U.S. patent application number 09/965760 was filed with the patent office on 2002-02-14 for treatment of skeletal disorders.
Invention is credited to Ke, HuaZhu, Steppan, Claire M., Swick, Andrew Gordon.
Application Number | 20020019351 09/965760 |
Document ID | / |
Family ID | 22126118 |
Filed Date | 2002-02-14 |
United States Patent
Application |
20020019351 |
Kind Code |
A1 |
Ke, HuaZhu ; et al. |
February 14, 2002 |
Treatment of skeletal disorders
Abstract
This invention relates to methods for treating bone loss in a
mammal by administering to the mammal a therapeutically effective
amount of leptin or a leptin mimetic. This invention also relates
to methods for treating bone fracture, enhancing bone healing
following facial reconstruction, maxillary reconstruction or
madibular reconstruction, enhancing long bone extension, enhancing
the healing rate of a bone graft, enhancing prosthetic growth and
inducing vertebral synostosis by administering a therapeutically
effective amount of leptin or a leptin mimetic. This invention
further relates to methods and compositions comprising leptin or a
leptin mimetic and estrogen, a selective estrogen receptor
modulator or a bisphonate for treating the above-recited diseases
and conditions.
Inventors: |
Ke, HuaZhu; (Ledyard,
CT) ; Steppan, Claire M.; (New London, CT) ;
Swick, Andrew Gordon; (East Lyme, CT) |
Correspondence
Address: |
Gregg C. Benson
Pfizer Inc.
Patent Department, MS 4159
Eastern Point Road
Groton
CT
06340
US
|
Family ID: |
22126118 |
Appl. No.: |
09/965760 |
Filed: |
September 27, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09965760 |
Sep 27, 2001 |
|
|
|
09253329 |
Feb 19, 1999 |
|
|
|
60075491 |
Feb 23, 1998 |
|
|
|
Current U.S.
Class: |
514/5.8 ;
514/16.9 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/2264 20130101; A61P 1/00 20180101; A61P 19/08 20180101;
A61P 19/10 20180101; A61K 38/2264 20130101; A61P 19/00 20180101;
A61P 1/02 20180101; A61K 2300/00 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Claims
1. A method for preventing bone loss or augmenting bone mass in a
mammal comprising administering to said mammal a therapeutically
effective amount of leptin, a fragment thereof or a leptin
mimetic.
2. A method of claim 1 wherein leptin or a fragment thereof is
administered.
3. A method for treating a mammal having a condition which presents
with low bone mass comprising administering to said mammal a
therapeutically effective amount of leptin, a fragment thereof or a
leptin mimetic.
4. A method of claim 3 wherein leptin or a fragment thereof is
adminstered.
5. The method of claim 4 wherein osteoporosis, osteotomy, childhood
idiopathic bone loss or bone loss associated with periodontitis is
treated.
6. The method of claim 5 wherein osteoporosis is treated.
7. The method of claim 4 wherein the mammal is a human.
8. The method of claim 7 wherein the human is a post-menopausal
woman or a man over the age of 60.
9. The method of claim 4 wherein secondary osteoporosis is
treated.
10. The method of claim 9 wherein glucocorticoid-induced
osteoporosis, hyperthyroidism-induced osteoporosis,
immobilization-induced osteoporosis, heparin-induced osteoporosis
or immunosuppressive-induced osteoporosis is treated.
11. A method for treating a bone fracture in a mammal by
administering to said mammal suffering from or susceptible to a
bone fracture a therapeutically effective amount of leptin, a
fragment thereof or a leptin mimetic.
12. A method of claim 11 wherein leptin or a fragment thereof is
administered.
13. The method of claim 12 wherein the mammal is a human.
14. A method for enhancing bone healing following facial
reconstruction, maxillary reconstruction or mandibular
reconstruction, enhancing long bone extension, enhancing the
healing rate of a bone graft, enhancing prosthetic ingrowth, or
inducing vertebral synostosis in a mammal by administering to said
mammal a therapeutically effective amount of leptin, a fragment
thereof or a leptin mimetic.
15. A method for treating low bone mass or bone fracture in a
mammal comprising administering to said mammal a therapeutically
effective amount of leptin, a fragment thereof or a leptin mimetic,
and estrogen.
16. A method for treating low bone mass or bone fracture in a
mammal comprising administering to said mammal a therapeutically
effective amount of leptin, a fragment thereof or a leptin mimetic,
and a selective estrogen receptor modulator.
17. A method of claim 16 wherein leptin or a fragment thereof is
adminstered.
18. A method of claim 17 wherein said selective estrogen receptor
modulator is droloxifene.
19. A method of claim 17 wherein said selective estrogen receptor
modulator is raloxifene.
20. A method of claim 17 wherein said selective estrogen receptor
modulator is idoxifene.
21. A method of claim 17 wherein said selective estrogen receptor
modulator is
(-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl)-ethoxy)-phenyl)-5,-
6,7,8-tetrahydronaphthalen-2-ol.
22. A method for treating low bone mass or bone fracture in a
mammal comprising administering to said mammal a therapeutically
effective amount of leptin, a fragment thereof or a leptin mimetic,
and a bisphosphonate.
23. A method of claim 22 wherein leptin or a fragment thereof is
administered.
24. A method of claim 23 wherein said bisphosphonate is
alendronate.
25. A method of claim 23 wherein said bisphosphonate is
risedronate.
26. A method for enhancing bone healing following facial
reconstruction, maxillary reconstruction or mandibular
reconstruction, enhancing long bone extension, enhancing the
healing rate of a bone graft, enhancing prosthetic ingrowth or
inducing vertebral synostosis in a mammal comprising administering
to said mammal a therapeutically effective amount of leptin, a
fragment thereof or a leptin mimetic, and estrogen.
27. A method of claim 26 wherein leptin or a fragment thereof is
adminstered.
28. A method for enhancing bone healing following facial
reconstruction, maxillary reconstruction or mandibular
reconstruction, enhancing long bone extension, enhancing the
healing rate of a bone graft, enhancing prosthetic ingrowth or
inducing vertebral synostosis in a mammal comprising administering
to said mammal a therapeutically effective amount of leptin, a
fragment thereof or a leptin mimetic, and a selective estrogen
receptor modulator.
29. A method of claim 28 wherein leptin or a fragment thereof is
adminstered.
30. A method for enhancing bone healing following facial
reconstruction, maxillary reconstruction or mandibular
reconstruction, enhancing long bone extension, enhancing the
healing rate of a bone graft, enhancing prosthetic ingrowth or
inducing vertebral synostosis in a mammal comprising administering
to said mammal a therapeutically effective amount of leptin, a
fragment thereof or a leptin mimetic, and a bisphosphonate.
31. A method of claim 30 wherein leptin or a fragment thereof is
administered.
32. A pharmaceutical composition comprising a leptin mimetic and a
pharmaceutically acceptable carrier or diluent.
33. A pharmaceutical composition comprising leptin, a fragment
thereof or a leptin mimetic, estrogen and a pharmaceutically
acceptable carrier or diluent.
34. A composition of claim 33 comprising leptin or a fragment
thereof.
35. A pharmaceutical composition comprising leptin, a fragment
thereof or a leptin mimetic, a selective estrogen receptor
modulator and a pharmaceutically acceptable carrier or diluent.
36. A composition of claim 35 comprising leptin or a fragment
thereof.
37. A pharmaceutical composition comprising leptin, a fragment
thereof or a leptin mimetic, a bisphosphonate and a
pharmaceutically acceptable carrier or diluent.
38. A composition of claim 37 comprising leptin or a fragment
thereof.
39. A kit comprising: a. an amount of leptin, a fragment thereof or
a leptin mimetic in a first unit dosage form; b. an amount of
estrogen in a second unit dosage form; and c. a container.
40. A kit of claim 39 comprising leptin or a fragment thereof.
41. A kit comprising: a. an amount of leptin, a fragment thereof or
a leptin mimetic in a first unit dosage form; b. an amount of a
selective estrogen receptor modulator in a second unit dosage form;
and c. a container.
42. A kit of claim 41 comprising leptin or a fragment thereof.
43. A kit comprising: a. an amount of leptin, a fragment thereof or
a leptin mimetic in a first unit dosage form; b. a bisphonate in a
second unit dosage form; and c. a container.
44. A kit of claim 43 comprising leptin or a fragment thereof.
Description
BACKGROUND OF INVENTION
[0001] This invention relates to the use of leptin and leptin
mimetics to augment bone mass including the prevention and
treatment of skeletal disorders such as osteoporosis in
vertebrates, e.g., mammals, including humans.
