U.S. patent application number 09/749342 was filed with the patent office on 2003-09-11 for pharmaceutical formulations for sustained drug delivery.
This patent application is currently assigned to Praecis Pharmaceuticals, Inc.. Invention is credited to Barker, Nicholas, Gefter, Malcolm L., Molineaux, Christopher J., Musso, Gary.
Application Number | 20030171296 09/749342 |
Document ID | / |
Family ID | 25065930 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030171296 |
Kind Code |
A1 |
Gefter, Malcolm L. ; et
al. |
September 11, 2003 |
Pharmaceutical formulations for sustained drug delivery
Abstract
Sustained delivery formulations comprising a water-insoluble
complex of a peptidic compound (e.g., a peptide, polypeptide,
protein, peptidomimetic or the like) and a carrier macromolecule
are disclosed. The formulations of the invention allow for loading
of high concentrations of peptidic compound in a small volume and
for delivery of a pharmaceutically active peptidic compound for
prolonged periods, e.g., one month, after administration of the
complex. The complexes of the invention can be milled or crushed to
a fine powder. In powdered form, the complexes form stable aqueous
suspensions and dispersions, suitable for injection. In a preferred
embodiment, the peptidic compound of the complex is an LHRH
analogue, preferably an LHRH antagonist, and the carrier
macromolecule is an anionic polymer, preferably
carboxymethylcellulose. Methods of making the complexes of the
invention, and methods of using LHRH-analogue-containing complexes
to treat conditions treatable with an LHRH analogue, are also
disclosed.
Inventors: |
Gefter, Malcolm L.;
(Lincoln, MA) ; Barker, Nicholas; (Southborough,
MA) ; Musso, Gary; (Hopkinton, MA) ;
Molineaux, Christopher J.; (Brookline, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Praecis Pharmaceuticals,
Inc.
|
Family ID: |
25065930 |
Appl. No.: |
09/749342 |
Filed: |
December 27, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09749342 |
Dec 27, 2000 |
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08988851 |
Dec 11, 1997 |
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6180608 |
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08988851 |
Dec 11, 1997 |
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08762747 |
Dec 11, 1996 |
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5968895 |
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Current U.S.
Class: |
514/10.3 ;
514/19.5; 514/9.8 |
Current CPC
Class: |
B82Y 5/00 20130101; Y10S
514/80 20130101; A61K 47/61 20170801; A61P 15/08 20180101; A61P
35/00 20180101; A61K 38/09 20130101; A61P 5/00 20180101; A61P 15/18
20180101; A61P 13/08 20180101; A61P 5/24 20180101; A61P 15/00
20180101; A61K 47/6949 20170801 |
Class at
Publication: |
514/13 |
International
Class: |
A61K 038/00 |
Claims
We claim:
1. A pharmaceutical composition comprising a water-insoluble
complex of a pharmaceutically active peptidic compound and a
carrier macromolecule.
2. The pharmaceutical composition of claim 1, wherein formation of
the water-insoluble complex is mediated at least in part by ionic
interactions between the pharmaceutically active peptidic compound
and the carrier macromolecule.
3. The pharmaceutical composition of claim 2, wherein the
pharmaceutically active peptidic compound is cationic and the
carrier macromolecule is anionic.
4. The pharmaceutical composition of claim 2, wherein the
pharmaceutically active peptic compound is anionic and the carrier
macromolecule is cationic.
5. The pharmaceutical composition of claim 1, wherein formation of
the water-insoluble complex is mediated at least in part by
hydrophobic interactions between the pharmaceutically active
peptidic compound and the carrier macromolecule.
6. The pharmaceutical composition of claim 1, wherein a single dose
of the water-insoluble complex provides sustained delivery of the
pharmaceutically active peptide to a subject for at least one week
after the pharmaceutical composition is administered to the
subject.
7. The pharmaceutical composition of claim 1, wherein a single dose
of the water-insoluble complex provides sustained delivery of the
pharmaceutically active peptide to a subject for at least two weeks
after the pharmaceutical composition is administered to the
subject.
8. The pharmaceutical composition of claim 1, wherein a single dose
of the water-insoluble complex provides sustained delivery of the
pharmaceutically active peptide to a subject for at least three
weeks after the pharmaceutical composition is administered to the
subject.
9. The pharmaceutical composition of claim 1, wherein a single dose
of the water-insoluble complex provides sustained delivery of the
pharmaceutically active peptide to a subject for at least four
weeks after the pharmaceutical composition is administered to the
subject.
10. The pharmaceutical composition of claim 1, wherein the
pharmaceutically active peptidic compound is a multivalent cationic
or anionic peptide.
11. The pharmaceutical composition of claim 1, wherein the peptide
is 5 to 20 amino acids in length.
12. The pharmaceutical composition of claim 1, wherein the peptide
is 8 to 15 amino acids in length.
13. The pharmaceutical composition of claim 1, wherein the peptide
is 8 to 12 amino acids in length.
14. The pharmaceutical composition of claim 1, wherein the carrier
macromolecule is an anionic polymer.
15. The pharmaceutical composition of claim 1, wherein the carrier
macromolecule is an anionic polyalcohol derivative, or fragment
thereof, or a pharmaceutically acceptable salt thereof.
16. The pharmaceutical composition of claim 1, wherein the carrier
macromolecule is an anionic polysaccharide derivative, or fragment
thereof, or a pharmaceutically acceptable salt thereof.
17. The pharmaceutical composition of claim 1, wherein the carrier
macromolecule is carboxymethylcellulose, or a pharmaceutically
acceptable salt thereof.
18. The pharmaceutical composition of claim 1, wherein the carrier
macromolecule is selected from the group consisting of algin,
alginate anionic acetate polymers, anionic acrylic polymers,
xantham gums, anionic carageenan derivatives, anionic
polygalacturonic acid derivatives, sodium starch glycolate, and
fragments, derivatives and pharmaceutically acceptable salts
thereof.
19. The pharmaceutical composition of claim 1, which is a dry
solid.
20. The pharmaceutical composition of claim 1, which is a liquid
suspension or semi-solid dispersion.
21. A pharmaceutical composition comprising a water-insoluble
complex, wherein the water-insoluble complex consists essentially
of a pharmaceutically active peptidic compound and a carrier
macromolecule.
22. A pharmaceutical composition comprising a water-insoluble
complex of an LHRH analogue and a carrier macromolecule.
23. The pharmaceutical composition of claim 22, wherein formation
of the water-insoluble complex is mediated at least in part by
ionic interactions between the LHRH analogue and the carrier
macromolecule.
24. The pharmaceutical composition of claim 22, wherein formation
of the water-insoluble complex is mediated at least in part by
hydrophobic interactions between the LHRH analogue and the carrier
macromolecule.
25. The pharmaceutical composition of claim 22, wherein a single
dose of the water-insoluble complex provides sustained delivery of
the LHRH analogue to a subject for at least one week after the
pharmaceutical composition is administered to the subject.
26. The pharmaceutical composition of claim 22, wherein a single
dose of the water-insoluble complex provides sustained delivery of
the LHRH analogue to a subject for at least two weeks after the
pharmaceutical composition is administered to the subject.
27. The pharmaceutical composition of claim 22, wherein a single
dose of the water-insoluble complex provides sustained delivery of
the LHRH analogue to a subject for at least three weeks after the
pharmaceutical composition is administered to the subject.
28. The pharmaceutical composition of claim 22, wherein a single
dose of the water-insoluble complex provides sustained delivery of
the LHRH analogue to a subject for at least four weeks after the
pharmaceutical composition is administered to the subject.
29. The pharmaceutical composition of claim 22 wherein the LHRH
analogue is an LHRH antagonist.
30. The pharmaceutical composition of claim 29 wherein the LHRH
antagonist comprises a peptidic compound, wherein a residue of the
peptidic compound corresponding to the amino acid at position 6 of
natural mammalian LHRH comprises a D-asparagine structure.
31. The pharmaceutical composition of claim 29 wherein the LHRH
antagonist comprises a peptidic compound comprising a structure:
A-B-C-D-E-F-G-H-I-J wherein A is pyro-Glu, Ac-D-Nal, Ac-D-Qal,
Ac-Sar, or Ac-D-Pal B is His or 4-Cl-D-Phe C is Trp, D-Pal, D-Nal,
L-Nal, D-Pal(N-O), or D-Trp D is Ser E is N-Me-Ala, Tyr, N-Me-Tyr,
Ser, Lys(iPr), 4-Ci-Phe, His, Asn, Met, Ala, Arg or Ile; F is
2wherein R and X are, independently, H or alkyl; and L comprises a
small polar moiety; G is Leu or Trp; H is Lys(iPr), Gln, Met, or
Arg I is Pro; and J is Gly-NH.sub.2 or D-Ala-NH.sub.2; or a
pharmaceutically acceptable salt thereof.
32. The pharmaceutical composition of claim 31, wherein F is
selected from the group consisting of D-Asn, D-Gln and D-Thr.
33. The pharmaceutical composition of claim 31, wherein F is
D-Asn.
34. The pharmaceutical composition of claim 31, wherein E is
tyrosine or N-methyl-tyrosine.
35. The pharmaceutical composition of claim 29, wherein the LHRH
antagonist has the following structure: Ac-D-Nal .sup.1,
4-Cl-D-Phe.sup.2, D-Pal.sup.3, N-Me-Tyr.sup.5, D-Asn.sup.6,
Lys(iPr).sup.8, D-Ala.sup.10-LHRH.
36. The pharmaceutical composition of claim 22, wherein the carrier
macromolecule is an anionic polymer.
37. The pharmaceutical composition of claim 22, wherein the carrier
macromolecule is an anionic polyalcohol derivative, or fragment
thereof, or a pharmaceutically acceptable salt thereof.
38. The pharmaceutical composition of claim 22, wherein the carrier
macromolecule is an anionic polysaccharide derivative, or fragment
thereof, or a pharmaceutically acceptable salt thereof.
39. The pharmaceutical composition of claim 22, wherein the carrier
macromolecule is carboxymethylcellulose, or a pharmaceutically
acceptable salt thereof.
