Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
`` 1332349
The present invention relates to a process for the production
of preparations for the rectal and vaginal use of
biologically active peptides in human and veterinary
medicine.
Ever since the introduction of insulin therapy, suitable
application forms that make it possible to supply the insulin
in quantities required, such as to pro~ide for a high level
of kiological availability and at the same time avoid the
parenteral route, have been sought.
The use of resorption mediators (DD 117 681) made the nasal
and rectal use of insulin preparations possible. However,
when resorption mediators are used alone, even with the
addition of protease inhibitors against proteases of the
pancreas (WP DD 254 881) and control of the rate of
liberation of the insulin from a polymer carrier in a
suitable ratio to the resorption speed (WP DD 252 539, DD 254
881, DD 257 197), there is still a considerable difference in
the biological availability of the insulin compared to that
seen when insulin is administered by injection. This entails
considerable economic losses, the cause ~or these being that
up to the present, no clinically usable results had been
obtained.
The problems connected with the instability of insulin in the
gastro-intestinal tract, on the one hand, and the poor
resorption characteristics of such large polar molecules, on
the other, also occur with various other biologically active
;~ peptides.
Attempts have been made to affect both the resorption
characteristics and the stability relative to enzymatic
breakdown by structural modification of the molecule. As a
rule, when this is done, the intended action of the peptide
is altered. The use of resorption mediators and protection
of the peptide against enzymatic breakdown by protease
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inhibitors are described. This also applies to the
controlled liberation by fixing the peptide on a carrier.
The stability of polypeptides such as insulin, as well as for
oligopeptides, and thereby the biological availability of
these peptides during rectal and vaginal resorption can also
be influenced positively by agents that act against anaerobic
bacteria.
None of the possibilities described above can alone provide
any all-embracing solution to the problem of inadequate
biological availability of peptides during rectal and vaginal
application. The combination of the possibilities according
to the present invention, set out above, in a pharmacological
preparation permits the economically meaningful rectal or
vaginal resorption of peptide ingredients.
The present invention discloses a process that permits the
production of preparations with which biologically active
peptides can be applied rectally or vaginally, in which a -~
persistent effect is to be ensured with a constant high level
of effectiveness of the peptide that is used.
The present invention describes a new process for the
25 ~ ~;produ¢tion of preparations for the application of
iologically active peptides by using individual elements, -~
nown per se,~that provide a combined effect.
More particularly, the invention provides a process in which
, 30 biologically aative peptides, such as insulin, GnRH and GnRH
analogues, substance P and derivatives thereof, oligopeptides
such os~thymus peptides and analogues thereof, as well as
microbiologically produced peptides such as cyclosporines are
fixed~to a high-molecular, inert carrier, and liberated
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13323A9
slowly and continuously, passage through the mucuous membrane
being made possible by an anionic resorption mediator such as
aminoacid, peptide or albumen fatty acid condensate, when the
biological activity of the peptide and the action of the
resorption mediator are maintained completely during the
duration of the slow release under the conditions of
rectal/vaginal application by the combined co-utilisation of
a protease inhibitor and an agent that is effective against
anaerobic bacteria.
The biologically active peptide is fixed to the carrier as -
described in DD 219 673 and DD 257 197.
The anionic resorption mediator is obtained by condensation
of aminoacids or albumen hydrolysate with fatty acids (BONDI,
S. Biochem. Z. 17/1909/545; IZAR, G. Biochem. Z.
40/1912/402; DD 107 783; DD 117 681). The extremely pure
resorption mediators obtained in this way are tolerated by
the mucuous membrane and are biologically degradable.
In order to prevent biochemical decomposition of both the
bioactive peptide and also of the resorption mediator
throughout the duration of the slow liberation in the area of
the rectum or vagina, it is important that a combination of
protease inhibitor and a substance that minimizes the effects
of the anaerobic bacteria that are active in the area of
application be present. Aprotinine or EAC, for example, can
be used as protease inhibitor. Within the scope of the
present invention, chemotherapeuticals and/or disinfection
agents such as p-hydroxibenzoic acid ester, for example, can
be used as substances that are effective against
anaerobically existant bacteria.
