Note: Descriptions are shown in the official language in which they were submitted.
, /' 1 33 57 ~ 1
PROCESS FOR INCREASING THE ANTAGONISTIC
EFFECT OF PEPTIDIC COMPOUNDS ON
HORMONE-DEPENDENT DISEASES
10 Technical Field
The invention i6 concerned generally with the field of
pharmacology and clinical medicine, and more particularly
relates to a process for significantly increasing the
15 antagonistic and paradoxical effect of certaln compounds with
a peptidic structure on hormone-dependent diseases.
Summary of the Invention
This invention relates to a sterilized sustained release
pharmacological formulation comprising a L~-RH analog and a
polylactide-glycolide copolymer which releases between about
20 and 55% of the analog within the first five days after
administration to a human, with the remainder of the analog
25 belng released within about 3 wee~s to 2 month~ thereafter.
Another embodiment of the lnvention is directed to a
process for increasing the paradoxical and antagonistic effect
of the releasing hormone of a LM-RH analog on hormone
30 dependent diseases in a human, characterized in that a
therapeuticaly effective amount of one of the pharmacological
formulations of the invention is administered to the human,
preferably by intramuscular in~ectlon. This process
stimulates LH and FSH in the human for the first few da~s,
.. ~
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followed by a substantial and almost complete suppression of
LH and FSH thereafter for at least 3 weeks to as long as 2
months.
Detailed Description of the Invention
One object of the invention is a process for increasing
the antagonistic effect of the releasing hormone of LH and FSH
or of one of its synthetic analogues on hormone-dependent
diseases characterized in that these compounds are formulated
with a copolymer of D,L-lactide and glycolide so that, when
the compounds are injected into a patient, about 20 to 50
percent of the active principle is released with the first
five days after the injection.
Yet another object of the invention is a process for
decreasing the initial stimulation of the releasing hormone of
LH and FSH or of one of its synthetic analogues characterized
in that said compounds are microencapsulated by means of a
20 copolymer of D,L-lactide and glycolide. The invention is
further defined in the Claims.
The releasing hormone of LH and FSH is a decapeptide of
the following structure:
(pyro) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.
This hormone and its synthetic analogs exert an
antagonistic effect on a number of biological processes which
are specific to mammals, and has been therefore suggested -
among other uses - as an ovulation regulating agent. In human
medicine, such compounds are advantageously used for the
30 treatment of certain endocrine disorders, such as for example
those of the menstrual cycle, or for contraceptive purposes
(for example, see Swiss Patent No. 615,662 on this subject).
Whether the hormone-dependent biological processes be of
a pathological or a natural origin, it is desirable in certain
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instances to administer the above-mentioned compounds in a
continuous manner over prolonged periods and preferably as
preparations with an activity extending in time, for example
through controlled release or the fo~mation of deposits. Such
formulations are known and widely used in various areas of
medicine, and can consist, for example of a salt with a low
degree of solubility in the body fluids or of a highly
viscuous liquid. Recently, the use of bio-compatible
polymers, such as the copolymer of D,L-lactide and glycolide
has been proposed for this purpose: it has been possible to
obtain with such material matrixes which can trap the
pharmacologically active ingredient to release it in a
controlled manner (see for example the European Patent
Application No. 0058481). The use of a copolymer such as
D,L-lactide and glycolide has also been suggested for the
coating of LH-RH and some of its analogues in the preparation
of microcapsules (see for example the European patent
Application No. 0052510). From these disclosures, medical
preparations are thus obtained which exhibit a delayed release
20 of the active principle against various hormone-dependent
diseases.
Independently from what precedes, it has been recently
proposed to use the LH-RH or some of its synthetic analogues
for the treatment of hormone-dependent diseases on which these
25 compounds exert a paradoxical and antagonistic effect (see for
example A.V. Schally et al. in "Frontiers of Medicine -
Implications for the Future", Human Science Press, New York
1983 : Biomed. Pharmacoether. 3~ No. 2, 120 1982). Among
such diseases, one can mention for example breast cancer,
30 prostate cancer, endometriosis or benign prostatic
hyperplasia. For such treatments, it has been found desirable
to improve the conditions under which these compounds are
administered to take into account the sometimes undesirable
side-effects of LH-RH and of its synthetic analogs: in fact,
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it is for example known that such compounds raise initially
the testosterone level, which is contrary to the desired
therapeutic action. The physician when facing such a problem
does not have at present the possibility of administrating
compounds with antagonistic properties in sufficiently high
doses without causing undesirable side-effects, such as pain.