40. The pharmaceutical composition of claim 22, wherein the carrier
macromolecule is selected from the group consisting of algin,
alginate, anionic acetate polymers, anionic acrylic polymers,
xantham gums, anionic carageenan derivatives, anionic
polygalacturonic acid derivatives, sodium starch glycolate, and
fragments, derivatives and pharmaceutically acceptable salts
thereof.
41. The pharmaceutical composition of claim 22, which is a dry
solid.
42. The pharmaceutical composition of claim 22, which is a liquid
suspension or semi-solid dispersion.
43. A packaged formulation for treating a subject for a condition
treatable with an LHRH analogue, comprising: a water-insoluble
complex of an LHRH analogue and a carrier macromolecule packaged
with instructions for using the water-insoluble complex for
treating a subject having a condition treatable with an LHRH
analogue.
44. The packaged formulation of claim 43, wherein the LHRH analogue
has the following structure: Ac-D-Nal.sup.1, 4-Cl-D-Phe.sup.2,
D-Pal.sup.3, N-Me-Tyr.sup.5, D-Asn .sup.6, Lys(iPr).sup.8,
D-Ala.sup.10-LHRH, and the carrier macromolecule is
carboxymethylcellulose, or a pharmaceutically acceptable salt
thereof.
45. In a syringe having a lumen, the improvement comprises,
inclusion of a liquid suspension of a water-insoluble complex of an
LHRH analogue and a carrier macromolecule in the lumen.
46. The syringe of claim 45, wherein the LHRH analogue has the
following structure: Ac-D-Nal.sup.1, 4-Cl-D-Phe.sup.2, D-Pal.sup.3,
N-Me-Tyr.sup.5, D-Asn.sup.6, Lys(iPr).sup.8, D-Ala.sup.10-LHRH, and
the carrier macromolecule is carboxymethylcellulose, or a
pharmaceutically acceptable salt thereof.
47. A method for preparing a pharmaceutical formulation,
comprising: providing a peptidic compound and a carrier
macromolecule; combining the peptidic compound and the carrier
macromolecule under conditions such that a water-insoluble complex
of the peptidic compound and the carrier macromolecule forms; and
preparing a pharmaceutical formulation comprising the water
insoluble complex.
48. The method of claim 47 wherein a solution of the peptidic
compound and a solution of the carrier macromolecule combined until
a water-insoluble complex of the peptidic compound and the carrier
macromolecule precipitates.
49. The method of claim 48, wherein the solution of the peptidic
compound and the solution of the carrier macromolecule are aqueous
solutions.
50. The method of claim 48, wherein the solution of the peptidic
compound and the solution of the carrier macromolecule are combined
and heated until a water-insoluble insoluble complex of the
peptidic compound and the carrier macromolecule precipitates.
51. The method of claim 47, further comprising sterilizing the
water-insoluble complex by gamma irradiation or electron beam
irradiation.
52. The method of claim 47, wherein the water-insoluble complex is
formed using aseptic procedures.
53. The method of claim 47, wherein the peptidic compound is
cationic and the carrier macromolecule is anionic.
54. The method of claim 47, wherein the peptidic compound is
anionic and the carrier macromolecule is cationic.
55. The method of claim 47, wherein the peptidic compound is a
multivalent cationic or anionic peptide.
56. The method of claim 47, wherein the peptidic compound is an
LHRH analogue
57. The method of claim 56, wherein the LHRH analogue is an LHRH
antagonist.
58. The method of claim 57, wherein the LHRH antagonist comprises a
peptidic compound, wherein a residue of the peptidic compound
corresponding to the amino acid at position 6 of natural mammalian
LHRH comprises a D-asparagine structure.
59. The method of claim 57, wherein the LHRH antagonist comprises a
peptidic compound comprising a structure: A-B-C-D-E-F-G-H-I-J
wherein A is pyro-Glu, Ac-D-Nal, Ac-D-Qal, Ac-Sar, or Ac-D-Pal B is
His or 4-Cl-D-Phe C is Trp, D-Pal, D-Nal, L-Nal, D-Pal(N-O), or
D-Trp D is Ser E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr),
4-Cl-Phe, His, Asn, Met, Ala, Arg or Ile; F is 3wherein R and X
are, independently, H or alkyl; and L comprises a small polar
moiety; G is Leu or Trp; H is Lys(iPr), Gln, Met, or Arg I is Pro;
and J is Gly-NH.sub.2 or D-Ala-NH.sub.2; or a pharmaceutically
acceptable salt thereof.
60. The method of claim 59, wherein F is selected from the group
consisting of D-Asn, D-Gln and D-Thr.
61. The method of claim 59, wherein F is D-Asn.
62. The method of claim 59, wherein E is tyrosine or
N-methyl-tyrosine.
63. The method of claim 57, wherein the LHRH antagonist has the
following structure: Ac-D-Nal.sup.1, 4-Cl-D-Phe.sup.2, D-Pal.sup.3,
N-Me-Tyr.sup.5, D-Asn.sup.6, Lys(iPr).sup.8, D-Ala.sup.10-LHRH.
64. The method of claim 47, wherein the carrier macromolecule is an
anionic polymer.
65. The method of claim 47, wherein the carrier macromolecule is an
anionic polyalcohol derivative, or fragment thereof, or a
pharmaceutically acceptable salt thereof.
66. The method of claim 47, wherein the carrier macromolecule is an
anionic polysaccharide derivative, or fragment thereof, or a
pharmaceutically acceptable salt thereof.
67. The method of claim 47, wherein the carrier macromolecule is
carboxymethylcellulose, or a pharmaceutically acceptable salt
thereof.
68. The method of claim 47, wherein the carrier macromolecule is
selected from the group consisting of algin, alginate, anionic
acetate polymers, anionic acrylic polymers, xantham gums, anionic
carageenan derivatives, anionic polygalacturonic acid derivatives,
sodium starch glycolate, and fragments, derivatives and
pharmaceutically acceptable salts thereof.
69. The method of claim 47, wherein the pharmaceutical formulation
is a dry solid.
70. The method of claim 47, wherein the pharmaceutical formulation
is a liquid suspension or semi-solid dispersion.
71. A pharmaceutical formulation prepared according to the method
of claim 47.
72. A method for treating a subject for a condition treatable with
an LHRH analogue, comprising administering to the subject a
pharmaceutical formulation comprising a water-insoluble complex of
an LHRH analogue and a carrier macromolecule.
73. The method of claim 72, wherein a single dose of the
water-insoluble complex provides sustained delivery of the LHRH
analogue to a subject for at least one week after the
pharmaceutical composition is administered to the subject.
74. The method of claim 72, wherein a single dose of the
water-insoluble complex provides sustained delivery of the LHRH
analogue to a subject for at least two weeks after the
pharmaceutical composition is administered to the subject.
75. The method of claim 72 wherein a single dose of the
water-insoluble noncovalent complex provides sustained delivery of
the LHRH analogue to a subject for at least three weeks after the
pharmaceutical composition is administered to the subject.
76. The method of claim 72, wherein a single dose of the
water-insoluble noncovalent complex provides sustained delivery of
the LHRH analogue to a subject for at least four weeks after the
pharmaceutical composition is administered to the subject.
77. The method of claim 72, wherein the LHRH analogue is an LHRH
antagonist.
78. The method of claim 77, wherein the LHRH antagonist has the
following structure: Ac-D-Nal.sup.1, 4-Cl-D-Phe.sup.2, D-Pal.sup.3,
N-Me-Tyr.sup.5, D-Asn.sup.6, Lys(iPr).sup.8, D-Ala.sup.10-LHRH.
79. The method of claim 72, wherein the carrier macromolecule is an
anionic polymer.
80. The method of claim 72, wherein the carrier macromolecule is an
anionic polyalcohol derivative, or fragment thereof, or a
pharmaceutically acceptable salt thereof.
81. The method of claim 72, wherein the carrier macromolecule is an
anionic polysaccharide derivative, or fragment thereof, or a
pharmaceutically acceptable salt thereof.
82. The method of claim 72, wherein the carrier macromolecule is
carboxymethylcellulose, or a pharmaceutically acceptable salt
thereof.
83. The method of claim 72, wherein the carrier macromolecule is
selected from the group consisting of algin, alginate, anionic
acetate polymers, anionic acrylic polymers, xantham gums, anionic
carageenan derivatives, anionic polygalacturonic acid derivatives,
sodium starch glycolate, and fragments, derivatives and
pharmaceutically acceptable salts thereof.
84. The method of claim 72, wherein the pharmaceutical formulation
is administered to the subject by a parenteral route.
85. The method of claim 72, wherein the pharmaceutical formulation
is administered to the subject orally.
86. The method of claim 72, wherein the pharmaceutical formulation
is administered by intramuscular injection or
subcutaneous/intradermal injection.
87. The method of claim 72, wherein the condition treatable with an
LHRH analogue is a hormone dependent cancer.
88. The method of claim 87, wherein the hormone dependent cancer is
prostate cancer.
89. The method of claim 72, wherein the condition treatable with an
LHRH analogue is selected from the group consisting of benign
prostatic hypertrophy, precocious puberty, endometriosis and
uterine fibroids.
90. The method of claim 72, wherein the LHRH analogue is
administered for in vitro fertilization or contraceptive purposes.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/762,747, filed Dec. 11, 1996, pending, the
entire contents of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] A variety of diseases and clinical disorders are treated by
the administration of a pharmaceutically active peptide. One such
example is prostate cancer, which is a sex hormone dependent cancer
and which can be treated by administration of a luteinizing hormone
releasing hormone (LHRH) analogue that disturbs the production of
luteinizing hormone (LH), which regulates the synthesis of male
hormones. In particular, to decrease LH production, peptidic
analogues of LHRH that act as superagonists of the luteinizing
hormone releasing hormone receptor, such as leuprolide and
goserelin, have been used.
[0003] In many instances, the therapeutic effectiveness of a
pharmaceutically active peptide depends upon its continued presence
in vivo over prolonged time periods. To achieve continuous delivery
of the peptide in vivo, a sustained release or sustained delivery
formulation is desirable, to avoid the need for repeated
administrations. One approach for sustained drug delivery is by
microencapsulation, in which the active ingredient is enclosed
within a polymeric membrane to produce microparticles. For example,
LHRH superagonists, such as leuprolide and goserelin, typically are
encapsulated within a microparticle comprising a
poly-lactide/poly-glycolide copolymer to prepare formulations
suitable for depot injection that provide sustained delivery of the
superagonist over several weeks to months (see e.g., U.S. Pat. Nos.