In respect to the solution that was discovered, it was
surprising that the bioavailability of the peptides in the
application form described not only corresponded to the sum
of the individual characteristics that could be achieved, but
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also went far beyond these. Whereas the bioavailabilty of
peptides could be increased to approximately 30% by an
addition of the individual features, in the process according
to the present invention it was possible to identify a
bioavailability of 60 to 75~ compared to an IM
(Intramuscular) application.
Example 1
20.000 g gelatine were allowed to swell in a mixture of
25.000 g glycerol, 19.0 ml water and 4.0 ml 2N NaOH for 60
minutes at room temperature, then homogenized at 60C and
after cooling to 40C, 20.0 g insulin-agar adduct as in the
first example, 1.000 g albumen fatty acid condensate, 5.0 g
nipagine, and 150,000 ATrE aprotinine were stirred in
homogeneously. Finally, this was poured into moulds.
Example 2
20.000 g of an insulin-aqar adduct with 5 IU insulin per 200
mg adduct were distributed together with 0.240 g
lauroylleucine, 6 mg metronidazol, and 150,000 ATrE
aprotinine homogeneously in a melted Rosupol R suppository
mass and moulded into 2.0-g suppositories. These were
removed from the moulds after cooling.
Example 3
::
20.000 g of an insulin-agar adduct with 5 IU insulin per 200
mg adduct were distributed together with 0.240 g
lauroylleucine, 6 mg metronidazol, and 150,000 ATrE
aprotinine homogeneously in a melted Rosupol R suppository
mass, picked up in glass capilliaries, and immediately cooled
to 0xC. The resulting cylinders were used for application to -
diabetic rats.
Example 4
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A 55-% insulin-polyglycine complex was produced by suspending
250 mg polyglycine (M-4000) in 7.5 ml 85-% formic acid with
the addition of 1.1 ml insulin solution (250 mg insulin/ml
85-~ formic acid). 5.7 mg of this complex were added to the
pouring batch ~7.5 g glycerol, 8.9 citrate-phosphate buffer,
pH 7.5, 9 g gelatine). In addition, analogously to Example
2, resorption mediators, conserving agent, and protease
inhibitor were added. This mass was moulded into
suppositories.
At an insulin dose of 1.2 IU/kg body mass, this resulted in a
blood glucose reduction by 48% that lasted 1 to 4 hours. The
starting level was achieved after 6 hours. The control group
of animals, that obtained only insulin sticks with resorption
mediator without fixing of the insulin on polyglycine,
exhibited a blood glucose reduction of 30% that lasted only 1
to 2 hours.
Example 5
Analogously to Example 4, 11.4 mg of a 55-% insulin-
polyglycine complex with 300 mg lauroylleucine were so
processed into sticks that suppositories containing 10 IU
insulin were obtained. After rectal application of an
insulin dose of 2.5 IU/kg body mass the test animals
exhibited a blood glucose that was reduced by 50% for a
period of 4 hours. The starting level was achieved after 7
hours.
Example 6
In order to produce a 55-% insulin polylysine complex, 450 mg
polylysine (M~4000) were dissolved in 20 ml distilled water
and 560 mg insulin were added to the solution. 11.4 mg of
the complex so obtained were processed into suppositories
with 300 mg laurolylleucine with the addition of 100 mg
methylhydroxybenzoate and 150,000 ATrE aprotinine as in
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Example 4. Out of an insulin dose of 2.5 IU/kg body mass,
the test animals displayed a 60-% blood glucose reduction for
three hours, the starting level being reached again after 6
hours.
Example 7
5.6 g dextrane (M-500,000) were stirred into a stiff mass
with 4.6 ml distilled water: 1000 ul insulin solution (250 mg
insulin/ml), 0.2 N Na2HP04 solution, pH 9.7 were added to
this mass and stirred. The gel was dried at 1 Torr and 25C.