[0002] Osteoporosis is a systemic skeletal disorder, characterized
by low bone mass and deterioration of bone tissue, with a
consequent increase in bone fragility and susceptibility to
fracture. In the U.S., the condition affects more than 25 million
people and causes more than 1.3 million fractures each year,
including 500,000 spine, 250,000 hip and 240,000 wrist fractures
annually. Hip fractures are the most serious, with 5-20% of
patients dying within one year, and over 50% of survivors being
incapacitated.
[0003] The elderly are at greatest risk of osteoporosis, and the
problem is therefore predicted to increase significantly with the
aging of the population. Worldwide fracture incidence is forecast
to increase three-fold over the next 60 years, and one study
estimates that there will be 4.5 million hip fractures worldwide in
2050.
[0004] Women are at greater risk of osteoporosis than men. Women
experience a sharp acceleration of bone loss during the five years
following menopause. Other factors that increase the risk include
smoking, alcohol abuse, a sedentary lifestyle and low calcium
intake.
[0005] In addition to hip fractures numbering approximately
250,000/year in the U.S., approximately, 20-25 million women and an
increasing number of men have detectable vertebral fractures. Hip
fracture is associated with a 12% mortality rate within the first
two years and with a 30% rate of patients requiring nursing home
care after the fracture. While this is already significant, the
economic and medical consequences of convalescence due to slow or
imperfect healing of these bone fractures is expected to increase,
due to the aging of the general population.
[0006] There are currently two main types of pharmaceutical therapy
for the treatment of osteoporosis and skeletal fractures. The first
is the use of anti-resorptive compounds to inhibit the resorption
of bone tissue and therefore prevent bone loss and reduce the
incidence of skeletal fractures.
[0007] Estrogen is an example of an anti-resorptive agent. It is
known that estrogen prevents post-menopausal bone loss and reduces
skeletal fractures. However, estrogen fails to restore bone to the
established osteoporotic skeleton. Furthermore, long-term estrogen
therapy, however, has been implicated in a variety of disorders,
including an increase in the risk of uterine cancer, endometrial
cancer and possibly breast cancer, causing many women to avoid this
treatment. The significant undesirable effects associated with
estrogen therapy support the need to develop alternative therapies
for osteoporosis.
[0008] A second type of pharmaceutical therapy for the treatment of
osteoporosis and bone fractures is the use of anabolic agents to
promote bone formation and increase bone mass. This class of agents
is expected to restore bone to the established osteoporotic
skeleton by stimulating osteoblastic bone formation. Currently,
such pharmaceutical therapy is not available for established
osteoporotic patents.
[0009] Leptin, a product of the obese gene, is a 16 kDa protein.
Leptin is produced by mature adipocytes and is secreted in plasma.
Leptin has been reported to increase lean body mass (Friedman et
al., UK Patent Application No. GB 2292382 and Pelleymounter et al.,
International Patent Application Publication Number WO97/18833) and
decrease fat body mass (Halaas et al., Science 269:543-546, 1997).
Further, a leptin receptor, OB-R, has been identified and cloned
(Tartaglia et al., Cell 83:1263-1271, 1995). Further, leptin has
been disclosed to stimulate cortical bone formation in ob/ob mice
(Liu et al., Americal Society for Bone and Mineral Research, 19th
Annual Meeting, Sep. 10-14, 1997, Cincinati, Ohio).
[0010] Skeletal disorders are highly prevalent diseases caused by
nutrition deficiency, sex steroid deficiency, aging, trauma or
other factors. All approved therapies and clinically advanced
candidates including calcitonin, estrogen replacement therapy,
bisphosphonates and tissue selective estrogen agonists act to
prevent bone loss by inhibiting bone resorption, but these agents
cannot restore bone mass. Thus, there is significant medical need
for agents that would increase bone mass and strength above a
critical threshold in established osteoporotic patients, fractured
patients, and other skeletal disorder patients.
SUMMARY OF THE INVENTION
[0011] This invention is directed to methods for augmenting bone
mass and preventing bone loss in a vertebrate, e.g., a mammal
(including humans) comprising administering to said vertebrate,
e.g., a mammal, a therapeutically effective amount of leptin or a
leptin mimetic.
[0012] This invention is also directed to methods for treating a
vertebrate, e.g. a mammal (including a human being) having a
condition which presents with low bone mass comprising
administering to said vertebrate, e.g., mammal, having a condition
which presents with low bone mass a therapeutically effective
amount of leptin or a leptin mimetic.
[0013] Yet another aspect of this invention is directed to methods
for treating osteoporosis, bone fractures, osteotomy, bone loss
associated with periodontitis, prosthetic ingrowth, or inducing
vertebral synostosis in a vertebrate, e.g. a mammal (including a
human being) by administering to said vertebrate, e.g., mammal,
suffering from or susceptible to osteoporosis, bone fractures,
osteotomy, bone loss associated with periodontitis, prosthetic
ingrowth or vertebral synostosis a therapeutically effective amount
of leptin or a leptin mimetic.
[0014] Yet another aspect of this invention is directed to methods
for treating osteoporosis in a vertebrate, e.g., a mammal
(including a human being), by administering to said vertebrate,
e.g., mammal, suffering from or susceptible to osteoporosis a
therapeutically effective amount of a leptin or a leptin
mimetic.
[0015] Yet another aspect of this invention is directed to methods
for treating osteotomy bone loss in a vertebrate, e.g., a mammal
(including a human being), by administering to said vertebrate,
e.g., a mammal, suffering from or susceptible to an osteotomy bone
loss a therapeutically effective amount of leptin or a leptin
mimetic.
[0016] Yet another aspect of this invention is directed to methods
for treating alveolar bone loss in a vertebrate, e.g., a mammal
(including a human being), by administering to said vertebrate,
e.g., mammal, suffering from or susceptible to an alveolar bone
loss a therapeutically effective amount of leptin or a leptin
mimetic.
[0017] Yet another aspect of this invention is directed to methods
for treating bone loss associated with periodontitis in a
vertebrate, e.g., a mammal (including a human being) by
administering to said vertebrate, suffering from or susceptible to
bone loss associated with periodontitis a therapeutically effective
amount of leptin or a leptin mimetic.
[0018] Yet another aspect of this invention is directed to methods
for treating childhood idiopathic bone loss in a child by
administering to a child suffering from or susceptible to childhood
idiopathic bone loss a therapeutically effective amount of leptin
or a leptin mimetic.
[0019] Yet another aspect of this invention is directed to methods
for treating "secondary osteoporosis", which includes
glucocorticoid-induced osteoporosis, hyperthyroidism-induced
osteoporosis, immobilization-induced osteoporosis, heparin-induced
osteoporosis or immunosuppressive-induced osteoporosis in a
vertebrate, e.g., a mammal (including a human being), by
administering to said vertebrate, suffering from or susceptible to
"secondary osteoporosis" a therapeutically effective amount of
leptin or a leptin mimetic.
[0020] Yet another aspect of this invention is directed to methods
for treating glucocorticoid-induced osteoporosis in a vertebrate,
e.g., a mammal (including a human being), by administering to said
vertebrate, suffering from or susceptible to glucocorticoid-induced
osteoporosis a therapeutically effective amount of leptin or a
leptin mimetic.
[0021] Yet another aspect of this invention is directed to methods
for treating hyperthyroidism-induced osteoporosis in a vertebrate,
e.g., a mammal (including a human being), by administering to said
vertebrate, e.g., mammal, suffering from or susceptible to
hyperthyroidism-induced osteoporosis a therapeutically effective
amount of leptin or a leptin mimetic.
[0022] Yet another aspect of this invention is directed to methods
for treating immobilization-induced osteoporosis in a vertebrate,
e.g., a mammal, (including a human being), by administering to said
vertebrate, suffering from or susceptible to immobilization-induced
osteoporosis a therapeutically effective amount of leptin or a
leptin mimetic.
[0023] Yet another aspect of this invention is directed to methods
for treating heparin-induced osteoporosis in a vertebrate, e.g.,
mammal, (including a human being), by administering to said
vertebrate, e.g., a mammal suffering from or susceptible to
heparin-induced osteoporosis a therapeutically effective amount of
leptin or a leptin mimetic.
[0024] Yet another aspect of this invention is directed to methods
for treating immunosuppressive-induced osteoporosis in a
vertebrate, e.g., a mammal (including a human being), by
administering to said vertebrate, e.g., mammal, suffering from or
susceptible to immunosuppressive-induced osteoporosis a
therapeutically effective amount of a leptin or a leptin
mimetic.
