bond i5 cleaved with the
use of strong acid conditions,:such as anhydrous HF,
~uch that the protecting groups are simultaneously
removed. In the FMOC-strategy, the peptide-resin i5
25 .- cleaved with the use of intermediate acid conditions,
such as TF~, ~uch that most or all of the protecting
: groups are simu Naneously removed. ~sing the novel
(or~hochloro)trityl resin, the conditions for cleaving
, the peptide-resin bond is so mild that even the most
¦ 30 acid-sensitive protecting groups commonly used in solid
phase peptide synthesis remain attached to the peptide.
. Consequently, the ability to retain the acid-sensitive
protecting groups has thus allowed the present
inventors to synthesize new and useful analogs
` ~0 93/030s~s P(~/C~92/0028 1
-36-
biolo~ically active ligand~ such ~s GnRH and opiate
peptides.
Using the (orthochloro)trltyl resln, ~tepwise
synthesis of the peptide with FMOC-~mino acid6
(2.5 equivalents) is carried out wlth DCC/N~T
(2.5 equivalents) as coupling agent(s) 1n a ~ultable
solvent such a~ DMF for 1-2 hour6, and the FMOC groups
removed by treatment with 20~ piperidine in DMF for
10-30 minutes. Acid-sen6itive sidechain protecting
group~ such as His(trityl), Ser(t-butyl) and
Cys(t-butyl), and the like, remain intact when the
peptide-resin bond is cleaved with
trifluoromethanol/acetic acid/methylene chloride in a
ratio of 7/1/2, re~pectively.
Additionally, a peptide ~mide may be prepared
by treating the liberated peptide with meth~nolic HCl
for 1-4 hour~ to yield the methyl ester followed by
- aminolysis in methanolic ammonia for 4-24 hours.
Alternatively, treatment of the received peptide with
~onoalkylamine in the presence of a suitable coupling
agent such as DCC/HBT in a suitable ~olvent such as DMF
for 1-4 hours affords the peptide N-alkylamide.
Removal of protecting groups may be accomplished with
50% TFA in chloroform for 30 min. When an acid-
sensitive group is to be maintained, such asD-His( m ), the synthesis i~ carried out with ~limitedH
sidechain protection, for example, Pyr-His-Trp-Ser(Me)-
Tyr(Me)-D-His( m )-Leu-Arg-Pro-resin, or alternatively
by fragment condensation of Pyr-His-Trp-Ser(Me)-
Tyr(Me)-D-Hi~(Trt)-LieuOH (prepared using the
(orthochloro)trityl resin) to Arg-Pro-resin or Arg-Pro-
NHEt using a suitable coupling agent such as DCC/HBT
and a suitable solvent such as DMF.
J
~.
W093/0~58 ~; PC~/CA~/~ ~l
-37-
Synthesis of tSer(Me)~Tyr(Me)5DHis(Trt)~-
ProNHEt9]-GnR~ i8 accomplished using FMOC-amino ~cid~
and (orthochloro)trityl resin by the protocol outllned
above to give: Pyr-Nis-Trp-Ser(He)-Tyr(Me)-D-His(Trt)-
Leu-Arg-Pro-re6in which i6 treated with
F3COH/CH3C~N/CH2Cl, (7:1:2) for 1 hour ~nd filtered. The
peptide i8 precipitated from the filtrate with ether,
filtered, dis~olved in DMF and treated with NH~Et and
DCC/HBT (2 equiv. of eaoh) for 2 hours. The desired
product i5 precipitated with ether and puri~ied by
reverse-pha6e HPLC.
The novel compounds produced according to the
novel process described above are an~logs of GnAH, and
other hor~onal peptides, in which an acid-sensitive
protecting group is maintained at the end of the
synthesi~ as ~ result of the use of an extremely labile
peptide-resin bond provided by the (orthochloro)trityl-
resin bond. Such analogs include, but are not limited
to, compounds having the Formula III:
Xl-X2-X3-~-X5-~-X~-X~-X9-X~O III.