4,675,189; 4,677,191; 5,480,656 and 4,728,721).
[0004] Additional sustained delivery formulations for administering
pharmaceutically active peptides in vivo continuously for prolonged
time periods are needed.
SUMMARY OF THE INVENTION
[0005] The present invention provides pharmaceutical compositions
comprising a stable water-insoluble complex composed of a peptidic
compound (e.g., a peptide, polypeptide, protein, peptidomimetic and
the like), preferably a pharmaceutically active peptidic compound,
and a carrier macromolecule that allow for sustained delivery of
the peptidic compound in vivo upon administration of the complex.
Accordingly, the complex of the invention can permit continuous
delivery of a pharmaceutically active peptidic compound to a
subject for prolonged periods of time, e g, one month. Moreover,
the association of the peptidic compound and the carrier
macromolecule in a tight, stable complex allows for loading of high
concentrations of the peptidic compound into the formulation.
[0006] The complex of the invention is formed by combining the
peptidic compound and the carrier macromolecule under conditions
such that a substantially water-insoluble complex is formed, e.g,
aqueous solutions of the peptidic compound and carrier
macromolecule are mixed until the complex precipitates. The complex
may be in the form of a solid (e.g., a paste, granules, a powder or
a lyophilizate) or the powdered form of the complex can be
pulverized finely enough to form stable liquid suspensions or
semi-solid dispersions.
[0007] In a preferred embodiment, the peptidic compound of the
water-insoluble complex is an LHRH analogue, more preferably an
LHRH antagonist, and the carrier macromolecule is an anionic
polymer, preferably carboxymethylcellulose. The complex of the
invention is suitable for sterilization, such as by gamma
irradiation or electron beam irradiation, prior to administration
in vivo.
[0008] Method for treating a subject for a condition treatable with
an LHRH analogue by administering to the subject an
LHRH-analogue-containing composition of the invention are also
provided. In a preferred embodiment, the treatment methods of the
invention are used in the treatment of prostate cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows graphs depicting the plasma testosterone levels
(in ng/ml; open black boxes) and plasma PPI-149 levels (in ng/ml;
closed boxes) in rats (left graph) and dogs (right graph) over time
following intramuscular injection of a complex of PPI-149 and
carboxymethylcellulose.
[0010] FIG. 2 is a graph depicting the plasma testosterone levels
(in ng/ml; open boxes) and plasma PPI-149 levels (in ng/ml; closed
boxes) in rats over time following intramuscular injection of a
complex of the LHRH antagonist PPI-149 and carboxymethylcellulose
on day 0 and injection of the LHRH agonist Lupron.TM. at day 30,
demonstrating suppression of the Lupron.TM.-induced testosterone
surge by the PPI-149 pretreatment.
[0011] FIGS. 3A-3C are a series of graphs depicting the plasma
testosterone levels (in ng/ml) in male Sprague-Dawley rats over
time, following intramuscular injection of a PPI-149-CMC (FIG. 3A),
PPI-258-CMC (FIG. 3B) or Cetrorelix.TM.-CMC (FIG. 3C).
[0012] FIG. 4 is a graph depicting the plasma testosterone levels
(in ng/ml; open boxes) and plasma PPI-149 levels (in ng/ml; closed
boxes) in dogs over time following subcutaneous injection of
PPI-149-CMC at the indicated dosages at 28 day intervals,
demonstrating prolonged suppression of plasma testosterone
levels.
[0013] FIG. 5 is a graph depicting the plasma testosterone levels
(in ng/ml; open boxes) and plasma PPI-149 levels (in ng/ml; closed
boxes) in dogs over time following intramuscular injection of
PPI-149-CMC at the indicated dosages at 28 day intervals,
demonstrating prolonged suppression of plasma testosterone
levels.
DETAILED DESCRIPTION OF THE INVENTION
[0014] This invention pertains to pharmaceutical compositions
comprising a stable water-insoluble complex composed of a peptidic
compound (e.g., a peptide, polypeptide, protein, peptidomimetic and
the like) and a carrier macromolecule, methods of making such
compositions and methods of using such compositions. The advantages
of the pharmaceutical compositions of the invention include the
ability for delivery of a pharmaceutically active peptidic
compound, either systemically or locally, for prolonged periods
(e.g., several weeks, one month or several months) and the ability
to load high concentrations of peptidic compound into the
complex.
[0015] In order that the invention may be more readily understood,
certain terms are first defined.
[0016] As used herein, the term "peptidic compound" is intended to
refer to compounds composed, at least in part, of amino acid
residues linked by amide bonds (i.e., peptide bonds). The term
"peptidic compound" is intended to encompass peptides, polypeptide
and proteins. Typically, a peptide will be composed of less than
about 100 amino acids, more typically less than about 50 amino acid
residues and even more typically, less than about 25 amino acid
residues. The term "peptidic compound" is further intended to
encompass peptide analogues, peptide derivatives and
peptidomimetics that mimic the chemical structure of a peptide
composed of naturally-occurring amino acids. Examples of peptide
analogues include peptides comprising one or more non-natural amino
acids. Examples of peptide derivatives include peptides in which an
amino acid side chain, the peptide backbone, or the amino- or
carboxy-terminus has been derivatized (e.g., peptidic compounds
with methylated amide linkages). Examples of peptidomimetics
include peptidic compounds in which the peptide backbone is
substituted with one or more benzodiazepine molecules (see e.g.,
James, G. L. et al. (1993) Science 260:1937-1942), "inverso"
peptide, in which all L-amino acids are substituted with the
corresponding D-amino acids, "retro-inverso" peptides (see U.S.
Pat. No. 4,522,752 by Sisto) in which the sequence of amino acids
is reversed ("retro") and all L-amino acids are replaced with
D-amino acids )"inverso") and other isosteres, such as peptide
back-bone (i.e., amide bond) mimetics, including modifications of
the amide nitrogen, the .alpha.-carbon, amide carbonyl, complete
replacement of the amide bond, extensions, deletions or backbone
crosslinks. Several peptide backbone modifications are known,
including .psi.[CH.sub.2S], .psi. [CH.sub.2NH], .psi.[CSNH.sub.2],
.psi.[NHCO], .psi.[COCH.sub.2], and .psi.[(E) or (Z) CH.dbd.CH]. In
the nomenclature used above,.psi. indicates the absence of an amide
bond. The structure that replaces the amide group is specified
within the brackets. Other possible modifications include an
N-alkyl (or aryl) substitution (.psi.[CONR]), backbone crosslinking
to construct lactams and other cyclic structures, and other
derivatives including C-terminal hydroxymethyl derivatives,
O-modified derivatives and N-terminally modified derivatives
including substituted amides such as aikylamides and
hydrazides.
[0017] As used herein, the term "pharmaceutically active peptidic
compound" is intended to refer to a peptidic compound that exhibits
pharmacologic activity, either in its present form or upon
processing in vivo (i.e., pharmaceutically active peptidic
compounds include peptidic compounds with constitutive
pharmacologic activity and peptidic compounds in a "prodrug" form
that have to be metabolized or processed in some way in vivo
following administration in order to exhibit pharmacologic
activity).
[0018] As used herein, the terms "multivalent cationic peptidic
compound" and "multivalent anionic peptidic compound" are intended
to refer to peptidic compounds comprising a multiplicity of
positive or negative charges, respectively. A "bivalent cationic"
or "bivalent anionic" peptidic compound is intended to refer to a
peptidic compound comprising two positive or negative charges,
respectively. A "trivalent cationic" or "trivalent anionic"
peptidic compound is intended to refer to a peptidic compound
comprising three positive or negative charges, respectively.
[0019] As used herein, the term "LHRH analogue" is intended to
encompass peptidic compounds that mimic the structure of
luteinizing hormone releasing hormone. An LHRH analogue may be an
LHRH agonist or an LHRH antagonist.
[0020] As used herein, an "LHRH agonist" is intended to refer to a
compound which stimulates the luteinizing hormone releasing hormone
receptor (LHRH-R) such that release of luteinizing hormone is
stimulated, or an "LHRH antagonist", which refers to a compound
that inhibits LHRH-R such that release of luteinizing hormone is
inhibited. Examples of LHRH agonists include leuprolide (trade
name: Lupron.RTM.; Abbott/TAP), goserelin (trade name:
Zoladex.RTM.; Zeneca), buserelin (Hoechst), triptorelin (also known
as Decapeptyl, D-Trp-6-LHRH and Debiopharm.RTM.; Ipsen/Beaufour),
nafarelin (trade name" Synarel.RTM.; Syntex), lutrelin (Wyeth),
cystorelin (Hoechst), gonadorelin (Ayerst) and histrelin
(Ortho).
[0021] As used herein, the term "LHRH antagonist" is intended to
refer to a compound that inhibits the luteinizing hormone releasing
hormone receptor such that release of luteinizing hormone is
inhibited. Examples of LHRH antagonists include Antide, Cetrorelix,
compounds described in U.S. Pat. No. 5,470,947 to Folkers et al;
PCT Publication No. WO 89/01944 by Folkers et al; U.S. Pat. No.
5,413,990 to Haviv; U.S. Pat. No. 5,300,492 to Haviv; U.S Pat. No.
5,371,070 to Koerberetal.; U.S. Pat. No. 5,296,468 to Hoeger et
al.; U.S. Pat. No. 5,171,835 to Janaky et al.; U.S. Pat. No.
5,003,011 to Coy et al; U.S. Pat. No. 4,431,635 to Coy; U.S. Pat.
No. 4,992,421 to De et aL; U.S. Pat. No. 4,851,385 to Roeske; U.S.
Pat. No. 4,801,577 to Nestor, Jr. et aL; and U.S. Pat. No.
4,689,396 to Roeske et al. and compounds disclosed in U.S. patent
application Ser. No. 08/480,494, entitled "LHRH Antagonist
Peptides", and a corresponding PCT application thereof (PCT
Application No. PCT/US96/09852), also entitled "LHRH Antagonist
Peptides", the entire contents of both of which are expressly
incorporated herein by reference. An especially preferred LHRH
antagonist comprises the structure: Ac-D-Nal.sup.1,
4-Cl-D-Phe.sup.2, D-Pal.sup.3, N-Me-Tyr.sup.5, D-Asn.sup.6,
Lys(iPr).sup.8, D-Ala.sup.10-LHRH, referred to herein as
PPI-149.