[0025] Yet another aspect of this invention is directed to methods
for enhancing bone fracture healing in a vertebrate, e.g., a mammal
(including a human being), by administering to said vertebrate,
suffering from or susceptible to a bone fracture a therapeutically
effective amount of a leptin or leptin mimetic. In one aspect of
this invention, leptin or a leptin mimetic is applied locally to
the site of bone fracture.
[0026] Yet another aspect of this invention is directed to methods
for enhancing bone healing following facial reconstruction or
maxillary reconstruction or mandibular reconstruction in a
vertebrate, e.g., a mammal (including a human being), by
administering to said vertebrate, which has undergone facial
reconstruction or maxillary reconstruction or mandibular
reconstruction a therapeutically effective amount of leptin or a
leptin mimetic. In one aspect of this invention, leptin or a leptin
mimetic is applied locally to the site of bone reconstruction.
[0027] Yet another aspect of this invention is directed to methods
for enhancing prosthetic ingrowth in a vertebrate, e.g., a mammal
(including a human being), by administering to said vertebrate, in
need of enhancing prosthetic ingrowth a therapeutically effective
amount of leptin or a leptin mimetic.
[0028] Yet another aspect of this invention is directed to methods
for inducing vertebral synostosis in a vertebrate, e.g., a mammal
(including a human being), by administering to said vertebrate,
undergoing surgery for vertebral synostosis a therapeutically
effective amount of leptin or a leptin mimetic.
[0029] Yet another aspect of this invention is directed to methods
for enhancing long bone extension in a vertebrate, e.g.,, a mammal
(including a human being), by administering to said vertebrate,
suffering from or susceptible to an insufficiently sized long bone
a therapeutically effective amount of leptin or a leptin
mimetic.
[0030] Yet another aspect of this invention is directed to methods
for treating a bone graft in a vertebrate, e.g., a mammal
(including a human being), by administering to said vertebrate,
e.g., a mammal, suffering from or susceptible to a bone graft a
therapeutically effective amount of leptin or a leptin mimetic. In
one aspect of this invention, leptin or a leptin mimetic is applied
locally to the site of the bone graft.
[0031] Yet another aspect of this invention is directed to methods
for treating low bone mass or bone fracture in a vertebrate, e.g.,
a mammal (including a human being), comprising administering to
said vertebrate, a therapeutically effective amount of leptin or a
leptin mimetic, and estrogen.
[0032] Yet another aspect of this invention is directed to methods
for treating low bone mass or bone fracture in a vertebrate, e.g.,
a mammal (including a human being), comprising administering to
said vertebrate, a therapeutically effective amount of a leptin or
a leptin mimetic, and a selective estrogen receptor modulator.
[0033] Preferred selective estrogen receptor modulators include
droloxifene, raloxifene, tamoxifen; 4-hydroxy-tamoxifen;
toremifene; centchroman; levormeloxifene; idoxifene;
6-(4-hydroxy-phenyl)-5-(4-(2-pip-
eridin-1-yl-ethoxy)benzyl)-naphthalen-2-ol;
(4-(2-(2-aza-bicyclo[2.2.1]hep-
t-2-yl)-ethoxy)-phenyl)-(6-hydroxy-2-(4-hydroxy-phenyl)-benzo[b]thiophen-3-
-yl)-methanone;
[0034] 3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid;
[0035]
2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]benzo[b-
]thiophen-6-ol;
[0036]
cis-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,-
7,8-tetrahydro-naphthalene-2-ol;
[0037]
(-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-te-
trahydronaphthalene-2-ol;
[0038]
cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrah-
ydronaphthalene-2-ol;
[0039]
cis-1-(6'-pyrrolodinoethoxy-3'-pyridyl)-2-phenyl-6-hydroxy-1,2,3,4--
tetrahydro-naphthalene;
[0040]
1-(4'-pyrrolidinoethoxyphenyl)-2-(4"-fluorophenyl)-6-hydroxy-1,2,3,-
4-tetrahydroisoquinoline;
[0041] cis-6-(4-hydroxyphenyl)-5-(4-(2-piperidin-1-yl-ethoxy)
-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
[0042]
1-(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahyd-
roisoquinoline; and the pharmaceutically acceptable salts
thereof.
[0043] Especially preferred selective estrogen receptor modulators
include raloxifene; droloxifene; idoxifene;
[0044] 3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid;
[0045]
2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]benzo[b-
]thiophen-6-ol;
[0046] cis-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)
-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
[0047]
(-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-te-
trahydro-naphthalene-2-ol;
[0048]
cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrah-
ydro-naphthalene-2-ol;
[0049]
cis-1-(6'-pyrrolodinoethoxy-3'-pyridyl)-2-phenyl-6-hydroxy-1,2,3,4--
tetrahydro-naphthalene;
[0050]
1-(4'-pyrrolidinoethoxyphenyl)-2-(4"-fluorophenyl)-6-hydroxy-1,2,3,-
4-tetrahydroisoquinoline;
[0051] cis-6-(4-hydroxyphenyl)-5-(4-(2-piperidin-1-yl-ethoxy)
-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
[0052]
1-(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahyd-
roisoquinoline; and the pharmaceutically acceptable salts
thereof.
[0053] Still more especially preferred selective estrogen receptor
modulators include raloxifene, droloxifene, idoxifene and
(-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)
phenyl)-5,6,7,8-tetrahyd- ro-naphthalen-2-ol.
[0054] Yet another aspect of this invention is directed to methods
for treating low bone mass or bone fracture in a vertebrate, e.g.,
a mammal (including a human being), comprising administering to
said vertebrate, a therapeutically effective amount of leptin or a
leptin mimetic, and a bisphosphonate.
[0055] Preferred bisphosphonates include tiludronic acid,
alendronic acid, zoledronic acid, ibandronic acid, risedronic acid,
etidronic acid, clodronic acid, and pamidronic acid and their
pharmaceutically acceptable salts. Those skilled in the art will
know that these compounds are often referred to as their ion form,
e.g., tiludronate, alendronate, zoledronate, ibandronate,
risedronate, etidronate, clodronate and pamidronate.
[0056] Especially preferred bisphosphonates include alendronate and
risedroate.
[0057] Yet another aspect of this invention is directed to methods
for enhancing bone healing following facial reconstruction,
maxillary reconstruction, mandibular reconstruction, enhancing long
bone extension, enhancing the healing rate of a bone graft,
enhancing prosthetic ingrowth or inducing vertebral synostosis in a
vertebrate, e.g., a mammal (including a human being), comprising
administering to said vertebrate, a therapeutically effective
amount of leptin or a leptin mimetic, and estrogen.
[0058] Yet another aspect of this invention is directed to methods
for enhancing bone healing following facial reconstruction,
maxillary reconstruction, mandibular reconstruction, enhancing long
bone extension, enhancing the healing rate of a bone graft,
enhancing prosthetic ingrowth or inducing vertebral synostosis in a
vertebrate, e.g., a mammal (including a human being), comprising
administering to said vertebrate, a therapeutically effective
amount of leptin or a leptin mimetic, and a selective estrogen
receptor modulator.
[0059] Yet another aspect of this invention is directed to methods
for enhancing bone healing following facial reconstruction,
maxillary reconstruction, mandibular reconstruction, enhancing long
bone extension, enhancing the healing rate of a bone graft,
enhancing prosthetic ingrowth or inducing vertebral synostosis in a
vertebrate, e.g., a mammal (including a human being), comprising
administering to said vertebrate, a therapeutically effective
amount of leptin or a leptin mimetic, and a bisphosphonate.
[0060] Yet another aspect of this invention is directed to a
pharmaceutical composition comprising a leptin mimetic and a
pharmaceutically acceptable carrier or diluent.
[0061] Yet another aspect of this invention is directed to a
pharmaceutical composition comprising leptin or a leptin mimetic,
estrogen and a pharmaceutically acceptable carrier or diluent.
[0062] Yet another aspect of this invention is directed to a
pharmaceutical composition comprising leptin or a leptin mimetic, a
selective estrogen receptor modulator; and a pharmaceutically
acceptable carrier or diluent.
[0063] Yet another aspect of this invention is directed to a
pharmaceutical composition comprising leptin or a leptin mimetic, a
bisphosphonate; and a pharmaceutically acceptable carrier or
diluent.
[0064] Yet another aspect of this invention is directed to a kit
comprising:
[0065] a. leptin or a leptin mimetic in a first unit dosage
form;
[0066] b. estrogen in a second unit dosage form; and
[0067] c. a container.