The merit of the applicants' parent application is to
provide a new and original solution to overcome the above-
described problem. Thanks to the object of this invention, it
is now possible to administer preparations, which although
containing an identical dose of pharmacologically active
ingredient (LH-RH or synthetic analogs) demonstrate an
antagonistic effect notably better than that obtained with the
same ingredient administered as a non-encapsulated or non-
coated preparation. Such an effect, which can be described assynergetic, is totally unexpected from the present knowledge
of the art. Moreover, such an effect is advantageously
accompanied by a simultaneous decrease of the initial
stimulating effect which is inherent to these compounds.
The objective of the present invention is to provide a
sustained release formulation comprising LH-RH or a LH-RH
analog and a polyactide-glycolide copolymer wherein between
about 20 and 5S% preferably between 23 and 50% of the active
principle of the analog is released within the first five days
25 after administration to a human, with the remainder of the
analog being released within about 3 to 6 weeks thereafter.
A preferred way of achieving this objective is by
proceeding to the coating or encapsulation of the LH-RH
analogs with a copolymer of D,L-lactide and glycolide. Such a
30 copolymer is generally known (see references), and can be
produced of the quality required for the present invention
according to known practices. According to the invention,
however, properties of the copolymer are specifically defined
to achieve the desired results. The molar proportion of D,L-
1 3357 1 1
lactide and of glycolide in the copolymer is preferablysituated between approximately 50:50 and 55:45, while the
copolymer preferably has an average molecular weight (Mw)
situated between approximately 30,000 and lO0,000, and
preferably between approximately 36,000 and 50,000, to which
corresponds a viscosity situated between approximately 0.5 and
0.8, preferably 0.5 to 0.7 dl/g. By coating micro-particles
according to usual practices with copolymers such as those
defined above, a high degree of coating is achieved which
amounts to approximately 70 to 90%, depending on the specific
processing conditions utilized.
In this manner, the shape and the size of the micro-
capsules are easily controlled, and also the proportion of
pharmacologically active ingredients can be accurately
controlled. Generally, the particle size is less than 2S0
microns. To achieve the desired effect according to the
invention, spherical microparticles are used which have a
diameter preferably situated between approximately 30 and
approximately 50 microns and which contain between approximat-
20 ely l.S and 3, preferably l.7 and 2.9~ (w/w) of coated activeingredient. Details of typical values for the microparticles
obtained according to the present invention are given in the
examples.
Microparticles having the characteristics given above
25 provide a product capable of releasing in vivo or in similar
conditions the active ingredient over a period of
approximately 2S to 30 days from a single administered dose.
The process of the invention has also the advantage that the
microparticles can be coated in almost sterile conditions,
30 because the encapsulation is carried out in a substantially
organic medium and the peptide to be encapsulated is dissolved
in sterile water or added in a dry state. However, for
administration to humans, the coated microparticles are
preferably sterilized by irradiating with gamma rays: it has
1 3357 1 1
been observed that this sterilization method does not alter
the properties of the product provided that this is
accomplished by exposure to gamma rays at a dosage of between
2.5 and 2.8 Megarads. Further, such microparticles can be
kept under the usual conditions of storage of similar
medicines for example from 4 to 6 months at approximately 4 to
21-C.
It is also possible to form a matrix compound of the
analog and copolymer wherein the matric compound is capable of
releasing the desired amounts of active principle in vivo.
Those skilled in the art are capable of varying the amount and
properties of the copolymer used in the matrix to achieve the
desired release characteristics.
Any of the LH-RH analogs disclosed in EP 0,052,510 are
suitable for use in the present invention in the form of
matrix or encapsulated compounds, but the most advantageous
formulations are the LH-RH analogs given below which are
encapsulated with a copolymer of D,L-lactide and glycolide in
the manner described herein:
(pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2,
(pyro) Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH2
or
(pyro) Glu-His-Trp-D-Ser-Tyr-D-Leu-Arg-Pro-Gly-NH Rl,
wherein Rl is an alkyl group.
To obtain the desired antagonistic effect, the
microcapsules are preferably administered as injectable
suspensions. The size of the particles (preferably 30 to 50
microns) obtained according to the process of the invention is
such that their injection offers no difficulty. The
30 biocompatible diluents used for this purpose are known in the
art. The injection is generally given by the intra-muscular
route. The amount thus injected depends to a considerable
extent on the disease which is treated, on the part of the
body which is treated, and on the general state of the
1 33571 1
patient. Expressed in weight of pharmacologically active
ingredient (or peptide), the dose injected can advantageously
be determined as between 0.1 and 10 micrograms per day per
kilogram of body weight (these doses are expressed in weight
of pharmacologically active ingredient released from one
single initial administered dose of the encapsulated product).