[0022] As used herein, the term "carrier macromolecule" is intended
to refer to a macromolecule that can complex with a peptidic
compound to form a water-insoluble complex. Prior to complexing
with the peptidic compound, the carrier macromolecule typically is
water-soluble. Preferably, the macromolecule has a molecular weight
of at least 5 kDa, more preferably 10 kDa. The term "anionic
carrier macromolecule" is intended to include negatively charged
high molecular weight molecules, such as anionic polymers. The term
"cationic carrier macromolecule" is intended to includes positively
charged high molecular weight molecules, such as cationic
polymers.
[0023] As used herein, the term "water-insoluble complex" is
intended to refer to a physically and chemically stable complex
that forms upon appropriate combining of a peptidic compound and
carrier macromolecule according to procedures described herein.
This complex typically takes the form of a precipitate that is
produced upon combining aqueous preparations of the peptidic
compound and carrier macromolecule. Although not intending to be
limited by mechanism, the formation of preferred water-insoluble
complexes of the invention is thought to involve (i.e., be mediated
at least in part by) ionic interactions in situations where the
peptidic compound is cationic and the carrier molecule is anionic
or vice versa. Additionally or alternatively, the formation of a
water-insoluble complex of the invention may involve (i.e., be
mediated at least in part by) hydrophobic interactions. Still
further, formation of a water-insoluble complex of the invention
may involve (i.e., be mediated at least in part by) covalent
interactions. Description of the complex as being "water-insoluble"
is intended to indicate that the complex does not substantially or
readily dissolve in water, as indicated by its precipitation from
aqueous solution. However, it should be understood that a
"water-insoluble" complex of the invention may exhibit limited
solubility (i.e., partial solubility) in water either in vitro or
in the aqueous physiological environment in vivo.
[0024] As used herein, the term "sustained delivery" is intended to
refer to continual delivery of a pharmaceutical agent in vivo over
a period of time following administration, preferably at least
several days, a week or several weeks. Sustained delivery of the
agent can be demonstrated by, for example, the continued
therapeutic effect of the agent over time (e.g., for an LHRH
analogue, sustained delivery of the analogue can be demonstrated by
continued suppression of testosterone synthesis over time).
Alternatively, sustained delivery of the agent may be demonstrated
by detecting the presence of the agent in vivo over time.
[0025] As used herein, the term "subject" is intended to include is
intended to include warm-blooded animals, preferably mammals, more
preferably primates and most preferably humans.
[0026] As used herein, the term "administering to a subject" is
intended to refer to dispensing, delivering or applying a
composition (e.g., pharmaceutical formulation) to a subject by any
suitable route for delivery of the composition to the desired
location in the subject, including delivery by either the
parenteral or oral route, intramuscular injection,
subcutaneous/intradermal injection, intravenous injection, buccal
administration, transdermal delivery and administration by the
rectal, colonic, vaginal, intranasal or respiratory tract
route.
[0027] As used herein, the term "a condition treatable with an LHRH
analogue" is intended to include diseases, disorders and other
conditions in which administration of an LHRH agonist or LHRH
antagonist has a desired effect, e.g., a therapeutically beneficial
effect. Examples of conditions treatable with an LHRH analogue
include hormone-dependent cancers (including prostate cancer,
breast cancer, ovarian cancer, uterine cancer and testicular
cancer), benign prostatic hypertrophy, precocious puberty,
endometriosis, uterine fibroids, infertility (through in vitro
fertilization) and fertility (ie., contraceptive uses).
[0028] One aspect of the present invention pertains to a
pharmaceutical composition comprising a water-insoluble complex of
a pharmaceutically active peptidic compound and a carrier
macromolecule. In a preferred embodiment, formation of the
water-insoluble complex is mediated at least in part by ionic
interactions between the pharmaceutically active peptidic and the
carrier macromolecule. In these embodiments, either the
pharmaceutically active peptidic compound is cationic and the
carrier macromolecule is anionic or the pharmaceutically active
peptidic compound is anionic and the carrier macromolecule is
cationic. In another embodiment, formation of the water-insoluble
complex is mediated at least in part by hydrophobic interactions
between the pharmaceutically active peptidic compound and the
carrier macromolecule. In a preferred embodiment, the peptidic
compound used in the complex is a multivalent cationic peptidic
compound, such as a bivalent or trivalent cationic peptidic
compound and the carrier macromolecule is an anionic
macromolecule.
[0029] The pharmaceutical compositions of the invention permit
sustained delivery of the peptidic compound to a subject in vivo
after administering the composition to the subject, wherein the
duration of the sustained delivery can be varied depending upon the
concentration of peptidic compound and carrier macromolecule used
to form the complex. For example, in one embodiment, a single dose
of the water-insoluble complex provides sustained delivery of the
peptidic compound to a subject for at least one week after the
pharmaceutical composition is administered to the subject. In
another embodiment, a single dose of the water-insoluble complex
provides sustained delivery of the peptidic compound to a subject
for at least two weeks after the pharmaceutical composition is
administered to the subject. In yet another one embodiment, a
single dose of the water-insoluble complex provides sustained
delivery of the peptidic compound to a subject for at least three
weeks after the pharmaceutical composition is administered to the
subject. In still another embodiment, a single dose of the
water-insoluble complex provides sustained delivery of the peptidic
compound to a subject for at least four weeks after the
pharmaceutical composition is administered to the subject.
Formulations that provide sustained delivery for longer or shorter
durations are also encompassed by the invention, such as
formulations that provide continuous delivery for 1 day, 1-7 days,
one month, two months, three months, and the like. Continuous
delivery of the peptidic compound for a period of several months
can be accomplished, for example, by repeated monthly dosages, each
of which provide sustained delivery of the peptidic compound for
approximately one month (see e.g., Example 14).
[0030] Any size peptidic compound may be suitable for use in the
complex as long as the peptidic compound has the ability to form a
water-insoluble noncovalent complex with the carrier macromolecule
upon combination of the peptidic compound and carrier
macromolecule. However, in certain preferred embodiments, the
peptidic compound is a peptide that is about 5 to about 20 amino
acids in length, about 8 to about 15 amino acids in length or about
8 to about 12 amino acids in length. A variety of pharmaceutically
active peptides may be used in the formulations, non-limiting
examples of which include LHRH analogues (discussed further below),
bradykinin analogues, parathyroid hormone, adenocorticotrophic
hormone (ACTH), calcitonin, and vasopressin analogues (e.g.,
1-deamino-8-D-arginine vasopressin (DDAVP)).
[0031] Although a variety of carrier macromolecules may be suitable
for formation of the water-insoluble complexes of the invention,
preferred macromolecules are polymers, preferably water-soluble
polymers. In a preferred embodiment, the carrier macromolecule is
an anionic polymer, such as an anionic polyacohol derivative, or
fragment thereof, and salts thereof (e.g., sodium salts). Anionic
moieties with which the polyalcohol can be derivatized include, for
example, carboxylate, phosphate or sulfate groups. A particularly
preferred anionic polymer is an anionic polysaccharide derivative,
or fragment thereof, and salts thereof (e.g., sodium salts). The
carrier macromolecule may comprise a single molecular species
(e.g., a single type of polymer) or two or more different molecular
species (e.g., a mixture of two types of polymers). Examples of
specific anionic polymers include carboxymethylcellulose, algin,
alginate, anionic acetate polymers, anionic acrylic polymers,
xantham gums, sodium starch glycolate, and fragments, derivatives
and pharmaceutically acceptable salts thereof, as well as anionic
carageenan derivatives, anionic polygalacturonic acid derivatives,
and sulfated and sulfonated polystyrene derivatives. A preferred
anionic polymer is carboxymethylcellulose sodium salt. Examples of
cationic polymers include poly-L-lysine and other polymers of basic
amino acids.
[0032] In a particularly preferred embodiment of the invention, the
peptidic compound of the water-insoluble complex is an LHRH
analogue, for example an LHRH agonist or, more preferably, an LHRH
antagonist. Such LHRH analogues typically are 10 amino acids in
length. Preferred LHRH antagonists include LHRH antagonists that
comprise a peptide compound, wherein a residue of the peptide
compound corresponding to the amino acid at position 6 of natural
mammalian LHRH comprises a D-asparagine (D-Asn) structure. As used
herein, the term "D-asparagine structure" is intended to include
D-Asn and analogues, derivatives and mimetic thereof that retain
the functional activity of D-Asn. Other preferred LHRH antagonists
include LHRH antagonists that comprise a peptidic compound
comprising a structure: A-B-C-D-E-F-G-H-I-J
[0033] wherein
[0034] A is pyro-Glu, Ac-D-Nal , Ac-D-Qal, Ac-Sar, or Ac-D-Pal
[0035] B is His or 4-Cl-D-Phe
[0036] C is Trp, D-Pal, D-Nal, L-Nal, D-Pal(N-O), or D-Trp
[0037] D is Ser
[0038] E is N-Me-Ala, Tyr, N-Me-Tyr, Ser, Lys(iPr), 4-Cl-Phe, His,
Asn, Met, Ala, Arg or Ile;
[0039] F is 1
[0040] wherein
[0041] R and X are, independently, H or alkyl; and
[0042] L comprises a small polar moiety;
[0043] G is Leu or Trp;
[0044] H is Lys(iPr), Gin, Met, or Arg
[0045] I is Pro; and
[0046] J is Gly-NH.sub.2 or D-Ala-NH.sub.2; or a pharmaceutically
acceptable salt thereof.
[0047] The term "small polar moiety" refers to a moiety which has
small steric bulk and is relatively polar. Polarity is measured as
hydrophilicity by the P scale. The partition coefficient, P,
between 1-octanol and water has been used as a reference for
measuring the hydrophilicity of a compound. Hydrophilicity can be
expressed as log P, the logarithm of the partition coefficient
(Hansch et al., Nature 194:178 (1962); Fujita et al., J. Am. Chem.