[0068] Yet another aspect of this invention is directed to a kit
comprising:
[0069] a. leptin or a leptin mimetic in a first unit dosage
form;
[0070] b. a selective estrogen receptor modulator in a second unit
dosage form; and
[0071] c. a container.
[0072] Yet another aspect of this invention is directed to a kit
comprising:
[0073] a. leptin or a leptin mimetic in a first unit dosage
form;
[0074] b. a bisphosphonate in a second unit dosage form; and
[0075] c. a container.
[0076] Preferably post-menopausal women and men over the age of 60
are treated.
[0077] Leptin and fragments thereof are particularly preferred
agents in the pharmaceutical compositions and methods of this
invention. Leptin is most particularly preferred.
[0078] A preferred dosage is about 0.0001 to 1000 mg/kg/day of
leptin or a leptin mimetic. An especially preferred dosage is about
0.001 to 100 mg/kg/day of leptin or a leptin mimetic.
[0079] The phrase "condition(s) which presents with low bone mass"
refers to a condition where the level of bone mass is below the age
specific normal as defined in standards by the World Health
Organization "Assessment of Fracture Risk and its Application to
Screening for Postmenopausal Osteoporosis (1994). Report of a World
Health Organization Study Group. World Health Organization
Technical Series 843". Included in "condition(s) which presents
with low bone mass" are primary and secondary osteoporosis.
Secondary osteoporosis includes glucocorticoid-induced
osteoporosis, hyperthyroidism-induced osteoporosis,
immobilization-induced osteoporosis, heparin-induced osteoporosis
and immunosuppressive-induced osteoporosis. Also included is
periodontal disease, alveolar bone loss, osteotomy bone loss and
childhood idiopathic bone loss. The "condition(s) which presents
with low bone mass" also includes long term complications of
osteoporosis such as curvature of the spine, loss of height and
prosthetic surgery.
[0080] The phrase "condition which presents with low bone mass"
also refers to a mammal known to have a significantly higher than
average chance of developing such diseases as are described above
including osteoporosis (e.g., post-menopausal women, men over the
age of 60.
[0081] Those skilled in the art will recognize that the term bone
mass actually refers to bone mass per unit area which is sometimes
(although not strictly correctly) referred to as bone mineral
density.
[0082] The term "treating", "treat" or "treatment" as used herein
includes preventative (e.g., prophylactic), palliative and curative
treatment.
[0083] The term "leptin mimetic" as used herein means any ligands
including recombinant products and small molecules which can bind
to and/or activate the leptin receptor (OB-R) and act as receptor
agonists. Methods for the recombinant production of leptin have
been described by Friedman et al., UK Patent Application No.
2292382.
[0084] A fragment of leptin or a leptin fragment is any active
portion of the leptin molecule.
[0085] By "pharmaceutically acceptable" it is meant the carrier,
diluent, excipients, and/or salt must be compatible with the other
ingredients of the formulation, and not deleterious to the
recipient thereof.
[0086] The expression "pharmaceutically-acceptable salt" refers to
nontoxic anionic salts containing anions such as (but not limited
to) chloride, bromide, iodide, sulfate, bisulfate, phosphate,
acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate,
gluconate, methanesulfonate and 4-toluene-sulfonate. The expression
also refers to nontoxic cationic salts such as (but not limited to)
sodium, potassium, calcium, magnesium, ammonium or protonated
benzathine (N,N'-dibenzylethylenediamine), choline, ethanolamine,
diethanolamine, ethylenediamine, meglamine (N-methylglucamine),
benethamine (N-benzylphenethylamine), piperazine or tromethamine
(2amino-2-hydroxymethyl-1,3-propanediol).
[0087] The term "mammal" is meant to include both companion animals
and humans. The phrase "companion animal" refers to a household pet
or other domesticated animal such as, but not limited to, cattle,
sheep, ferrets, swine, horses, poultry, fish, rabbits, goats, dogs,
cats and the like. Particularly preferred companion animals are
dogs and cats.
[0088] The methods of this invention result in bone formation
resulting in decreased fracture rates. This invention makes a
significant contribution to the art by providing compounds and
methods that increase bone formation resulting in prevention,
retardation, and/or regression of osteoporosis and related bone
disorders.
[0089] Other features and advantages of this invention will be
apparent from the specification and appendant claims which describe
the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0090] Leptin is a 16 kiloDalton peptide hormone produced by
adipose tissue. Leptin can be prepared according to the methods
disclosed in Murakami et al., Biochem Biophys Res Com. 209:944-952.
Further, the mouse and human OB genes can be cloned as described in
Friedman et al., UK Patent Application No. 2292382 and Zhang et
al., Nature, 1994, 372:425431. Leptin receptor, OB-R, can be can be
cloned as described in Tartaglia et al., Cell, 1995,
83:1263-1271.
[0091] Certain leptin mimetics and leptin fragments may be prepared
as described in International Patent Application Publication No.
WO97/46585, which is incorporated herein by reference.
[0092] Many of the compounds of this invention are acidic and they
form a salt with a pharmaceutically acceptable cation. Some of the
compounds of this invention are basic and they form a salt with a
pharmaceutically acceptable anion. All such salts are within the
scope of this invention and they can be prepared by conventional
methods. For example, they can be prepared simply by contacting the
acidic and basic entities, usually in a stoichiometric ratio, in
either an aqueous, non-aqueous or partially aqueous medium, as
appropriate. The salts are recovered either by filtration, by
precipitation with a non-solvent followed by filtration, by
evaporation of the solvent, or, in the case of aqueous solutions,
by lyophilization, as appropriate.
[0093] The compounds of this invention, prodrugs thereof and
pharmaceutically acceptable salts of said compounds and prodrugs
are all adapted to therapeutic use as agents that stimulate bone
formation and increase bone mass in a vertebrates, e.g., mammals,
and particularly humans. Since bone formation is closely related to
the development of osteoporosis and bone related disorders, these
compounds, prodrugs thereof and pharmaceutically acceptable salts
of said compounds and said prodrugs, by virtue of their action on
bone, prevent, arrest and/or regress osteoporosis.
[0094] The compounds of this invention may be combined with a
mammalian selective estrogen receptor modulator. Any selective
estrogen receptor modulator may be used as the second compound of
this invention. The term selective estrogen receptor modulator
refers to compounds which bind with the estrogen receptor, inhibit
bone turnover and/or prevent bone loss. A variety of these
compounds are described and referenced below. The disclosure of
each of the U.S. patents listed below is incorporated herein by
reference.
[0095] A preferred selective estrogen receptor modulator is
droloxifene: (phenol,
3-(1(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenyl-1-butenyl)-, (E)-)
and related compounds which are disclosed in U.S. Pat. No.
5,047,431.
[0096] Another preferred selective estrogen receptor modulator is
3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid, which is
disclosed in Willson et al., Endocrinology, 1997, 138,
3901-3911.
[0097] Another preferred selective estrogen receptor modulator is
tamoxifen: (ethanamine, 2-(-4-(1,2-diphenyl-1-butenyl)
phenoxy)-N,N-dimethyl, (Z)-2-,
2-hydroxy-1,2,3-propanetricarboxylate(1:1)- ) and related compounds
which are disclosed in U.S. Pat. No. 4,536,516.
[0098] Another related compound is 4-hydroxy tamoxifen which is
disclosed in U.S. Pat. No 4,623,660.
[0099] A preferred selective estrogen receptor modulator is
raloxifene: (methanone,
(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl)(4-(2-(1
-piperidinyl)ethoxy)phenyl)hydrochloride) which is disclosed in
U.S. Pat. No. 4,418,068.
[0100] Another preferred selective estrogen receptor modulator is
toremifene: (ethanamine,
2-(4-(4-chloro-1,2-diphenyl-1-butenyl)phenoxy)-N- ,N-dimethyl-,
(Z)-, 2hydroxy-1,2,3-propanetricarboxylate (1:1) which is disclosed
in U.S. Pat. No. 4,996,225.
[0101] Another preferred selective estrogen receptor modulator is
centchroman:
1-(2((4-(-methoxy-2,2,dimethyl-3-phenyl-chroman-4-yl)-phenox-
y)-ethyl)-pyrrolidine, which is disclosed in U.S. Pat. No.
3,822,287. Also preferred is levormeloxifene.
[0102] Another preferred selective estrogen receptor modulator is
idoxifene:
(E)-1-(2(4-(1-(4-iodo-phenyl)-2-phenyl-but-1-enyl)-phenoxy)-et-
hyl)-pyrrolidinone, which is disclosed in U.S. Pat. No. 4,839,.