After reduction, an insulin content of 3.5% relative to the
dry mass could be determined.
Example 8
A 7.5-% agar-agar solution was produced after dissolving 450
mg agar-agar in 4.5 ml distilled water at up to 90C.
Shortly before the agar-agar solution that had cooled to 40C
gelled, 1000 microl. of a GnRH solution (100 mg GnRH in 1 ml
distilled water, pH 6.0) was added and stirred in. Reduction
and then drying followed. The gel so obtained had a GnRH
component of 13%, relative to the dry mass.
Example 9
r *
1.8 g sephadex G100 and 7.2 ml distilled water were
homogenized and left to stand for 24 hours. Then 1 ml
insulin solution (250 mg/ml, 0.2 mol/litre Na2HP04 solution,
pH 9~7) was added, stirred well for 1 hour and allowed to
stand for 8 hours. This was then precipitated in 200 ml
absolute acetone, suctioned off, and dried for 36 hours over
P2O5 until the weight remained constant (1 Torr, 25C).
The gels so obtained with a content of 10 IU/3.4 mg Sephadex
were processed as follows:
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49.415 g rosupol were added, together with 500 mg
lauroylleucine at 40C, 85 mg of the insulin-sephadex adduct
were added, and poured so as to form suppositories. At an
insulin dose of 10 IU/suppositorium this resulted in a blood
glucose reduction by 50% with the starting level achieved
after 6~5 hours.
Biological Test Results
1. Material and Methods
Rabbits
The test was conducted with male rabbits having normal
metabolism, with a live weight of 3.5 to 4.0 kg of a crossed
Deutsche Widder-Chincilla strain. The animals were pretested
for normal insulin sensitivity.
For 18 hours prior to the start of the test, the animals
received no food, but were given water on demand. They were
housed in wire cages, no bedding, at 100 g standard pellet
feed per day at a room temperature of 20C +/- 2C without
any light-darkness regime. For special test arrangements,
see AB of the German Democratic Republic.
~: :
The application was made subcutaneously under the skin on the
neck. -Ten animals per test group were used. Ten animals
formed the control group (group 1). Blood was taken from the
ear veins. Glucose determination was effected my means of a
Fermognost test apparatus.
Mouse Ovulation Test
Infant, 3-week old mice were given 0.5 IU PMSG and 53 to 54
hours later this was followed by the ovulation-initiating
second injection. Eighteen to nineteen hours later, these
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i3323~9
mice were killed and microscopic examination for the presence
of ovulated egg cells in the oviducts was carried out.
Injection volume: each 0.2 ml
Solvent physiological: NaCl solution
Micenumber/group: 9 to 11
Mathematical calculation of the mean effective dose 50 (ED
50) was effected by means of probil analysis.
2. Plasma glucose - reduction time profile
2.1 Test results for example 1, test animals:
normoglycaemic rabbits
_________________________________________________________________
~ t 20' 40' 60' 80' 120' 180' 240' 300'
_________________________________________________________________
x 58.6 49.3 52.3 65.8 78.4 84.3 92.7 90.9
1 +
sx 15.3 9.72 11.12 15.7 19.3 20.63 18.77 15.69
~? 55.3 52.9 44.7 45.2 53.7 56.5 67.2 76.8
~: 2 + `
: ~~ 13.62 11.59 13.72 9.36 14.84 17.61 14.3 18.8
~; -- ----------------------------______________________
Gelatine sticks with 5 IU insulin-agar adduct + 5%
albumen fatty acid condensate
2: Gelatine sti~ks as in example 1
2.2 Test results for example 2, test animals: normoglycaemic
;~ rabbits
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t 20' 40' 60' 80' 120' 180' 240' 300'
_________________________________________________________________
x 65.5 50~5 50.6 46.7 63.5 80.4 91.5 86.6
+
sx 19.57 12.34 9.03 13.57 18.20 17.63 19.58 19.03
x 63.4 55.3 50.3 42.3 51.9 50.3 50.6 47.9
2 +
sx 18.20 15.73 12.80 15.70 13.50 16.80 18.40 20.30
_________________________________________________________________
1: Rosupol sticks with 5 IU insulin-agar adduct and 7.5 x 10-
5 mol lauroylleucine (1.2%)
2: Rosupol sticks as in example 2.