Soc. 86:5175 (1964)). Standard tables of hydrophilicity for many
molecules, and lipophilicity (hydrophobicity) substituent constants
(denoted.pi.) for many functional groups, have been compiled (see,
e.g., Hansch and Leo, "Substituent Constants for Correlation
Analysis in Chemistry and Biology," Wiley, New York, N.Y., (1979)).
The hydrophilicity of a vast range of candidate hydrophilicity
moieties can be quite accurately predicted with the aid of these
tables. For example, the measured log P (octanol/water) of
naphthalene is 3.45. The substituent constant p for--OH is -0.67.
Therefore, the predicted log P for .beta.-naphthol is 3.45
+(-0.67)=2.78. This value is in good agreement with the measured
log P for .beta.-naphthol, which is 2.84. As used herein, the term
"small polar moiety" refers to moieties that have a log P between
-1 and +2 and a steric bulk that is less than the steric bulk of
Trp.
[0048] In certain embodiments, L comprises a small polar moiety
with the proviso that F is not D-Cit, D-Hci or a lower alkyl
derivative of D-Cit or D-Hci. Preferably, F is selected from the
group consisting of D-Asn, D-Gln and D-Thr. More preferably, F is
D-Asn. Preferably, E is tyrosine (Tyr) or N-methyl-tyrosine
(N-Me-Tyr). In a particularly preferred embodiment, the LHRH
antagonist has the following structure: Ac-D-Nal.sup.1,
4-Cl-D-Phe.sup.2, D-Pal.sup.3, N-Me-Tyr.sup.5, D-Asn.sup.6,
Lys(iPr).sup.8, D-Ala.sup.10-LHRH (referred to herein as PPI-149).
A particularly preferred complex of the invention comprises PPI-149
and carboxymethylcellulose.
[0049] In addition to the water-insoluble complex, the
pharmaceutical formulations of the invention can comprise
additional pharmaceutically acceptable carriers and/or excipients.
As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Preferably, the carrier
is suitable for intravenous, intramuscular, subcutaneous or
parenteral administration (e.g., by injection). Excipients include
pharmaceuticlly acceptable stabilizers and disintegrants.
[0050] In addition to pharmaceutical formulations of LHRH analogues
complexed with a carrier macromolecule, the invention further
encompasses packaged formulations containing such complexes and
syringes containing such complexes. For example, the invention
provides a packaged formulation for treating a subject for a
condition treatable with an LHRH analogue, comprising a
water-insoluble complex of an LHRH analogue (preferably PPI-149)
and a carrier macromolecule (preferably carboxymethylcellulose),
packaged with instructions for using the water-insoluble complex
for treating a subject for a condition treatable with an LHRH
analogue. In another embodiment, the invention provides a syringe
having a lumen, wherein a water-insoluble complex of an LHRH
analogue (preferably PPI-149) and a carrier macromolecule
(preferably, carboxymethyl-cellulose) is included in the lumen.
[0051] The complex of the invention is prepared by combining the
peptidic compound and the carrier macromolecule under conditions
such that a water-insoluble complex of the peptidic compound and
the carrier macromolecule forms. Accordingly, another aspect of the
invention pertains to methods for preparing pharmaceutical
formulations. In one embodiment, the method comprises:
[0052] providing a peptidic compound and a carrier
macromolecule;
[0053] combining the peptidic compound and the carrier
macromolecule under conditions such that a water-insoluble complex
of the peptidic compound and the carrier macromolecule forms;
and
[0054] preparing a pharmaceutical formulation comprising the
water-insoluble complex. For example, a solution of the peptidic
compound and a solution of the carrier macromolecule are combined
until a water-insoluble complex of the peptidic compound and the
carrier macromolecule precipitates out of solution. In certain
embodiments, the solutions of the peptidic compound and the carrier
macromolecule are aqueous solutions. Alternatively, if the peptidic
compound or the carrier molecule (or both) is not substantially
water soluble prior to combination the two, then the peptidic
compound and/or carrier macromolecule can be dissolved in a
water-miscible solvent, such as an alcohol (e.g., ethanol) prior to
combining the two components of the complex. In another embodiment
of the method of preparing the water-insoluble complex, the
solution of the peptidic compound and the solution of the carrier
macromolecule are combined and heated until a water-insoluble
complex of the peptidic compound and the carrier macromolecule
precipitates out of solution. The amounts of peptidic compound and
carrier macromolecule necessary to achieve the water-insoluble
complex may vary depending upon the particular peptidic compound
and carrier macromolecule used, the particular solvent(s) used
and/or the procedure used to achieve the complex. Typically,
however, the peptidic compound will be in excess relative to the
carrier macromolecule on a molar basis. Often, the peptidic
compound also will be in excess on a weight/weight basis, as
demonstrated in the Examples. In certain embodiments, the carrier
macromolecule, preferably carboxymethylcellulose sodium, and the
peptidic compound, preferably PPI-149, are combined at a ratio of
0.2:1 (w/w) of carrier macromolecule:peptidic compound. In various
other embodiments, the ratio of carrier macromolecule to peptidic
compound (w/w) can be, for example, 0.5:1, 0.4:1, 0.3:1, 0.25:1,
0.15:1 or 0.1:1. Non-limiting examples of conditions and procedures
for preparing a water-insoluble complex of the invention are
described further in Example 1-5 Sand 8-9.
[0055] Once the peptidic compound/macromolecule complex
precipitates out of solution, the precipitate can be removed from
the solution by means known in the art, such as filtration (e.g.,
through a 0.45 micron nylon membrane), centrifugation and the like.
The recovered paste then can be dried (e.g., in vacuo or in a
70.degree. C. oven) and the solid can be milled or pulverized to a
powder by means known in the art (e.g., hammer or gore milling, or
grinding in mortar and pestle). Following milling or pulverizing,
the powder can be sieved through a screen (preferably a 90 micron
screen) to obtain a uniform distribution of particles. Moreover,
the recovered paste can be frozen and Iyophilized to dryness. The
powder form of the complex can be dispersed in a carrier solution
to form a liquid suspension or semi-solid dispersion suitable for
injection. Accordingly, in various embodiments, a pharmaceutical
formulation of the invention is a dry solid, a liquid suspension or
a semi-solid dispersion. Examples of liquid carriers suitable for
use in liquid suspensions include saline solutions, glycerin
solutions and lecithin solutions.
[0056] In another embodiment, the pharmaceutical formulation of the
invention is sterile formulation. For example, following formation
of the water-insoluble complex, the complex can be sterilized,
optimally by gamma irradiation or electron beam sterilization.
Accordingly, the method of the invention for preparing a
pharmaceutical formulation described above can further comprise
sterilizing the water-insoluble complex by gamma irradiation or
electron beam irradiation. Preferably, the formulation is
sterilized by gamma irradiation using a gamma irradiation dose of
at least 15 KGy. In other embodiments, the formulation is
sterilized by gamma irradiation using a gamma irradiation dose of
at least 19 KGy or at least 24 KGy. As demonstrated in Example 11,
the formulations of the invention remain acceptably stable upon
gamma irradiation.
[0057] Alternatively, to prepare a sterile pharmaceutical
formulation, the water-insoluble complex can be isolated using
conventional sterile techniques (e.g., using sterile starting
materials and carrying out the production process aseptically).
[0058] Accordingly, in another embodiment of the method for
preparing a pharmaceutical formulation described above, the
water-insoluble complex is formed using aseptic procedures.
[0059] Methods of forming a water-insoluble complex of the
invention are described further in Examples 1-5 and 8-9.
Pharmaceutical formulations, including powders, liquid suspensions,
semi-solid dispersions, dry solids (e.g., lyophilized solids), and
sterilized forms thereof (e.g., by gamma irradiation), prepared
according to the methods of the invention, are also encompassed by
the invention.
[0060] Yet another aspect of the invention pertains to methods of
using the pharmaceutical formulations of the invention to treat a
subject suffering from a condition treatable by the
pharmaceutically active peptidic compound included in the
water-insoluble complex. Accordingly, in a preferred embodiment,
the invention provides a method for treating a subject for a
condition treatable with an LHRH analogue, comprising administering
to the subject a pharmaceutical formulation comprising a
water-insoluble complex of an LHRH analogue and a carrier
macromolecule.
[0061] The pharmaceutical formulation can be administered to the
subject by any route suitable for achieving the desired therapeutic
result(s), although preferred routes of administration are
parenteral routes, in particular intramuscular (i.m.) injection and
subcutaneous/intradermal (s.c./i.d.) injection. Alternatively, the
formulation can be administered to the subject orally. Other
suitable parental routes include intravenous injection, buccal
administration, transdermal delivery and administration by the
rectal, vaginal, intranasal or respiratory tract route. It should
be noted that when a formulation that provides sustained delivery
for weeks to months by the i.m or s.c./i.d. route is administered
by an alternative route, there may not be sustained delivery of the
agent for an equivalent length of time due to clearance of the
agent by other physiological mechanisms (i.e., the dosage form may
be cleared from the site of delivery such that prolonged
therapeutic effects are not observed for time periods as long as
those observed with i.m or s.c./i.d. injection).
[0062] The pharmaceutical formulation contains a therapeutically
effective amount of the LHRH analogue. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired result. A therapeutically
effective amount of an LHRH analogue may vary according to factors
such as the disease state, age, and weight of the individual, and
the ability of the LHRH analogue (alone or in combination with one
or more other drugs) to elicit a desired response in the
individual. Dosage regimens may be adjusted to provide the optimum
therapeutic response. A therapeutically effective amount is also
one in which any toxic or detrimental effects of the antagonist are
outweighed by the therapeutically beneficial effects. A
non-limiting range for a therapeutically effective amount of an
LHRH analogue is 0.01 to 10 mg/kg. A preferred dosage of the LHRH
analogue PPI-149 for sustained reduction of plasma testosterone
levels for 28 days is approximately 0.1-10 mg/kg, more preferably
0.3-1.2 mg/kg (expressed as free peptide) in a liquid suspension
volume of approximately 1 mL or less. It is to be noted that dosage
values may vary with the severity of the condition to be
alleviated. It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions, and that dosage ranges set forth herein are exemplary
only and are not intended to limit the scope or practice of the
claimed composition.
[0063] The treatment method of the invention can be applied to the
treatment of various conditions, diseases and disorders in which
administration of an LHRH analogue has a desired clinical effect.