[0103] Another preferred selective estrogen receptor modulator is
2-(4-methoxyphenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]-benzo[b]thiop-
hen-6-ol which is disclosed in U.S. Pat. No. 5,488,058.
[0104] Another preferred selective estrogen receptor modulator is
6-(4-hydroxyphenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-benzyl)-naphthalen-2-o-
l which is disclosed in U.S. Pat. 5,484,795.
[0105] Another preferred selective estrogen receptor modulator is
(4-(2-(2-azabicyclo[2.2.1]hept-2-yl)-ethoxy)-phenyl)-(6-hydroxy-2-(4-hydr-
oxy-phenyl)benzo[b]thiophen-3-yl)-methanone which is disclosed,
along with methods of preparation, in PCT publication no. WO
95/10513 assigned to Pfizer Inc and designating, inter alia, the
United States. The disclosure thereof is incorporated herein by
reference.
[0106] Other preferred selective estrogen receptor modulators
include compounds as described in commonly assigned U.S. Pat. No.
5,552,412, the disclosure of which is incorporated herein by
reference. Especially preferred compounds described therein
are:
[0107] cis-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)
-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
[0108]
(-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-te-
trahydronaphthalene-2-ol;
[0109]
cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrah-
ydro-naphthalene-2-ol;
[0110]
cis-1-(6'-pyrrolodinoethoxy-3'-pyridyl)-2-phenyl-6-hydroxy-1,2,3,4--
tetrahydro-naphthalene;
[0111]
1-(4'-pyrrolidinoethoxyphenyl)-2-(4"-fluorophenyl)-6-hydroxy-1,2,3,-
4-tetrahydroisoquinoline;
[0112]
cis-6-(4-hydroxyphenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,-
7,8-tetrahydro-naphthalene-2-ol; and
[0113]
1-(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahyd-
roisoquinoline.
[0114] Other selective estrogen receptor modulators are described
in U.S. Pat. No. 4,133,814, which discloses derivatives of
2-phenyl-3-aroyl-benzothiophene and
2-phenyl-3-aroylbenzothiophene-1-oxid- e.
[0115] The utility of the compounds of the present invention as
medical agents in the treatment of conditions which present with
low bone mass (e.g., osteoporosis) in a vertebrates, e.g., mammals
(e.g. humans, particularly the female) is demonstrated by the
activity of the compounds of this invention in conventional assays,
including a receptor binding assay, in vivo assays, and a fracture
healing assay. A leptin receptor binding assay is described in
Tartaglia et al., Cell 83:1263-1271, 1995. The in vivo bone
formation assay may be used to determine the activity of the
compounds of this invention. The in vivo anti-resorption assay may
be used to determine the anti-resorptive activity of the compounds
of this invention. The combination and sequential treatment assay
described below is useful for demonstrating the utility of the
combinations of the bone forming agents (e.g., the compounds of
this invention) and anti-resorptive agents (e.g., estrogen
agonists/antagonists) described herein. The fracture healing assays
described below are useful for demonstrating the utility of the
compounds in this invention for enhancing the healing of a bone
fracture. Such assays also provide a means whereby the activities
of the compounds of this invention (or the other anabolic agents
and anti-resorptive agents described herein) can be compared to
each other and with the activities of other known compounds. The
results of these comparisons are useful for determining dosage
levels in a vertebrates, e.g., mammals, including humans, for the
treatment of such diseases.
In Vivo Bone Formation Assay
[0116] The activity of the compounds in this invention in
stimulating bone formation and increasing bone mass can be tested
in intact male or female rats, sex hormone deficient male
(orchidectomy) or female (ovariectomy) rats.
[0117] Male or female rats at different ages (such as 3 months of
age) can be used in the study. The rats are either intact or
castrated (ovariectomized or orchidectomized), and treated p.o.,
s.c. or i.p. with a test compound at different doses (such as 1, 3,
or 10 mg/kg/day) for 30 days. In the castrated rats, treatment is
started at the next day after surgery (for the purpose of
preventing bone loss) or at the time bone loss has already occured
(for the purpose of restoring bone mass). During the study, all
rats are allowed free access to water and a pelleted commercial
diet (Teklad Rodent Diet #8064, Harlan Teklad, Madison, Wis.)
containing 1.46% calcium, 0.99% phosphorus and 4.96 IU/g of Vitamin
D.sub.3. All rats are given subcutaneous injections of 10 mg/kg
calcein on days 12 and 2 before sacrifice. The rats are sacrificed.
The following endpoints are determined:
Femoral Bone Mineral Measurements
[0118] The right femur from each rat is removed at autopsy and
scanned using dual energy X-ray absorptiometry (DXA, QDR 1000/W,
Hologic Inc., Waltham, Mass.) equipped with "Regional High
Resolution Scan" software (Hologic Inc., Waltham, Mass.). The scan
field size is 5.08.times.1.902 cm, resolution is
0.0254.times.0.0127 cm and scan speed is 7.25 mm/second. The
femoral scan images are analyzed and bone area, bone mineral
content (BMC), and bone mineral density (BMD) of whole femora (WF),
distal femoral metaphyses (DFM), femoral shaft (FS), and proximal
femora (PF) are determined.
Tibial Bone Histomorphometric Analyses
[0119] The right tibia is removed at autopsy, dissected free of
muscle, and cut into three parts. The proximal tibia and the tibial
shaft are fixed in 70% ethanol, dehydrated in graded concentrations
of ethanol, defatted in acetone, then embedded in methyl
methacrylate (Eastman Organic Chemicals, Rochester, N.Y.).
[0120] Frontal sections of proximal tibial metaphyses at 4 and 10
.mu.m thickness are cut using a Reichert-Jung Polycut S microtome.
The 4 .mu.m sections are stained with modified Masson's Trichrome
stain while the 10 .mu.m sections remained unstained. One 4 .mu.m
and one 10 .mu.m section from each rat is used for cancellous bone
histomorphometry.
[0121] Cross sections of tibial shaft at 10 .mu.m thickness are cut
using a Reichert-Jung Polycut S microtome. These sections are used
for cortical bone histomorphometric analysis.
[0122] Cancellous bone histomorphometry: A Bioquant OS/2
histomorphometry system (R&M Biometrics, Inc., Nashville,
Tenn.) is used for the static and dynamic histomorphometric
measurements of the secondary spongiosa of the proximal tibial
metaphyses between 1.2 and 3.6 mm distal to the growth
plate-epiphyseal junction. The first 1.2 mm of the tibial
metaphyseal region needs to be omitted in order to restrict
measurements to the secondary spongiosa. The 4 .mu.m sections are
used to determine indices related to bone volume, bone structure,
and bone resorption, while the 10 .mu.m sections are used to
determine indices related to bone formation and bone turnover.
[0123] I) Measurements and calculations related to trabecular bone
volume and structure: (1) Total metaphyseal area (TV, mm.sup.2):
metaphyseal area between 1.2 and 3.6 mm distal to the growth
plate-epiphyseal junction. (2) Trabecular bone area (BV, mm.sup.2):
total area of trabeculae within TV. (3) Trabecular bone perimeter
(BS, mm): the length of total perimeter of trabeculae. (4)
Trabecular bone volume (BV/TV, %): BV/TV.times.100. (5) Trabecular
bone number (TBN, #/mm): 1.199/2.times.BS/TV. (6) Trabecular bone
thickness (TBT, .mu.m): (2000/1.199).times.(BV/BS). (7m) Trabecular
bone separation (TBS, .mu.m): (2000.times.1.199).mu.(TV-BV).
[0124] II) Measurements and calculations related to bone
resorption: (1) Osteoclast number (OCN,#): total number of
osteoclast within total metaphyseal area. (2) Osteoclast perimeter
(OCP, mm): length of trabecular perimeter covered by osteoclast.
(3) Osteoclast number/mm (OCN/mm, #/mm): OCN/BS. (4) Percent
osteoclast perimeter (%OCP, %): OCP/BS.times.100.
[0125] III) Measurements and calculations related to bone formation
and turnover: (1) Single-calcein labeled perimeter (SLS, mm): total
length of trabecular perimeter labeled with one calcein label. (2)
Double-calcein labeled perimeter (DLS, mm): total length of
trabecular perimeter labeled with two calcein labels. (3)
Inter-labeled width (ILW, .mu.m): average distance between two
calcein labels. (4) Percent mineralizing perimeter (PMS, %):
(SLS/2+DLS)/BS.times.100. (5) Mineral apposition rate (MAR,
.mu.m/day): ILW/label interval. (6) Bone formation rate/surface
ref. (BFR/BS, .mu.m.sup.2/d/.mu.m): (SLS/2+DLS).times.MAR/BS. (7)
Bone turnover rate (BTR,
%/y):(SLS/2+DLS).times.MAR/BV.times.100.