In Formula III, above, X6 is D-His having the
protecting group trityl, tosyl, dansyl, dinitrophenyl,
benzyloxymethyl or optionally substituted benzyl;
D-Ser/Thr having the protecting group trityl, t-butyl,
acyl, optionally substituted benzyl or
benzyloxycarbonyl; D-~ys having the protecting group
trityl, t-butyl, tosyl, or optionally substituted
benzyl; D-Tyr having the protecting group tosyl,
trityl, acyl, t-butyl, optionally substituted benzyl or
benzyloxycarbonyl; D-Arg having the protecting group
Tos, Mtr or Pmc; D-Orn/Lys having the protecting group
t-Boc, trityl or optionally substituted
benzyloxycarbonyl; D-Asp/Glu having the protecting
~' ' '' ' . .
~ r~
l~:
~W~93/O~X ~ CI`/C~92/00~l
-38-
group t-butyl, trityl or optionally 6ubstituted benzyl;
D-Asn/Gln having the protecting group trityl or
xanthyl; D-Trp having the protecting group formyl or
t-sOc; X~ is Pyr, N-acyl-amino acid, N-acyl-~-C-alkyl-
amino acid, ~-C-alkyl-Pyr; X2, X3, ~, X7, X~are
independently natural, synthetic, protected or
~-C-alkyl- amino acid6; ~ i~ imino Acid; X~0 is -N(H)R
wherein R i6 GlyNH2, azaGlyNH2, alkyl, alkenyl,
cycloalkyl, hydroxyalkyl, hnloalkyl, nryl, wherein each
alkyl or alkenyl group has from 1-7 carbon atoms; and X5
is according to Formulas I or II, as described ~bove,
with the proviso that the same compounds excluded from
~ormulas I and II are likewise excluded from the
compounds ~ccording to Formula III.
Additionally, analogs of opiate peptides
j containing ~he acid-sensitive groups listed above
together with modification of Tyr or His according to
Formulas I and II are included.
A particularly preferred set of compounds
prepared by the novel process according to the present
~ invention are the compounds according to Formula III,
r~ wherein ~ comprises an amino acid which contains an
acid-sensitive sidechain protecting group. The amino
- acids containing acid-sensitive protecting groups are a
particularly preferred set of novel compounds which can
be prepared by the novel synthesis method of the
present invention as a result of the use of the
ex*remely labile peptide-resin bond provided by the
~ (orthochloro~trityl-resin bond. Thus, the particularly
i3, 30 preferred acid-sensitive protecting groups which are
uniquely useful in the novel method described above
are, for example, trityl, t-Boc, t-butyl, Mtr, Pmc or
formyl. The terms "acid-sensitive" and "sidechain
protecting group" are used in accordance with their art
~, '
:,
.,
~ .
,......
W1~93/0305~ l`/(A92/~2~l
recognized definition, to the extent not inconsietent
with the principles of the present invention.
The novel method of the pre~ent invention i~
also particularly well suited for the preparation of
S nnalogs of a biologic~lly active ligand having the
Formula III, wherein X~ i8 Pyr (L or D), N-acyl-~ino
- acid, ~-C-Alkyl-Pyr or N-acyl-~-C-alkyl-~mino ~c~d; X2,
X3, X4, ~, X7 and X~ are independently n~tural,
synthetic, protected or ~-C-alkyl- amino acid, or ~-X~
optionally contains a ~-lactam; ~ i5 imino acid or
~-C-alkyl-i~ino acid; X~0 is -N(H)R wherein R is GlyNH2,
azaGlyNH2, alkyl, alkenyl, haloalkyl, hydroxyalkyl,
cycloalkyl or aryl, wherein each alkyl, alkenyl or aryl
group hac from 1 to 7 carbon atoms; and X5 iS selected
according to Formulas I or II, as described above, with
the proviso that the same compounds excluded from
Formula~ I and II are likewise excluded from the
compounds according to Formula III.
The invention will be illustrated in qreater
detail by the fsllowing specific examples. It i8
understood that these example~-are given by way.of
illustration and are not meant to limit the disclosure
of the claims to follow.
- Example l
Desensitization effects of anqiotensin
antagonists in the rat isolated uterus assay:
Analog Recovery Time
[Sar~ Iles]ANG II 180 min
Sarl Tyr(0-CH3)4 Ile8]ANG II 20 min
30[Sar~ Phe(~'-F)4 Ile8]ANG II 20 min
~':
,
:
~,,.j
~"~ 93/0305X ~ 2/002~ 1
-40-
Ti~sues were treated with the particular an~log (lo ~)
for 2 min followed by washing out; tlme for recovery of
the response to an ED50 dose of ANG II wa~ determined.