Different comparative tests carried out in appropriate
conditions showed that at the same daily levels of
administration of the active ingredient, the therapeutical
effect of the encapsulated preparation was significantly
higher. The same observation was made when the dose of active
ingredient which had been encapsulated was lower by 50% than
the dose of non-encapsulated active ingredient.
When comparative tests were carried out in vivo with
15 rats for example, it was found that the administration of
the same doses of the pharmacologically active ingredient
(peptide) resulted in a decrease of the weight of a prostatic
tumor of approximately 27% in the case of the non-encapsulated
compound and of approximately 80~ in the case of the compound
20 encapsulated, according to the invention. Moreover, a
decrease of approximately 80% was recorded in the testosterone
level of the rats receiving the encapsulated compound.
The invention will be further illustrated in a more
detailed manner with the following examples.
Example 1
Encapsulation of a Decapeptide
The various operations for preparing a pharmacologically
active preparation were carried out with the compound of the
following formula (compound A):
(pyro) Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2
This compound was obtained according to the process
described for example in the Swiss Patent No. 615,662. The
peptide content of the preparation was approximately of 80%
(w/w) .
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Encapsulation
A copolymer of D,L-lactide and glycolide with a 50/50
molar ratio of D,L-lactide to glycolide and with an average
molecular weight of 53,000 is first dissolved in an appropri-
ate organic phase and the solution is introduced into areaction vessel provided with an agitator. A separate
solution of compound A in sterile water is prepared. This
solution is poured slowly into the vessel while stirring the
mixture with the agitator rotating at approximately 2,000 rpm.
Alternately, it is possible to add Compound A as a dry solid,
rather than as a solution. A phase separation inducer is
added to the mixture under agitation to cause the coacervation
of the poly-(D,L-lactide-co-glycolide) and the coating of
compound A. The mixture now containing the embryonic
15 microcapsules is next poured into a hardening liquid under
agitation, which continues for 30 minutes at approximately 800
rpm. After filtration, the product is dried under reduced
pressure for 24 hours.
The product was obtained by this procedure with a yield
20 amounting to 76% of the theoretical yield.
Characterization
- Spherical particles having a diameter in the range from 30
to 50 microns (the measurements were made on photographs taken
with a scanning electron microscope).
25 ~ Content of encapsulated compound, 2.07% (w/w). The
efficiency of the encapsulation was of 70%. To determine the
content of encapsulated compound, the microparticles are
dissolved in methylene chloride, the methylene chloride
solution is extracted with a pH 7.4 phosphate buffer, and the
30 amount of decapeptide measured by High Pressure Liquid
Chromatography.
The microparticles obtained according to this method can
be when desired irradiated with gamma rays at about 2.5 Mrad
before being administered in vivo.
1 33 57 1 1
before being administered in vivo.
Example 2
By varying the conditions under which the encapsula-
tion of compound A described in Example 1 was performed, we
5 obtained the results shown in Table 1. The compound A which
was used had a peptide content of about 80% (w/w). Table 1
gives the physical characteristics of the preparation thus
obtained. The various preparations of microparticles were
tested for the release of Compound A into a 0.01 molar aqueous
10 solution of monosodium phosphate pH 7.4 at 37-C.
Example 3
The antagonistic effect of the encapsulated compound A
(see the previous examples) and of the non-encapsulated
compound A was studied on rats having the prostatic tumor R-
3327-H according to Dunning. (DUNNING R-3327-H PROSTRATE
CARCINOMA).
In vivo tests were carried out on groups of 7 to 10
animals during a period of 30 days.
The administration of the non-encapsulated Compound A
20 adequately dissolved in a biocompatible solvent was achieved
by the subcutaneous injection of two doses every day, each
dose containing 25 mic~G~.ams of compound A (Table 2) or 12.5
micrograms of compound A (Table 3).
The administration of th'e encapsulated compound A was
~ 25 achieved by the daily intra-muscular injection of doses of the
preparation suspended in a biocompatible solvent, each such
dose containing 25 micrograms of compound A.
The results of the observation which were made are given
in Tables 2 and 3. The evolution of testosterone was measured
30 during 30 day period for those Ani~ls receiving the daily
dose of 25 micrograms of compound A. Fig. 1 clearly
illustrates the enhanced antagonistic effect (i.e. the
decrease of the testosterone blood content) of the compound A
when encapsulated.
--10--
1 3357 1 1
: Administration of non-encapsulated compound A
- - - - - - -: Administration of encapsulated compound A.