[0126] Cortical bone histomorphometry: A Bioquant OS/2
histomorphometry system (R&M Biometrics, Inc., Nashville,
Tenn.) is used for the static and dynamic histomorphometric
measurements of tibial shaft cortical bone. Total tissue area,
marrow cavity area, periosteal perimeter, endocortical perimeter,
single labeled perimeter, double labeled perimeter, and
interlabeled width on both periosteal and endocortical surface are
measured, and cortical bone area (total tissue area-marrow cavity
area), percent cortical bone area (cortical area/total tissue
area.times.100), percent marrow area (marrow cavity area/total
tissue area.times.100), periosteal and endocortical percent labeled
perimeter [(single labeled perimeter/2+double labeled
perimeter)/total perimeter.times.100], mineral apposition rate
(interlabeled width/intervals), and bone formation rate [mineral
apposition rate x [(single labeled perimeter/2+double labeled
perimeter)/total perimeter] are calculated.
Statistics
[0127] Statistics can be calculated using StatView 4.0 packages
(Abacus Concepts, Inc., Berkeley, Calif.). The analysis of variance
(ANOVA) test followed by Fisher's PLSD (Stat View, Abacus Concepts
Inc., 1918 Bonita Ave, Berkeley, Calif. 94704-1014) is used to
compare the differences between groups.
FRACTURE HEALING ASSAYS
ASSAY FOR EFFECTS ON FRACTURE HEALING AFTER SYSTEMIC
ADMINISTRATION
[0128] Fracture Technique: Sprage-Dawley rats at 3 months of age
are anesthetized with Ketamine. A 1 cm incision is made on the
anteromedial aspect of the proximal part of the right tibia or
femur. The following describes the tibial surgical technique. The
incision is carried through to the bone, and a 1 mm hole is drilled
4 mm proximal to the distal aspect of the tibial tuberosity 2 mm
medial to the anterior ridge. Intramedullary nailing is performed
with a 0.8 mm stainless steel tube (maximum load 36.3 N, maximum
stiffness 61.8 N/mm, tested under the same conditions as the
bones). No reaming of the medullary canal is performed. A
standardized closed fracture is produced 2 mm above the
tibiofibular junction by three-point bending using specially
designed adjustable forceps with blunt jaws. To minimize soft
tissue damage, care is taken not to displace the fracture. The skin
is closed with monofilament nylon sutures. The operation is
performed under sterile conditions. Radiographs of all fractures
are taken immediately after nailing, and rats with fractures
outside the specified diaphyseal area or with displaced nails are
excluded. The remaining animals are divided randomly into the
following groups with 10-12 animals per each subgroup per time
point for testing the fracture healing. The first group receives
daily gavage of vehicle (water: 100% Ethnanol=95:5) at 1 ml/rat,
while the others receive daily gavage from 0.01 to 100 mg/kg/day of
the compound or compounds to be tested (1 ml/rat) for 10,20,40 and
80 days.
[0129] At 10,20,40 and 80 days, 10-12 rats from each group are
anesthetized with Ketamine and sacrificed by exsanguination. Both
tibiofibular bones are removed by dissection and all soft tissue is
stripped. Bones from 5-6 rats for each group are stored in 70%
ethanol for histological analysis, and bones from another 5-6 rats
for each group are stored in Ringer's solution (+4.degree. C., pH
7.4) for radiographs and biomechanical testing which is
performed.
[0130] Histological Analysis: The methods for histologic analysis
of fractured bone have been previously published by Mosekilde and
Bak (The Effects of Growth Hormone on Fracture Healing in Rats: A
Histological Description. Bone, 14:19-27, 1993). Briefly, the
fracture side is sawed 8 mm to each side of the fracture line,
embedded undecalcified in methymethacrylate, and cut frontals
sections on a Reichert-Jung Polycut microtome in 8 .mu.m thick.
Masson-Trichrome stained mid-frontal sections (including both tibia
and fibula) are used for visualization of the cellullar and tissue
response to fracture healing with and without treatment. Sirius red
stained sections are used to demonstrate the characterisitics of
the callus structure and to differentiate between woven bone and
lamellar bone at the fracture site. The following measurements are
performed: (1) fracture gap--measured as the shortest distance
between the cortical bone ends in the fracture, (2) callus length
and callus diameter, (3) total bone volume area of callus, (4) bony
tissue per tissue area inside the callus area, (5) fibrous tissue
in the callus, and (6) cartilage area in the callus.
[0131] Biomechanical Analysis: The methods for biomechanical
analysis have been previously published by Bak and Andreassen (The
Effects of Aging on Fracture Healing in Rats. Calcif Tissue Int
45:292-297, 1989). Briefly, radiographs of all fractures are taken
prior to the biomechanical test. The mechanical properties of the
healing fractures are analyzed by a destructive three- or
four-point bending procedure. Maximum load, stiffness, energy at
maximum load, deflection at maximum load, and maximum stress are
determined.
ASSAY FOR EFFECTS ON FRACTURE HEALING AFTER LOCAL
ADMINISTRATION
[0132] Fracture Technique: Female or male beagle dogs at
approximately 2 years of age are used under anesthesia in the
study. Transverse radial fractures are produced by slow continuous
loading in three-point bending as described by Lenehan et al.
(Lenehan, T. M.; Balligand, M.; Nunamaker, D. M.; Wood, F. E.:
Effects of EHDP on Fracture Healing in Dogs. J. Orthop Res
3:499-507; 1985). The wire is pulled through the fracture site to
ensure complete anatomical disruption of the bone. Thereafter,
local delivery of the compound or compounds being tested to the
fracture site is achieved by slow release of compound(s) delivered
by slow release pellets or by administration of the compound(s) in
a suitable formulation such as a paste gel solution or suspension
for 10, 15, or 20 weeks.
[0133] Histological Analysis: The methods for histologic analysis
of fractured bone have been previously published by Peter et al.
(Peter, C. P.; Cook, W. O.; Nunamaker, D. M.; Provost, M. T.;
Seedor, J. G.; Rodan, G. A. Effects of alendronate on fracture
healing and bone remodeling in dogs. J. Orthop. Res. 14:74-70,
1996) and Mosekilde and Bak (The Effects of Growth Hormone on
Fracture Healing in Rats: A Histological Description. Bone,
14:19-27, 1993). Briefly, after sacrifice, the fracture side is
sawed 3 cm to each side of the fracture line, embedded
undecalcified in methymethacrylate, and cut on a Reichert-Jung
Polycut microtome into 8 .mu.m thick frontal sections.
Masson-Trichrome stained mid-frontal sections (including both tibia
and fibula) are used for visualization of the cellullar and tissue
response to fracture healing with and without treatment. Sirius red
stained sections are used to demonstrate the characterisitics of
the callus structure and to differentiate between woven bone and
lamellar bone at the fracture site. The following measurements are
performed: (1) fracture gap-measured as the shortest distance
between the cortical bone ends in the fracture, (2) callus length
and callus diameter, (3) total bone volume area of callus, (4) bony
tissue per tissue area inside the callus area, (5) fibrous tissue
in the callus and (6) cartilage area in the callus.
[0134] Biomechanical Analysis: The methods for biomechanical
analysis have been previously published by Bak and Andreassen (The
Effects of Aging on Fracture Healing in Rats. Calcif Tissue Int
45:292-297, 1989) and Peter et al. (Peter, C. P.; Cook, W. O.;
Nunamaker, D. M.; Provost, M. T.; Seedor, J. G.; Rodan, G. A.
Effects of Alendronate On Fracture Healing And Bone Remodeling In
Dogs. J. Orthop. Res. 14:74-70, 1996). Briefly, radiographs of all
fractures are taken prior to the biomechanical test. The mechanical
properties of the healing fractures are analyzed by a destructive
three- or four-point bending procedures. Maximum load, stiffness,
energy at maximum load, deflection at maximum load, and maximum
stress are determined.
In Vivo Anti-Resorption Assay
[0135] Anti-resorptive agents are a class of compounds which
inhibit bone turnover and prevent bone loss. The ovariectomized rat
bone loss model has been widely used as a model of postmenopausal
bone loss. Using this model, one can test the efficacy of the
compounds of this invention in preventing bone loss and inhibiting
bone resorption.