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2.3 Test results for example 3, test animals: diabetic rats
_________ __ _ _ __ ____ __ _ _ ___ _ ___ __ _ .____________ _ _ __ _ __ _ _ ______ ___ _
t 10 20 30 40 5060 80
x 88.93 65.3757.68 52.69 48.73 51.19 46.28
sx 11.32 12.3210.9 9.05 10.7612.15 10.01
x 88.33 67.0055.51 47.B2 42.16 44.14 36.15
2 +
sx 9.72 12.7711.88 10.92 9.82 9.97 6.29
___ __ _____ ____ ____~_____________________________ _______ ____
. _. _, ~ . _.., .... _.. .
___ ______ ____ ______ __ __ _________ ____________ ___ _______ ____ _______
t 100 ' 120 ' 135 ' 150 ' 180 ' 210 '
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ __ _ _ _
x 57.37 75.53 82.0391.52110.2~ 103.62
1 +
sx 15.26 17.02 15.6819.2521.34 24.63
41.67 38.55 45.7154.8856.~1 S7.66
` ~2 +
, sx 18.57 13.53 17.9525.5727.96 29.40
_______ __ _______ _____ ____________________________________________
~ 2.4 IU insulin-agar adduct/sticks + 7.5 x 10 J mol N-
r~ lauroylleucine (1.2%)
~ 2: Exampl~ 1, but 2.4 IU insulin-agar adduct/sticks n = 8
j~
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~ .4 Test results for example 4
:; ~ _____________________________________
'2' t 20' 40' 60' 80' 100' 120' 180' 240' 300' 360'
~ ~ j _____________ _________________ _________________________________
x 100.55 84.82 72.51 71.69 71.38 82.11 85.97 93.9 94.92 98.10
1 +
sx 17.32 13.26 12.43 14.59 15.08 10.88 10.91 11.37 14.01 12.03
x 82.56 68.32 62.31 58.92 57.32 63.25 67.79 69.08 78.92 92.18
-~ 2 +
2~ SX 12.46 14.01 il~01 8.g6 10.32 15.77 14.32 10.77 15.61 12.31
~ ~ ------------------------_------_________________
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1332349
1: Gelatine sticks with 5IU insulin/suppositories and 5%
albumen fatty acid condensate
2: Gelatine sticks as in Example 4.
2.5 Test results for example 8, mouse ovulation test Group
_________________________________________________________________
In~ectlon
Group 1 2 3 4 5
_________________________________________________________________
x ln ng 2.6 5.9 13.2 29.6 66.7
S 9 10 10 11 11
R 0 0 2 10 10
R% 0 0 20 90.91 90.91
ED 50 26.85 +/- 8.77
________________ __________
In~ection
Group 1 2 3 4 5
x ln ng 1.7 2.6 3.9 5.9 8.8
S `10 10 10 10 10
R 8 8 9 9 10
RS 80 80 90 90 100
ED 50 2.71 +/- 8.06
:~: --------------------------------____--_________________________________~_______
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In the first test, in the control group, an ED 50 of 26.85
ng, and in the test groups an ED 50 of 2.71 was measured.
~: Thus the dose required to bring about ovulation was 1 power
of tsn lower.
3. Comparison of the area integers of Examples 1 to 4 (Table
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Table 1: AUC: (Area integers) of the glycose profile in mm of
Examples 1 to 3
AUC: in mm
Example According to Accordlng to % Increase ln
Comparative Invention Effect
Recelpe
______________________ _____ ____________________________________
1 3721 6910 85
2 4643 8094 75
3 3034 5740 90
4 2238 5319 138
_________________________________________________________________
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