.
Example 2
Desensitization propertie~ of GnRH ngonist~
continuously ~uperfused in goldfish pituitaries in
vitro.
Pe.ptide Response (na LH/ml~
5 min 10 min 20 min 40 min 60 min
10 GnRH (lO~M) 50 12 15 14 13
tTYr(Me)53GnRH 50 50 47 47 46
(5xlO~M)
Methods were as described by Habibi, 1991 (Biology of
Reproduction 44, 275-283)~
More specifically, the relative
responsiveness of goldfish pituitary fragments to
various salmon and mammalian GnRH snalogs were
determined by using a superfusion system based on that
described previously (Mackenzie et al., 1984; Chang et
al., 1984; Marcha~t et al., 1387). Briefly, goldfish
pituitaries were removed, pars distalis were sep~rated,
and fragments were prepared (<0.5 mm2) and placed
: between two l90~ layers of Cytodex carrier beads in a
300-~1 superfusion chamber (3 pituitary equivalents per
chamber). The fragments were superfused (5 ml/h)
overnight (10-12 h) with Medium 199 containing Hanks'
basic salts (Gibco Laboratories, Grand Island, N.Y.) 25
mM 4-(1-hydroxyethyl)-1-piperazineethanesulfonic acid
~HEPES) and 56 U/ml nystatin. Two hours prior to the
experiment, the medium was switched to Hanks' basic
salts solution, supplemented with 25 mM HEPES buffer
and 0.1% BSA (HBSS), and the flow rate was increased to
15 ml/h. Using a 3-way valve system, we exposed
pituitary fragments to 2-min pulses of various
~,.j",. . .
~;.' , ' '
~;
,:
!. . '
-~ WO 93/03058 PC'r/CA92/0028 1
251 ~r,f~;
-41-
concentrations of various GnRH analog~ (10~, 10~, And s
x 105 M~ everv 60 min. All expQriments lnvolv~d
.. running 4 or 8 c01umn8 ~i~ultaneously, with 5-min
fractions collected automAtically; samples were ~rozen
5 at -25C until determination of GTH by a
A radioimmunoa6say, as described previou~ly (Peter et
al., 1984).
Example 3
Desensitization propertie6 of GnRH agonist~
10 aPter 2 ~in pulses every 20 min in superfused goldfish
pituitaries n vitro according to the methods de~cribed
~! in Example 2.
Peptide ResDonse rna LH/ml)
. Pulse ~1 ~2 ~4 ~7
GnRH (104M) 60 14 13 10
~I tTyr~Me)5~GnRH 36 32 33 38
;;~ (5xlO~M)
~,
Exa~Dle 4
Release of LH during superfusion of goldfish
pituitaries n vitro induced by tTyr(Me)s]GnRH in the
absence (fiolid bar; S ~in) and presence (open bar; 3
min) of GnRH. Methods were as described by Habibi,
' 1991.
.~ _ .
' ,~ 25 .
~ _
lS .
¦ h lo --
o~
r ~ 211 ~0 IID ~¦0
: ~ ` :
~.
W093/0305~ 'C'r/C'A~2/~ ~l
-42-
Exam~le 5
Effect of subcut~neous in~Qct~on of l.S ~g/ln~ctlon o~
GnRH, [Tyr(Me)5]G~, or Gn~H+[Tyr(Me}~]GnRH on ~erum LH
levels in rats determ~ned by radiolmmuno~say
LH (Units/S0 ~1 ~erum
-
Tlme fter ln~ectlon CnRB ~Tyr(l~c)~]CnRH CnRH~ITyr(~)~lCnRB
120 min 0.7 0 2.5
150 min 0.1 0 1.9
180 min 1.2 0 3.6
225 min - o 3.3
240 min - o 3,9
__
Rats were injected every 30 min with 1.5 ~g of GnRH,
tTyr(Me)5~GnRH, or GnRH+tTyr(Me)5]GnRH. The values
are corrected ~or basal levels (1.1 units/50 ~1) by
subtraction.
It w:ill be noted that the analog of GnRH,
tTyr(Me)5]GnRH~, administered alone was not effective
due to the low dosaqe administered.
7: - - .
~ . ,
: .
.
.