[0136] Sprague-Dawley female rats (Charles River, Wilmington,
Mass.) at different ages (such as 5 months of age) are used in
these studies. The rats are singly housed in 20 cm.times.32
cm.times.20 cm cages during the experimental period. All rats are
allowed free access to water and a pelleted commercial diet (Agway
ProLab 3000, Agway County Food, Inc., Syracuse, N.Y.) containing
0.97% calcium, 0.85% phosphorus, and 1.05 IU/g of Vitamin
D.sub.3
[0137] A group of rats (8 to 10) are sham-operated and treated p.o.
with vehicle (10% ethanol and 90% saline, 1 ml/day), while the
remaining rats are bilaterally ovariectomized (OVX) and treated
with either vehicle or with a test compound or compounds of this
invention at different doses (such as 5, 10, or 20 mg/kg, daily
p.o., s.c. or i.p.) for a certain period (such as 4 weeks). All
rats are given subcutaneous injections of 10mg/kg calcein
(fluorochrome bone marker) 12 and 2 days before being sacrificed in
order to examine the dynamic changes in bone tissue. After 4 weeks
of treatment, the rats are sacrificed and autopsied. The following
endpoints are determined:
[0138] Body Weight Gain: Body weight at autopsy minus body weight
at surgery.
[0139] Total Serum Cholesterol: Blood is obtained by cardiac
puncture and allowed to clot at 4.degree. C., and then centrifuged
at 2,000 g for 10 min. Serum samples are analyzed for total serum
cholesterol using a high performance cholesterol calorimetric assay
(Boehringer Mannheim Biochemicals, Indianapolis, Ind.).Femoral Bone
Mineral Measurements: The right femur from each rat is removed at
autopsy and scanned using dual energy X-ray absorptiometry (DEXA,
QDR 1000/W, Hologic Inc., Waltham, Mass.) equipped with "Regional
High Resolution Scan" software (Hologic Inc., Waltham, Mass.). The
scan field size is 5.08.times.1.902 cm, resolution is 0.0254
.times.0.0127 cm and scan speed is 7.25 mm/second. The femoral scan
images are analyzed and bone area, bone mineral content (BMC), and
bone mineral density (BMD) of whole femora (WF), distal femoral
metaphyses (DFM), femoral shaft (FS), and proximal femora (PF) are
determined.
[0140] Proximal Tibial Metaphyseal Cancellous Bone
Histomorphometric Analyses: The right tibia is removed at autopsy,
dissected free of muscle, and cut into three parts. The proximal
tibia is fixed in 70% ethanol, dehydrated in graded concentrations
of ethanol, defatted in acetone, then embedded in methyl
methacrylate (Eastman Organic Chemicals, Rochester, N.Y.). Frontal
sections of proximal tibial metaphyses at 4 and 10 .mu.m thickness
are cut using a Reichert-Jung Polycut S microtome. One 4 .mu.m and
one 10 .mu.m section from each rat is used for cancellous bone
histomorphometry. The 4 .mu.m sections are stained with modified
Masson's Trichrome stain while the 10 .mu.m sections remained
unstained.
[0141] A Bioquant OS/2 histomorphometry system (R&M Biometrics,
Inc., Nashville, Tenn.) is used for the static and dynamic
histomorphometric measurements of the secondary spongiosa of the
proximal tibial metaphyses between 1.2 and 3.6 mm distal to the
growth plate-epiphyseal junction. The first 1.2 mm of the tibial
metaphyseal region is omitted in order to restrict measurements to
the secondary spongiosa. The 4 .mu.m sections are used to determine
indices related to bone volume, bone structure, and bone
resorption, while the 10 .mu.m sections are used to determine
indices related to bone formation and bone turnover.
[0142] I. Measurements and Calculations Related to Trabecular Bone
Volume and Structure
[0143] 1. Total metaphyseal area (TV, mm.sup.2): metaphyseal area
between 1.2 and 3.6 mm distal to the growth plate-epiphyseal
junction.
[0144] 2. Trabecular bone area (BV, mm.sup.2): total area of
trabeculae within TV.
[0145] 3. Trabecular bone perimeter (BS, mm): the length of total
perimeter of trabeculae.
[0146] 4. Trabecular bone volume (BV/TV, %): BV/TV.times.100.
[0147] 5. Trabecular bone number (TBN, #/mm):1.199/2
.times.BS/TV.
[0148] 6. Trabecular bone thickness (TBT,
.mu.m):(2000/1.199).times.(BV/BS- ).
[0149] 7. Trabecular bone separation (TBS,
.mu.m):(2000.times.1.199).times- .(TV-BV).
[0150] II. Measurements and Calculations Related to Bone
Resorption
[0151] 1. Osteoclast number (OCN, #): total number of osteoclast
within total metaphyseal area.
[0152] 2. Osteoclast perimeter (OCP, mm): length of trabecular
perimeter covered by osteoclast.
[0153] 3. Osteoclast number/mm (OCN/mm, #/mm): OCN/BS.
[0154] 4. Percent osteoclast perimeter (%OCP, %):
OCP/BS.times.100.
[0155] III. Measurements and Calculations Related to Bone Formation
and Turnover
[0156] 1. Single-calcein labeled perimeter (SLS, mm): total length
of trabecular perimeter labeled with one calcein label.
[0157] 2. Double-calcein labeled perimeter (DLS, mm): total length
of trabecular perimeter labeled with two calcein labels.
[0158] 3. Inter-labeled width (ILW, .mu.m): average distance
between two calcein labels.
[0159] 4. Percent mineralizing perimeter (PMS,
%):(SLS/2+DLS)/BS.times.100- .
[0160] 5. Mineral apposition rate (MAR, .mu.m/day): ILW/label
interval.
[0161] 6. Bone formation rate/surface ref. (BFR/BS,
.mu.m.sup.2/d/.mu.m):(SLS/2+DLS).times.MAR/BS.
[0162] 7. Bone turnover rate (BTR, %/y):(SLS/2+DLS).times.MAR
BV.times.100.
Statistics
[0163] Statistics are calculated using StatView 4.0 packages
(Abacus Concepts, Inc., Berkeley, Calif.). The analysis of variance
(ANOVA) test followed by Fisher's PLSD (Stat View, Abacus Concepts
Inc. 1918 Bonita Ave, Berkeley, Calif. 94704-1014) is used to
compare the differences between groups.
Combination and Sequential Treatment Assay
[0164] The following protocols can of course be varied by those
skilled in the art. For example, intact male or female rats; sex
hormone deficient male (orchidectomy) or female (ovariectomy) rats
may be used. In addition, male or female rats at different ages
(such as 12 months of age) can be used in the studies. The rats can
be either intact or castrated (ovariectomized or orchidectomized),
and a compound or compounds of this invention are adminstered at
different doses (such as 0.1 to 10 mg/kg/day) for a certain period
(such as two weeks to two months), and followed by administration
of an anti-resorptive agent at different doses (such as 0.1 to 10
mg/kg/day) for a certain period (such as two weeks to two months),
or a combination treatment with both anabolic agent and
anti-resorptive agent at different doses for a certain period (such
as two weeks to two months). In the castrated rats, treatment can
be started on the next day after surgery (for the purpose of
preventing bone loss) or at the time bone loss has already occurred
(for the purpose of restoring bone mass).
[0165] The rats are sacrificed under ketamine anesthesia. The
following endpoints are determined:
[0166] Femoral bone mineral measurements are performed as described
above in the protocol for the in vivo antiresorption assay.
[0167] Lumbar Vertebral Bone Mineral Measurements: Dual energy
X-ray absorptiometry (QDR 1000/W, Hologic, Inc., Waltham, Mass.)
equipped with a "Regional High Resolution Scan" software (Hologic,
Inc., Waltham, Mass.) is used to determined the bone area, bone
mineral content (BMC), and bone mineral density (BMD) of whole
lumbar spine and each of the six lumbar vertebrae (LV1-6) in the
anesthetized rats. The rats are anesthetized by injection (i.p.) of
1 ml/kg of a mixture of ketamine/rompun (ratio of 4 to 3), and then
placed on a rat platform. The scan field sized is 6.times.1.9 cm,
resolution is 0.0254.times.0.0127 cm, and scan speed is 7.25
mm/sec. The whole lumbar spine scan image is obtained and analyzed.
Bone area (BA), and bone mineral content (BMC) is determined, and
bone mineral density is calculated (MBC divided by BA) for the
whole lumbar spine and each of the six lumbar vertebrae
(LV1-6).