Example 4
The inhibition of the initial stimulatdry effects of
compound A was tested in vivo with two separate groups
including 5 patients each. The hormones which were determined
were:
: testosterone (in ng/ml)
- - - - - - : LH (in mIU/ml)
-.-.-.-.-.- : FSH (in mIU/ml)
Fig. 2 shows the results obtained with the first
group of patients treated with one daily dose of lO0 gamma of
non-encapsulated compound A during 5 days, and thereafter with
one daily dose of about lO0 gamma of encapsulated compound A.
Fig. 3 shows the results obtained with the second
group of patients treated with one monthly injection which is
equivalent to a daily dose of lO0 gamma of encapsulated
compound A. Fig. 3 shows clearly the total absence of the
initial stimulatory effect on the testosterone level.
Example 5
Compound A was encapsulated with a copolymer of D,L
lactide and glycolide having a molecular weight of 39,127
daltons, a molar ratio of lactide to glycolide of 52:48 and a
25 viscosity of 0.65.
3 mg of this encapsulated compound in suspension was
injected intramuscularly into dogs to assess the delivery of
the active ingredient over time. Fig. 4 shows the release
pattern of the active principle, wherein a rapid release of
30 the peptide was found at day l followed by a plateau between
day 4 and 31, the broken line representing the basal level of
the decapeptide-like immunoreactivity in dog plasma before
injection. Stimulation of gonadotropin cells led to a rise in
testosterone plasma concentration at one day, but castration
3S level was obtained between day 4 and day 7 and maintained
until day 30.
1 3357 1 1
Example 6
Three encapsulated compound A formulations.(F1, F2, F3)
were administered to beagle dogs by intramuscular injection to
determine the release of active principle over time with once
a month dosing. Additionally, the relative bioequivalence of
the three formulations was ~x~;ned as reflected by plasma
concentrations of the active principle and by their ability to
reduce plasma testosterone. Statistically significant
differences in the total amount fr drug released from the
formulations were not present. However, the course of release
of the active principle was different, as was the profile of
testosterone suppression, for the different formulations. Fl
and F3 demonstrated similar patterns of release of the active
5 principle and testosterone suppressions. Both released
approximately 50% of their total by day 1 and demonstrated
peak plasma concentrations at the first (2 hour) sampling
time. F2 released less decapeptide on day 1 (23% of total
released) and also had lower peak plasma concentrations than
20 the other formulations. Testosterone concentration profiles
showed an initial elevation, lasting for 1-5 days (greatest in
F2), followed by depression and total suppression lasting from
30 to 60 days postdose (greatest in F1 and F3). A positive
correlation was established between the amount of decapeptide
25 released on day 1 and testosterone suppression. The cessation
of testosterone suppression was associated with the lack of
detectable decapeptide concentrations.
These results surprisingly demonstrate that the greatest
release of the active principle within the first 5 days after
30 injection, preferably within the first 3 days and most
preferably, by day 1, provides the best therapeutic activity,
with a short initial stimulation phase of the hormone in the
body, followed by a depression and total suppression for a
period of between about 3 and 6 weeks to 2 months, usually for
-12-
1 33 57 ~ 1
at least about 25-30 days. It is believed that the initial
release of a substantial amount of the analog produces the
greater and more rapid suppression that is observed. One
reason for the increased initial release of the active
5 principle is believed to be the low hydrophobic character of
coatings formed on the LH-RH compound as described above,
which enables the resulting compositions to be more soluble in
the body fluids. Also, since the initial stimulation is
short, higher amounts of the active principle can be used with
a lesser painful reaction in the patient.
While it is apparent that the invention herein disclosed
in well calculated to fulfill the desired results, it will be
appreciated that numerous modifications and embodiments may be
devised by those skilled in the art such as, for instance,
15 those claimed in U.S. Patent Nos. 3,887,699 and 3,976,071, and
it is intended that the appended claims cover all such
modifications and embodiments as fall within a the true spirit
and scope of the present invention.
1 3357 1 1
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133571 1
TABLE NO. 2
5 Type of Control Compound A
measure (unit) * encapsulated non-encapsulatec
Final body weight (g) 339+13 342+14 347+28
Ventral portion of337+48 44+6 54+5
prostate (mg)
Testicles (g) 2.81+0.131.15+0.08 1.36+0.14
Tumor* (mg) 922+150 186+95 546+0.14
Increase of tumor227+32 106+21 85+19
volume (%)
TABLE NO. 3
Type of Control Compound A
measure (unit) * encapsulated non-encapsulatec
Final body weight (g) 362+9 363+11 361+11
25 Ventral portion of419+39 47+5 88+7
prostate (mg)
Testicles (g) 3.14+0.071.26+0.06 1.83+0.07
Tumor* (mg) 357+118 68+32 260+68
30 Increase of tumor157+23 81+17 110+28
volume (%)
* (Dunning R-3327-H Prostate Carcinoma)