Examples of disease and disorders include hormone-dependent
cancers, such as prostate cancer, breast cancer, ovarian cancer,
uterine cancer and testicular cancer, benign prostatic hypertrophy,
precocious puberty, endometriosis and uterine fibroids.
Accordingly, the invention provides methods of treating these
diseases and disorders by administering a pharmaceutical
formulation of the invention. Additionally, LHRH analogues can be
used to alter fertility. Accordingly, the methods of the invention
also can be used in vitro fertilization and contraceptive
purposes.
[0064] In a particularly preferred embodiment, the method is used
to treat prostate cancer, the LHRH analogue used in the formulation
is an LHRH antagonist, most preferably PPI-149, and the method
allows for sustained delivery of the LHRH analogue in vivo for at
least four weeks after administration by intramuscular or
subcutaneous administration. An LHRH analogue, preferably PPI-149,
formulated according to the invention can be used to inhibit growth
of prostate cancer cells by administering the LHRH analogue to a
subject suffering from prostate cancer. Moreover, an LHRH
antagonist, preferably PPI-149, formulated according to the
invention, can be used to inhibit the testosterone surge that
accompanies the use of an LHRH agonist by preadministering the LHRH
antagonist, preferably PPI-149, to a subject suffering from
prostate cancer before initiating LHRH agonist therapy. Methods for
inhibiting LHRH agonist-induced testosterone surge, and other
methods for treating prostate cancer using LHRH antagonist, to
which the formulations of the present invention can be applied, are
described further in U.S. patent application Ser. No. 08/573,109,
entitled "Methods for Treating Prostate Using LHRH Antagonists",
filed Dec. 15, 1995, and a continuation-in-part patent application
thereof, Ser. No. 08/755,593, also entitled "Methods for Treating
Prostate Using LHRH Antagonists", filed Nov. 25, 1996, the contents
of both of which are incorporated in published PCT application WO
97/22357. The entire contents of the U.S. applications and
published PCT application are expressly incorporated herein by
reference.
[0065] Specific processes for complexing a pharmaceutically active
peptidic compound with a carrier macromolecule are set forth in
Examples 1-5 and 8-9 below. Also described are test results that
demonstrate that an LHRH antagonist-containing complex can enable
sustained delivery of the pharmaceutically active peptide in vivo
(Example 6) and can inhibit LHRH-agonist induced testosterone surge
(Example 7). The following examples, which further illustrate the
invention, should not be construed as limiting. The contents of all
references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
EXAMPLE 1
[0066] A 100 ml solution of the LHRH antagonist PPI-149 was
prepared by dissolving 6.25 mg/ml of PPI-149 in water. An equal
sample (100 ml minimum) of USP carboxymethylcellulose sodium (CMC)
(low viscosity grade, Hercules Chemical Co.) was prepared at 0.125%
w/v and mixed until dissolved. Equal portions of the PPI-149 and
CMC solutions were mixed (giving a CMC:peptide ratio of 0.2:1
(w/w)) and a solid material was obtained. The solid material was
stirred overnight and then collected by filtration over a 0.45
micron nylon filter. HPLC evaluation of the solution filtrate
indicated at least 95% of the PPI-149 compound was converted to the
solid complex. was removed from solution. The recovered white paste
was rinsed twice with water and then transferred to a vial and
dried in vacuo. Upon drying for 72 hours, 633 mg of a white powder
was obtained. The solid material was then powdered in a mortar and
pestle. Elemental analysis indicated 57% peptide in the
complex.
EXAMPLE 2
[0067] 25 mg of PPI-149 was dissolved in 1 ml of water. To this was
added 1 ml of a 0.5% carboxymethylcellulose solution. The mixture
formed a silky white solid upon mixing. The mixture was heated to
reflux for five minutes and a flocculent white precipitate was
formed. This material was isolated by centrifugation/decantation.
The solid was resuspended in water and collected by repeated
centrifugation. HPLC evaluation of the solution filtrate indicated
at least 90% of the PPI-149 compound was converted to the solid
complex. The white precipitate was dried in vacuo and the solid
material was comminuted in a mortar and pestle. Elemental analysis
indicated 77% peptide in the complex.
EXAMPLE 3
[0068] 50 mg of PPI-149 was dissolved in 2 mL of 5% mannitol and
mixed with 2 mL of 0.5% carboxymethylcellulose (low viscosity, USP,
Spectrum Quality Chemicals). The mixture was stirred and
immediately yielded a white precipitate. The suspension was frozen
and lyophilized to dryness to yield a PPI-149 sustained delivery
complex.
EXAMPLE 4
[0069] 25 mg of PPI-149 was dissolved in 1 mL water. To this was
added 1 mL of 0.5% sodium alginate, USP (Spectrum). The mixture
immediately formed a white precipitate upon mixing. This material
was isolated by centrifugation/decantation. The solid was
resuspended in water and collected by repeated centrifugation. The
white precipitate was dried in vacuo. Elemental analysis was
performed to obtain a peptide content of 66%.
EXAMPLE 5
[0070] 25 mg of PPI-149 was dissolved in 1 mL water. Ammonia was
added to adjust the pH to 11.0. To this was added 1 mL of 0.5%
alginic acid, USP (Spectrum). The mixture immediately formed a
white precipitate upon mixing. This material was isolated by
centrifugation/decantation. The solid was resuspended in water and
collected by repeated centrifugation. The white precipitate was
dried in vacuo. Elemental analysis was performed to obtain a
peptide content of 79%.
EXAMPLE 6
[0071] A water-insoluble complex of the LHRH antagonist PPI-149 and
carboxymethylcellulose was prepared according to the preceding
examples. A suspension of the PPI-149/CMC complex was prepared and
a single dose was injected intramuscularly into rats and dogs. The
dosage for the rats was 50 .mu.g/kg/day X 60 days and the dosage
for the dogs was 40 .mu.g/kg/day X 28 days. Plasma testosterone
levels (in ng/ml) were determined at various time points as a
measure of the activity of the LHRH antagonist in the animal.
Representative results, shown in the graph of FIG. 1, demonstrate
that intramuscular injection of the PPI-149/CMC complex leads to
sustained suppression of plasma testosterone levels for at least 42
days in the rats and at least 28 days in the dogs (indicated by the
open boxes in FIG. 1), demonstrating sustained delivery of the LHRH
antagonist. Plasma levels of PPI-149 (in ng/ml) were also monitored
in the animals (indicated by the closed boxes in FIG. 1). An
initial spike of PPI-149 was observed for about the first eight
days, after which time PPI-149 was essentially undetectable in the
plasma. Despite the inability to detect PPI-149 in the plasma
beyond about day 8, the testosterone level results demonstrate that
PPI-149 was still therapeutically active in vivo over the course of
the experiment.
EXAMPLE 7
[0072] A water-insoluble complex of the LHRH antagonist PPI-149 and
carboxymethylcellulose was prepared according to the preceding
examples. A suspension of the PPI-149/CMC complex was prepared and
a single dose was injected intramuscularly into rats on day 0. On
day 30, the LHRH agonist Lupron.TM. (leuprolide) was injected into
the rats. Plasma testosterone levels (in ng/ml; indicated by the
open boxes in FIG. 2) were determined at various time points as a
measure of the activity of the LHRH antagonist in the animal.
Plasma levels of PPI-149 (in ng/ml) were also monitored in the
animals (indicated by the closed boxes in FIG. 2). Representative
results, shown in the graph of FIG. 2, demonstrate that
pretreatment with the PPI-149/CMC complex rapidly reduces plasma
testosterone to castration levels and, moreover, blocks the LHRH
agonist-induced testosterone surge. Despite the inability to detect
PPI-149 in the plasma beyond about day 8, the testosterone level
results demonstrate that PPI-149 was still therapeutically active
in vivo over the course of the experiment.
EXAMPLE 8
[0073] In this example, an insoluble complex was formed between the
LHRH analogue PPI-258 and carboxymethylcellulose (CMC). PPI-258 has
the structure:
acetyl-D-napthylalanyl-D-4-Cl-phenylalanyl-D-pyridylalanyl-L-s-
eryl-L-tyrosyl-D-asparaginyl-L-leucyl-L-N.sup.e-isopropyl-lysyl-L-propyl-D-
-alanyl-amide. To prepare a PPI-258/CMC depot, 174.8 mg (148.6 mg
net) of PPI-258 was added to 29.72 mL of water and the material was
stirred to suspend and dissolve the peptide. To this stirred
solution was added 1.85 mL of a 2% sodium CMC solution (Hercules).
A solid precipitate was immediately observed. Upon heating to
reflux, the suspension became translucent and then appeared as
white precipitate. After a 5 minute reflux, the reaction was cooled
and the solid was isolated by centrifugation. The solid was rinsed
with water, and dried in vacuo overnight. The dried power was
powdered in a mortar and pestle and sieved through a 90 micron
stainless steel screen. The sieved powder (90 micron sieve) was
collected and characterized. Total yields were 198.4 mg of dried
solid which yielded 110.8 mg of sized powder after the milling
step. Characterization provided the following compositional makeup
of the complex: Peptide PPI-258 -80%, CMC -18.8%, water -6.6%.
EXAMPLE 9
[0074] In this example, an insoluble complex was formed between the
LHRH analogue Cetrorelix.TM. (also known as SB-75) and
carboxymethylcellulose (CMC). Cetrorelix.TM. has the structure:
acetyl-D-napthylalanyl-D-4-Cl-ph-
enylalanyl-D-pyridylalanyl-L-seryl-L-tyrosyl-D-citrulyl-L-leucyl-L-arginyl-
-L-prolyl-D-alanyl-amide. To prepare a Cetrorelix/CMC depot, 102.8
mg (87 mg net) of Cetrorelix.TM. was added to 17.4 mL of water and
the material was stirred to suspend and dissolve the peptide. To
this stirred solution was added 1.1 mL of a 2% sodium CMC solution
(Hercules). A clumpy white precipitate was immediately observed.
The suspension was heated to reflux for 5 minutes and cooled to
yield a solid white precipitate. The solid was isolated by
centrifugation, was rinsed with water, and dried in vacuo
overnight. The dried powder was powdered in a mortar and pestle and
sieved through a 90 micron stainless steel screen. The powder was
collected and characterized. Total yields were 95 mg of dried solid
which yielded 60 mg of sized powder after the milling step.