[0168] Proximal tibial metaphyseal cancellous bone
histomorphometric analyses are performed as described above above
in the protocol for the in vivo antiresorption assay.
[0169] Measurements and calculations related to trabecular bone
volume and structure, bone resorption, and bone formation and
turnover are performed as described above above in the protocol for
the in vivo antiresorption assay. Further, the data thus obtained
is analyzed using the statistical manipulations described above
above in the protocol for the in vivo antiresorption assay.
[0170] Administration of leptin for four weeks increased femur
length by 6% and increased cortical bone volumetric content by 18%,
as determined by peripheral quantitative computerized tomography.
Bone volumetric density was unchanged, suggesting that
leptin-induced bone was normal. These results show that stature of
the ob/ob mouse is due to a developmental defect and is reversible
upon administration of leptin or a leptin mimetic.
[0171] Administration of the compounds of this invention can be via
any method which delivers a compound of this invention systemically
and/or locally (e.g., at the site of the bone fracture, osteotomy,
or orthopedic surgery. These methods include oral routes,
parenteral routes, intraduodenal routes, etc. Generally, the
compounds of this invention are administered orally, but parenteral
administration (e.g., intravenous, intramuscular, subcutaneous or
intramedullary or on sites of bone fracture) may be utilized, for
example, where oral administration is inappropriate for the instant
target or where the patient is unable to ingest the drug.
[0172] The compounds are used for the treatment and promotion of
healing of bone fractures and osteotomies by the local application
(e.g., to the sites of bone fractures of osteotomies) of the
compounds of this invention or compositions thereof. The compounds
of this invention are applied to the sites of bone fractures or
osteotomies, for example, either by injection of the compound in a
suitable solvent (e.g., an oily solvent such as arachis oil) to the
cartilage growth plate or, in cases of open surgery, by local
application thereto of such compounds in a suitable carrier such as
bone-wax, demineralized bone powder, polymeric bone cements, bone
sealants etc. Alternatively, local application can be achieved by
applying a solution or dispersion of the compound in a suitable
carrier onto the surface of, or incorporating it into solid or
semi-solid implants conventionally used in orthopedic surgery, such
as dacron-mesh, gel-foam and kiel bone, or prostheses.
[0173] Two different compounds of this invention can be
co-administered simultaneously or sequentially in any order, or a
single pharmaceutical composition comprising, for example, leptin
or a leptin mimetic and a second compound as described above in a
pharmaceutically acceptable carrier can be administered.
[0174] In any event the amount and timing of a compound
administered will, of course, be dependent on the subject being
treated, on the severity of the affliction, on the manner of
administration and on the judgement of the prescribing physician.
Thus, because of patient to patient variability, the dosages given
below are a guideline and the physician may titrate doses of the
compound to achieve the treatment (e.g., bone mass augmentation or
bone loss prevention) that the physician considers appropriate for
the patient. In considering the degree of treatment desired, the
physician must balance a variety of factors such as bone mass
starting level, age of the patient, presence of preexisting
disease, as well as presence of-other diseases (e.g.,
cardiovascular).
[0175] In general an amount of a compound or compounds of this
invention is used that is sufficient to augment bone mass to a
level which is above the bone fracture threshold (as detailed in
the World Health Organization Study previously cited herein).
[0176] In general an effective dosage for the leptin or leptin
mimetic described above is in the range of 0.0001 to 1000
mg/kg/day, preferably 0.001 to 100 mg/kg/day.
[0177] The compounds of the present invention are generally
administered in the form of a pharmaceutical composition comprising
at least one of the compounds of this invention together with a
pharmaceutically acceptable carrier or diluent. Thus, the compounds
of this invention can be administered individually or together in
any conventional oral, parenteral, rectal or transdermal dosage
form.
[0178] For oral administration a pharmaceutical composition can
take the form of solutions, suspensions, tablets, pills, capsules,
powders, and the like. Tablets containing various excipients such
as sodium citrate, calcium carbonate and calcium phosphate are
employed along with various disintegrants such as starch and
preferably potato or tapioca starch and certain complex silicates,
together with binding agents such as polyvinylpyrrolidone, sucrose,
gelatin and acacia. Additionally, lubricating agents such as
magnesium stearate, sodium lauryl sulfate and talc are often very
useful for tabletting purposes. Solid compositions of a similar
type are also employed as fillers in soft and hard-filled gelatin
capsules; preferred materials in this connection also include
lactose or milk sugar as well as high molecular weight polyethylene
glycols. When aqueous suspensions and/or elixirs are desired for
oral administration, the compounds of this invention can be
combined with various sweetening agents, flavoring agents, coloring
agents, emulsifying agents and/or suspending agents, as well as
such diluents as water, ethanol, propylene glycol, glycerin and
various like combinations thereof.
[0179] For purposes of parenteral administration, solutions in
sesame or peanut oil or in aqueous propylene glycol can be
employed, as well as sterile aqueous solutions of the corresponding
water-soluble salts. Such aqueous solutions may be suitably
buffered, if necessary, and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal injection purposes. In this
connection, the sterile aqueous media employed are all readily
obtainable by standard techniques well-known to those skilled in
the art.
[0180] For purposes of transdermal (e.g.,topical) administration,
dilute sterile, aqueous or partially aqueous solutions (usually in
about 0.1% to 5% concentration), otherwise similar to the above
parenteral solutions, are prepared.
[0181] Methods of preparing various pharmaceutical compositions
with a certain amount of active ingredient are known, or will be
apparent in light of this disclosure, to those skilled in this art.
For examples of methods of preparing pharmaceutical compositions,
see Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 18th Edition (1990).
[0182] Pharmaceutical compositions according to this invention may
contain 0.1%-95% of the compound(s) of this invention, preferably
1%-70%. In any event, the composition or formulation to be
administered will contain a quantity of a compound(s) according to
this invention in an amount effective to treat the
disease/condition of the subject being treated, e.g., a bone
disorder.
[0183] Since the present invention has an aspect that relates to
the augmentation and maintenance of bone mass by treatment with a
combination of active ingredients which may be administered
separately, the invention also relates to combining separate
pharmaceutical compositions in kit form. The kit comprises two
separate pharmaceutical compositions: leptin or a leptin mimetic, a
prodrug thereof or a pharmaceutically acceptable salt of said
compound or said prodrug and a second compound as described above.
The kit comprises a container for containing the separate
compositions such as a divided bottle or a divided foil packet,
however, the separate compositions may also be contained within a
single, undivided container. Typically the kit comprises directions
for the administration of the separate components. The kit form is
particularly advantageous when the separate components are
preferably administered in different dosage forms (e.g., oral and
parenteral), are administered at different dosage intervals, or
when titration of the individual components of the combination is
desired by the prescribing physician.
[0184] An example of such a kit is a so-called blister pack.
Blister packs are well known in the packaging industry and are
being widely used for the packaging of pharmaceutical unit dosage
forms (tablets, capsules, and the like). Blister packs generally
consist of a sheet of relatively stiff material covered with a foil
of a preferably transparent plastic material. During the packaging
process recesses are formed in the plastic foil. The recesses have
the size and shape of the tablets or capsules to be packed. Next,
the tablets or capsules are placed in the recesses and the sheet of
relatively stiff material is sealed against the plastic foil at the
face of the foil which is opposite from the direction in which the
recesses were formed. As a result, the tablets or capsules are
sealed in the recesses between the plastic foil and the sheet.
Preferably the strength of the sheet is such that the tablets or
capsules can be removed from the blister pack by manually applying
pressure on the recesses whereby an opening is formed in the sheet
at the place of the recess. The tablet or capsule can then be
removed via said opening.
[0185] It may be desirable to provide a memory aid on the kit,
e.g., in the form of numbers next to the tablets or capsules
whereby the numbers correspond with the days of the regimen which
the dosage form so specified should be ingested. Another example of
such a memory aid is a calendar printed on the card e.g., as
follows "First Week, Monday, Tuesday, . . . etc., Second Week,
Monday, Tuesday, . . . " etc. Other variations of memory aids will
be readily apparent. A "daily dose" can be a single tablet or
capsule or several tablets or capsules to be taken on a given day.
Also, a daily dose of leptin or a leptin mimetic, a prodrug thereof
or a pharmaceutically acceptable salt of said compound or said
prodrug can consist of one tablet or capsule while a daily dose of
the second compound can consist of several tablets or capsules and
vice versa. The memory aid should reflect this.
[0186] It should be understood that the invention is not limited to
the particular embodiments described herein, but that various
changes and modifications may be made without departing from the
spirit and scope of this novel concept as defined by the following
claims.
* * * * *