Characterization provided the following compositional makeup of the
complex: Peptide Cetrorelix.TM. -75%, CMC -20.7%, water -6.5%
EXAMPLE 10
[0075] In this example, the sustained release of three different
LHRH analogues, PPI-149, PPI-258 and Cetrorelix.TM., prepared as
CMC depot formulations as described in three previous examples, was
examined in vivo. Three different formulation vehicles were tested,
saline, glycerin (15% glycerin/4% dextrose) and lecithin.
Sprague-Dawley rats (25 males, weight range 300-325 g) were used
and the efficacy of the LHRH analogue was determined based on
reduction in plasma testosterone levels.
[0076] The dosages and routes of administration were as
follows:
1 Dose Dose (.mu.g/kg/ Dose Route Group Compound (mg/kg) day)
(mg/rat) Vehicle Admin. A PPI-149 9 300 2.7 saline IM B PPI-149 9
300 2.7 glycerin IM C PPI-149 9 300 2.7 glycerin SC D PPI-149 9 300
2.7 lecithin IM E PPI-258 9 300 2.7 saline IM F Cetrorelix .TM. 9
300 2.7 saline IM
[0077] The actual dose of peptide was 300 .mu.g/kg/day for 30 days,
which was 2.7 mg/rat given as a single 200 .mu.L intramuscular (IM)
or subcutaneous (SC) injection. The total volume required to inject
5 rats/group was 1.3 mL at a concentration of 13.5 mg/mL active
peptide. The volume of injection was kept constant and the weight
of the powder was adjusted for total peptide content, as
follows:
2 Weight Vol. Req. Req. mg Weight used Group mL Powder mg Powder
Vol. used mL A 1.3 22.5 29.5 1.7 mL saline .sup. B, C 2.6 45 71.1
4.1 mL glycerin/dextrose D 1.3 22.5 35.2 2.03 mL 0.5%
lecithin/mannitol E 1.3 22.5 31 1.79 mL saline F 1.3 22.5 20.9 1.21
mL saline
[0078] A single 200 .mu.L intramuscular, or subcutaneous injection
of test article was made into the upper flank of the left hind limb
or under the skin between the scapulae, respectively, on Day 0
under anesthesia.
[0079] To test plasma testosterone levels, approximately 0.4 mL of
blood was removed from the retro-orbital sinus on Day 1 after
dosing and at days 3, 7, 14, 21, 28 and 35. Blood was processed to
plasma and frozen on dry ice for determination of testosterone
plasma levels by standard methods.
[0080] Representative results, shown in FIGS. 3A-3C, demonstrate
that plasma testosterone levels in male Sprague-Dawley rats were
reduced and maintained at low levels for at least 28 days and as
long as 50 days in response to sustained release of the LHRH
analogues PPI-149, PPI-258 and Centrolix.TM. prepared as CMC depot
formulations (shown in FIGS. 3A, 3B and 3C, respectively). These
results indicate that all three formulations are effective in
reducing plasma testosterone levels in vivo and maintaining reduced
plasma testosterone levels over time.
EXAMPLE 11
[0081] In this example, PPI-149-CMC formulations were exposed to
gamma irradiation for purposes of sterilization, followed by
evaluation of both physical and chemical properties of the
irradiated formulations. Data described below indicate that
.gamma.-irradiation is a viable means of sterilization of
PPI-149-CMC depot.
[0082] Peptide Stability
[0083] Approximately 40 mg of each of two separate PPI-149-CMC lots
was packed separately (under an air headspace) in to a number of
Type 1 Glass vials, sealed with rubber stoppers and aluminum seals.
Vials were then subjected to a variety of nominal doses of
gamma-irradiation. Two vials were analyzed for peptide purity
(expressed as %) at each level of .gamma.-irradiation exposure for
each of the two lots. The results indicated that at
.gamma.-irradiation doses up to and including 24 KGy, PPI-149-CMC
consistently exhibited less than a 2% reduction in peptide purity
(as determined by HPLC impurity profile). A second study utilizing
higher doses of gamma exposure was performed on an additional
laboratory lot of PPI-149-CMC. PPI-149-CMC demonstrated remarkably
good chemical stability when exposed to high .gamma.-irradiation
doses.
[0084] A subsequent preformulation study was implemented to compare
the degradation profile obtained following PPI-149-CMC
.gamma.-irradiation with that obtained following autoclaving of
PPI-149 injectable solution (1 mg/mL). Two samples were prepared:
a) PPI-149-CMC exposed to 19 KGy .gamma.-irradiation; b) A PPI-149
Solution (1 mg/mL) subjected to autoclaving (121.degree. C./20
minutes). The HPLC chromatograms of the two samples demonstrated
that the degradation profile for the two samples appeared to be
qualitatively similar (given similar relative retention times of
the major peaks).
[0085] Stressed Stability Storage Following Gamma-Irradiation
[0086] Stress-storage preformulation studies were also performed on
vials post-gamma-irradiation. Sealed vials from two laboratory lots
of PPI-149-CMC were exposed to 19KGy gamma-irradiation and stored
at 25.degree. C., 37.degree. C. and 50.degree. C. for up to one
month. The chemical stability data in these preformulation studies
indicated that .gamma.-irradiation at a dose of 19 KGy followed by
stressed-storage stability did not result in major chemical
instability even under highly stressed-storage conditions (e.g., 1
week at 50.degree. C.). The data indicate at .gamma.-irradiation
doses up to and including 19 KGy, storage of PPI-149-CMC for up to
28 days at or below 50.degree. C., consistently exhibited less than
a 2% reduction in peptide purity (as determined by HPLC impurity
profile). Despite an apparent difference in initial moisture
content between the two lots studied, no significant difference in
peptide purity was determined in either initial preformulation
stability samples or those stored for up to a month.
[0087] PPI-149-CMC Particle Size Analysis
[0088] A particle size method using laser light scattering was
developed, that is applicable to sizing studies of PPI-149-CMC. To
illustrate the utility of the method, a preformulation experiment
is presented, which was performed to investigate the effect of
gamma-irradiation on the particle size of PPI-149-CMC. This
experiment was conceived with the prior understanding that
amorphous solid materials may be predisposed to particle
consolidation, upon storage. Two samples of a laboratory lot of
PPI-149-CMC were packed in type I glass vials, closed with gray
butyl rubber stoppers and sealed with aluminum seals. Particle
evaluation was performed prior to and following exposure to a gamma
irradiation dose of 15.5KGy. Particle evaluation was performed by
laser light scattering (utilizing a Malvern Mastersizer S.TM.
equipped with a reverse fourier lens). 20 mg samples for particle
size analysis by laser light scattering were dispersed in
approximately 0.5 mL deionised water by vigorous shaking, then
sonicated in a bath at ambient temperature for 5 minutes. After
running a background count, a method qualification experiment was
performed. Sample dispersion was added drop-wise to the continuous
feed reservoir (approximately 60 mL nominal volume) until
approximately 20% obscuration was obtained. The mixer rotation
speed was held at 2700 rpm throughout the experiment (plus
background check). At this speed no vortex-induced bubbles were
generated, but an adequately stable dispersion was maintained.
Eight scans were performed, analysis of acquired data indicated a
standard deviation of <0.03% as the extreme of any data point
taken. When the sample dispersion was held in the reservoir for 15
minutes and then re-run, no significant change resulted, indicating
the absence of particle dissolution over the course of the
experiment.
[0089] Samples were analyzed using the experimental parameters
given above. Eight scans were performed and mean particle diameter
data was determined. Two distinct size distributions were noted,
and all had a clean cut-off at the high-end particle size,
indicating the absence of particle aggregation. One lot of
PPI-149-CMC had apparently lower mean volume diameter prior to
gamma irradiation than the sample post-irradiation. This
preformulation study would seem to indicate some particle
consolidation occurred during the sterilization process.
EXAMPLE 12
[0090] In this example, various preformulation experiments were
performed to investigate the effect of both gamma-irradiation and
temperature/humidity stress on the solid state form of
PPI-149-CMC.
[0091] X-Ray Powder Diffraction
[0092] In the initial experiment, two 60 mg samples of PPI-149-CMC
were packed (under an air headspace) in type I glass vials, closed
with gray butyl rubber stoppers and sealed with aluminum seals. One
sample was then exposed to a gamma-irradiation dose of 19.0 KGy.
The solid state form of the two 60 mg Samples was then studied by
X-ray powder diffraction. Diffractograms were compared prior to and
following exposure to a gamma irradiation dose of 19.0 Kgy.
[0093] In a subsequent study, a 60 mg sample of PPI-149-CMC (post
gamma-irradiation) was placed in a type I glass vial and placed in
a pre-equilibrated constant humidity incubator at 50.degree. C./75%
Relative Humidity for 5 days. Immediately after withdrawal from the
incubator, the sample container was closed with a gray butyl rubber
stopper and sealed with an aluminum seal. The X-ray powder
diffractogram of this stressed sample was then compared to another
sample of the same lot that had been held at room temperature in a
closed container. The samples were analyzed using a Siemens D500
automated Powder Diffractometer equipped with a graphite
monochromator and a Cu (.lambda.=1.54 .ANG.) X-Ray source operated
at 50 kV, 40 mA. The two-theta scan range was 4-40.degree. using a
step scan window of 0.05.degree./1.2 second step. Beam slits were
set at No. (1) 1.degree., (2) 1.degree.,(3) 1.degree., (4)
0.15.degree. and (5) 0.15.degree. widths. Two-theta calibration was
performed using an NBS mica standard (SRM 675). The samples were
analyzed using a zero background sample plate.
[0094] The data indicated that prior to gamma irradiation,
PPI-149-CMC had no apparent crystalline or pseudo-crystalline
structure. In fact, it gave an X-ray powder diffraction pattern
characteristic of an amorphous solid (a broad hump between
2-20.degree.20, with no significant peaks in the diffractogram).
The PPI-149-CMC sample post-irradiation generated a very similar
diffraction pattern to the non-irradiated sample, indicating that
gamma-irradiation processing (at doses up to and including 19KGy)
does not apparently induce a solid-state polymorphic transition
within the material. In a similar manner, the temperature/humidity
stressed sample of PPI-149-CMC generated a very similar diffraction
pattern to both the non-irradiated sample and the irradiated
sample, which strongly suggests that PPI-149-CMC is not unduly
prone to induction of solid-state polymorphic transitions within
the material.
[0095] Hygroscopicity
[0096] Preformulation studies on PPI-149-CMC (post-irradiation)
were performed to determine the equilibrium moisture uptake
(measured by weight gain) at constant temperature (25.degree. C.)
under various conditions of relative humidity. Analysis of the
equilibrium moisture (% water) as a function of relative humidity
(% RH) indicated that moisture content gradually increased up to
approximately 80% relative humidity. At high relative humidity (95%
RH) PPI-149-CMC was capable of significant moisture sorption. At
relative humidities at or below 80% RH, significant precautions in
terms of protection from moisture are deemed unnecessary; thus
certain manufacturing steps may be undertaken under ambient
humidity conditions (provided humidity extremes are avoided).
EXAMPLE 13
[0097] In this example, dissolution studies on PPI-149-CMC were
performed. Experiments were performed utilizing both sink and
non-sink conditions. PPI-149-CMC has an approximate solubility of
100 .mu.g/mL (measured and expressed as free peptide) at 25.degree.
C. in 0.1 M phosphate buffered saline at pH 7.3. Under sink
conditions (defined as <10% of the saturated solubility in the
system at a given temperature), even in the absence of stirring,
PPI-149-CMC dissolved rapidly (measured and expressed as free
peptide). In a similar experiment, the equilibrium solubility of
PPI-149-CMC was determined (measured and expressed as free peptide)
at 25.degree. C. in 0.1 M phosphate buffered saline at pH 7.3,
using three samples: PPI-149-CMC alone, PPI-149-CMC in the presence
of 10% additional (by weight) PPI-149 (expressed as free peptide,
but introduced as PPI-149 with associated acetate) and PPI-149-CMC
in the presence of 50% additional (by weight)
Carboxymethylcellulose sodium USP. All three samples gave
ostensibly a similar peptide equilibrium solubility. As the buffer
system selected approximates physiological conditions, the presence
of additional free Carboxymethylcellulose or peptide species
present in PPI-149-CMC seems unlikely to affect solubility.
EXAMPLE 14
[0098] In this example, the pharmacokinetics, pharmacodynamics and
safety of repeated subcutaneous (SC) and intramuscular (IM) doses
of PPI-149-CMC were characterized in dogs.
[0099] In a first study, conducted for three months, forty male
beagle dogs were evaluated, using monthly IM or SC injections of
PPI-149-CMC at 1.2 mg/kg (Day 1), 0.3 or 0.6 mg/kg (Day 29) and 1.2
mg/kg (Day 57) in a variety of reconstitution vehicles. Eight
groups of five dogs were assigned to the study as shown below:
3 Dose.sup.c Route Reconstitution Vehicle.sup.a,b (mg/kg) of Group
N Day 1 Day 29 Day 57 Day 1 Day 29 Day 57 Admin. A.sup.d 5 Saline
Glycerin Lecithin 0 0 0 IM B 5 Glycerin Glycerin Lecithin 1.2 0.3
1.2 IM C 5 Glycerin Glycerin Lecithin 1.2 0.6 1.2 IM D 5 PEG
Glycerin Lecithin 1.2 0.3 1.2 IM E 5 PEG Glycerin Lecithin 1.2 0.3
1.2 SC F.sup.d 5 Lecithin Glycerin Lecithin 1.2 0.6 1.2 IM G.sup.d
5 Lecithin Glycerin Lecithin 1.2 0.6 1.2 SC H 5 Glycerin Glycerin
Lecithin 1.2 0.3 1.2 SC .sup.aReconstitution vehicles are used to
reconstitute PPI-149-CMC as a particular suspension. They contain
the following (in water): 1. Glycerin = 15% glycerin/5% dextrose 2.
PEG = 4% polyethylene glycol-3350/4% mannitol 3. Lecithin = 0.5%
lecithin/5% mannitol .sup.bNote: the reconstitution vehicles to be
used in clinical studies is 0.9% sodium chloride USP .sup.cAll
doses are expressed in terms of peptide (PPI-149) content.
.sup.dThree animals were sacrificed at Day 85 for complete
anatomical and microscopic histology.
[0100] This study was designed such that the efficacy of
PPI-149-CMC at an initial dose in different vehicles was assessed
during the first month of treatment. During the second month
on-study, the dogs received lower doses of PPI-149-CMC in an
attempt to determine an efficacious "maintenance" dose. The third
month was scheduled to evaluate the long term safety and efficacy
characteristics of PPI-149-CMC.
[0101] IM or SC doses of PPI-149-CMC formulated in one of the
reconstitution vehicles, or IM doses of control article, were
administered on each dosing day into the upper flank of the right
hind limb (IM) or in the mid-scapular region (SC). Material was
drawn into a Icc tuberculin syringe with a 23 g short bevel needle.
The injection site was wiped with an alcohol swab immediately prior
to dosing. The volume injected was based on a specific dose of
peptide/kg body weight. It should be noted that all doses refer to
the amount of PPI-149 peptide administered.
[0102] Each animal was observed at least twice daily during the
entire study for overt signs of toxic or pharmacologic effect and
changes in general behavior and appearance. All abnormal clinical
observations were recorded.
[0103] Blood was collected prior to administration of the first
dose and at various times following dosing, for complete blood
counts (CBC), serum chemistry analysis, and determination of
PPI-149 and testosterone concentrations twice weekly by
radioimmunoassays.
[0104] After three months on-study, nine animals were sacrificed
and their tissues collected for gross pathological and
histopathological analysis. Animals were selected for sacrifice
from the vehicle control group, one of the IM dosing groups and one
of the SC dosing groups. The tissues collected for gross pathology
and histopathology at the 3 month sacrifice were: administration
Site (SC or IM), adrenal glands, aorta, bone, bone marrow, brain,
diaphragm, epididymis, esophagus, eyes with optic nerve, heart,
kidneys, large intestine (cecum, colon), liver with gall bladder,
lungs with bronchi, lymph nodes, pancreas, pituitary gland,
prostate gland with urethra, salivary glands, sciatic nerve,
skeletal muscle, skin, small intestine (duodenum, jejunum, ileum),
spinal cord, spleen, stomach, testes, thymus, thryoid gland with
parathyroid, tongue, trachea, urinary bladdr and gross lesions.
[0105] There were no significant changes in hematology or blood
chemistry from baseline during the study for either treated or
control animals. Gross and histological evaluation at the three
month sacrifice showed no apparent differences between PPI-149-CMC
treated dogs and control (vehicle-treated) animals, with the
exception of changes in the testes and prostate, as expected with
this LHRH antagonist.
[0106] Regarding PPI-149-CMC pharmacokinetics, all dogs treated
with 1.2 mg/kg PPI-149-CMC resuspended in a variety of
reconstitution vehicles and administered IM or SC showed similar
plasma PPI-149 pharmacokinetic profiles, with plasma concentration
peaking within the first 2 days and then decreasing slowly in an
exponential manner over the following month. PPI-149-CMC gave
similar plasma distribution of PPI-149 when suspended in any of the
three reconstitution vehicles used in the study.
[0107] Regarding PPI-149-CMC endocrine efficacy, castrate levels of
testosterone (<0.6 ng/mL) were observed within 24 hours of
initiation of PPI-149-CMC dosing in all dogs, and levels generally
remained in the castrate range throughout the first month
regardless of the route of administration or choice of
reconstitution vehicle. Twenty-six (26) of 35 dogs (75%) had
castrate levels of testosterone in a blood sample obtained
immediately prior to administration of the second dose of
PPI-149-CMC on Day 29. These results indicate that an initial dose
of 1.2 mg/kg in dogs successfully induces a rapid, long-lasting
suppression (>28 days) in plasma testosterone. In the second
month of dosing, when the efficacy of a "maintenance" dose (a dose
lower than the initial dose) was investigated, the results
indicated that administration of 0.3 or 0.6 mg/kg of PPI-149-CMC
maintained castrate levels of testosterone for more than 20 days in
30 out of 35 dogs. At the end of the second month of treatment (Day
57), 21 of 35 dogs (60%) remained castrate, while 14 animals had
testosterone in the normal range (>0.6% ng/mL). A dose of 1.2
mg/kg was administered in the beginning of the third month. Plasma
concentrations of PPI-149 were sustained for the following
twenty-eight day period while plasma levels of testosterone were
again "castrate." By the end of the third month (Day 85), plasma
levels of testosterone were shown to be in the castrate range in 30
of 35 PPI-149-CMC-treated dogs.
[0108] In summary, thirty-five (35) dogs received 1.2 mg/kg
PPI-149-CMC on Day 1, 0.3 or 0.6 mg/kg PPI-149-CMC on Day 29 and
1.2 mg/kg PPI-149-CMC on Day 57, using IM or SC dosing with a
variety of reconstitution vehicles. Of these 35 dogs, 19 animals
(54%) had plasma testosterone levels which remained in the castrate
range throughout the entire course of therapy. Thus, administration
of PPI-149-CMC at 28 day intervals was able to result in complete
suppression of plasma testosterone which is rapid (all animals had
castrate levels within 24 hours) and long-lasting (maintained
throughout the course of administration).
[0109] A similar study to that described above was conducted for
six months in dogs to further evaluate the long term safety and
efficacy characteristics of PPI-149-CMC. Animals received an
initial dose of 1.2 mg/kg PPI-149-CMC either IM or SC and five
subsequent doses (at a concentration of either 0.3 mg/kg, 0.6 mg/kg
or 1.2 mg/kg) at 28 day intervals. Plasma testosterone and PPI-149
levels were evaluated by radioimmunoassay at regular intervals.
Representative results are shown in FIG. 4 (for SC treatment) and
FIG. 5 (for IM treatment), which illustrate plasma testosterone
levels (open boxes) and PPI-149 levels (closed boxes). The
particular dosages used at each administration of PPI-149-CMC are
shown on the graphs. The results illustrated in FIGS. 4 and 5
further demonstrate that administration of PPI-149-CMC at 28 day
intervals was able to result in complete suppression of plasma
testosterone which is rapid and long-lasting, with reduced plasma
testosterone levels being maintained for as long as six months.
EQUIVALENTS
[0110] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *