Title: Methods for accelerating bone and cartilage growth and repair
Abstract: The present invention provides improved methods, kits, and compositions for enhancing bone, cartilage and cartilage repair, bone and prosthesis implantation, and attachment and fixation of cartilage and cartilage to bone or other tissues, and chondrocyte proliferation composing the administration of an effective amount of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists.
Patent Number: 6,916,783 Issued on 07/12/2005 to Rodgers, et al.
| Inventors:
|
Rodgers; Kathleen E. (Long Beach, CA);
DiZerega; Gere S. (Pasedena, CA)
|
| Assignee:
|
University of Southern California (Los Angeles, CA)
|
| Appl. No.:
|
772819 |
| Filed:
|
January 30, 2001 |
| Current U.S. Class: |
514/2 |
| Intern'l Class: |
A61K 038/00 |
| Field of Search: |
514/2
|
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| |
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|
Primary Examiner: Tate; Christopher R.
Assistant Examiner: Audet; Maury
Attorney, Agent or Firm: McDonnell Boehnen Hulbert & Berghoff, Harper; David S.
Parent Case Text
CROSS REFERENCE
This application is a continuation of U.S. patent application Ser. No. 09/352,191
filed Jul. 12, 1999 now U.S. Pat. No. 6,258,778, which claims priority from U.S.
Patent Application Ser. Nos. 60/092,653 filed Jul. 13, 1998 and U.S. Pat. No. 60/130,855
filed Apr. 22, 1999.
Claims
1. A method for treating a bone disorder that results in weakened bones, wherein
the bone disorder is selected from the group consisting of osteoporosis, osteoarthritis,
Paget's disease, osteohalisteresis, osteomalacia, periodontal disease, bone loss
resulting from cancer, bone loss resulting from steroid use, and age-related loss
of bone mass, comprising the administration of an amount effective for treating
the bone disorder in a patient in need thereof at least one active agent comprising
a sequence consisting of at least seven contiguous amino acids of groups R
1-R
8
in the sequence of general formula I
wherein
R
1 is selected from H and Asp,
R
2 is selected from Arg and Lys;
R
3 is selected from the group consisting of Val and norLeu;
R
4 is selected from the group consisting of Tyr and homoSer;
R
5 is selected from the group consisting of Ile, Ala, and Leu;
R
6 is His;
R
7 is Pro; and
R
8 is Phe or is absent.
2. The method of claim 1 wherein the active agent consists of an amino acid sequence
selected from the group consisting of SEQ ID NO:18.
3. The method of claim 1 wherein the active agent does not consist of SEQ ID NO:1.
4. The method of claim 1 wherein R8 is absent.
5. The method of claim 1 wherein the bone disorder is osteoporosis.
6. The method of claim 1 wherein the bone disorder is osteoarthritis.
7. The method of claim 1 wherein the bone disorder is Paget's disease.
8. The method of claim 1 wherein the bone disorder is osteohalisteresis.
9. The method of claim 1 wherein the bone disorder is osteomalacia.
10. The method of claim 1 wherein the bone disorder is periodontal disease.
11. The method of claim 1 wherein the bone disorder is bone loss resulting from cancer.
12. The method of claim 1 wherein the bone disorder is age-related loss of bone mass.
Description
FIELD OF THE INVENTION
The present invention relates to methods, compositions, and kits for the repair,
regeneration, and implantation of bone and cartilage.
BACKGROUND OF THE INVENTION
Natural mechanisms of repair, healing and augmentation are similar for bone
and cartilage. (U.S. Pat. No. 5,686,116) Although repair, healing and augmentation
require a complex series of events that are not well defined, it is known that
specific, naturally occurring factors are required to achieve these objectives.
Such factors are released or migrate into the injured area, and stimulate osteoblasts
and chondrocytes and odontoblasts in bone and cartilage to stimulate matrix formation
and remodeling of the wounded area. (ten Dijke et al., Bio/Technology, 7:793-798 (1989))
Living bone tissue is continuously being replenished by the processes of resorption
and deposition of bone matrix and minerals. This temporally and spatially coupled
process, termed bone remodeling, is accomplished largely by two cell populations,
the osteoclasts and osteoblasts. (U.S. Pat. No. 5,656,598, incorporated by reference
herein in its entirety) The remodeling process is initiated when osteoclasts are
recruited from the bone marrow or the circulation to the bone surface and remove
a disk-shaped packet of bone. The bone matrix and mineral is subsequently replaced
by a team of osteoblasts recruited to the resorbed bone surface from the bone marrow.
Osteoblasts are derived from local mesenchymal (stromal) precursors which differentiate
into osteoblasts.
New bone can be formed by three basic mechanisms: osteogenesis, osteoconduction
and osteoinduction. (U.S. Pat. No. 5,464,439 incorporated by reference herein in
its entirety) In osteogenic transplantation, viable osteoblasts and peri-osteoblasts
are moved from one body location to another where they establish centers of bone
formation. Cancellous bone and marrow grafts provide such viable cells. TGF-beta
has been shown to stimulate proliferation and matrix synthesis of osteoblastic
cells (Centrella, et al. (1987) J. Biol. Chem. 262:2869-2874) and to inhibit the
formation and activity of osteoclastic cells (Chenu, et al. (1988) Proc. Natl.
Acad. Sci. U.S.A. 85:683-5687; Kiebzak et al. (1988) J. Bone Min. Res. 3:439-446),
and to stimulate local bone formation in vivo. (Joyce, et al. (1990) J. Cell. Biol.
110.2195-2207; Noda and Camilliere (1989) Endocrinology 124:2991-2294). Other factors
reported to stimulate bone growth include bone morphogenetic proteins (WO 88/00205),
insulin-like growth factor (IGF) (Endocrinol. Metab. 13:E367-72,1986), and parathyroid
hormone (J. Bone & Min. Res. 1:377-381, 1986).
Members of the bone morphogenetic protein family have been shown to be useful
for induction of cartilage and bone formation. For example, BMP-2 has been shown
to be able to induce the formation of new cartilage and/or bone tissue in vivo
in a rat ectopic implant model, see U.S. Pat. No. 5,013,649; in mandibular defects
in dogs, see Toriumi et al., Arch. Otolaryngol Head Neck Surg., 117:1101-1112 (1991);
and in femoral segmental defects in sheep, see Gerhart et al., Trans Orthop Res
Soc, 16:172 (1991). Other members of the BMP family have also been shown to have
osteogenic activity, including BMP-4, -6 and -7 (see Wozney, Bone Morphogenetic
Proteins and Their Gene Expression, in Cellular and Molecular Biology of Bone,
pp. 131-167 (Academic Press, Inc. 1993)). BMP proteins have also been shown to
demonstrate inductive and/or differentiation potentiating activity on a variety
of other tissues, including cartilage. (U.S. Pat. No. 5,700,774, hereby incorporated
by reference in its entirety.
In the transplantation of large segments of cortical bone or allogenic banked
bone, direct osteogenesis does not occur. Rather, osteoconduction occurs wherein
the dead bone acts as a scaffold for the ingrowth of blood vessels, followed by
the resorption of the implant and deposition of new bone. This process is very
slow however, often requiring years to reunite a large segmental defect.
Osteoinduction is the phenotypic conversion of connective tissue into
bone by an appropriate stimulus. As this concept implies, formation of bone can
be induced at even non-skeletal sites. Osteoinduction is preferred over osteoconduction,
as grafts of this type are typically incorporated into the host bone within a two-week
period. In contrast, osteoconductive grafts have been found to be non-incorporated
as long as one year after implantation. In order to provide an environment suitable
for osteoinduction, a material should be selected which is not only capable of
inducing osteogenesis throughout its volume, but is also biocompatible, non-inflammatory,
and possesses the ability to be ultimately resorbed by the body and replaced with
new, natural bone.
Among the pathological conditions associated with abnormal bone cell function
are osteoporosis, osteoarthritis, Paget's disease, osteohalisteresis, osteomalacia,
periodontal disease, bone loss resulting from multiple myeloma and other forms
of cancer, bone loss resulting from side effects of other medical treatment (such
as steroids), and age-related loss of bone mass. Inadequate organic matrix mass
places an individual at risk of skeletal failure such that bone fractures can result
from the minimal trauma of everyday life. Such fractures cause significant illness,
or morbidity, inasmuch as there is insufficient repair or healing of the fractures.
In certain pathologic conditions, osteoclast-mediated resorption is not regulated
by osteoblasts but is driven by cancer cells, infecting organisms or the host's
immune cells. In those disease conditions, resorption of bone far exceeds bone
formation. Such accelerated osteoclastic activity leads to excessive release of
calcium from the inorganic mineral in bone, with a concomitant net loss of skeletal
mass, often with an attendant disturbance in calcium homeostasis in the form of
elevated blood levels of calcium. (U.S. Pat. No. 5,686,116, incorporated by reference
herein in its entirety.)
Although methods for directing new bone formation are known, improved methods
that provide for accelerated bone growth are needed. For example, currently approved
therapeutic agents for osteoporosis are antiresorptives. As such, they are not
as effective in patients with established osteoporosis of either type (decreased
bone density with fractures of the vertebrae and/or hip), or in patients with Type
II osteoporosis. In addition, the most accepted preventive agent for osteoporosis
currently in use is estrogen therapy, which is not an acceptable therapeutic agent
for women with a history of breast cancer or endometrial cancer or for men with osteoporosis.
Similarly, successful implantation and function of bone implants depends
on bonding of the adjacent bone to the implant. (U.S. Pat. No. 5,686,116) Such
bonding requires bone repair by the formation of new matrix components at the interface
between the implant and the bone proximate to the implant. An estimated ten percent
of bone and joint prosthetic devices that are placed in people fail to function
due to non-bonding of the bone to an implant. The resulting disability often requires
reoperation and reimplantation of the device. Furthermore, five to ten percent
of all bone fractures are never repaired. Although many methods have been proposed
to cure these non-healing bone fractures, none has yet proven to be satisfactory.
Based on all of the above, there clearly exists a need in the art for improved
methods that provide for accelerated bone growth.
Cartilage is a specialized type of dense connective tissue consisting of
cells embedded in a matrix. There are several kinds of cartilage. (U.S. Pat. No.
5,736,372, herein incorpoirated by reference in its entirety.) Translucent cartilage
having a homogeneous matrix containing collagenous fibers is found in articular
cartilage, in costal cartilages, in the septum of the nose, in larynx and trachea.
Articular cartilage is hyaline cartilage covering the articular surfaces of bones.
Costal cartilage connects the true ribs and the sternum. Fibrous cartilage contains
collagen fibers. Yellow cartilage is a network of elastic fibers holding cartilage
cells which is primarily found in the epiglottis, the external ear, and the auditory
tube. Cartilage is tissue made up of extracellular matrix primarily comprised of
the organic compounds collagen, hyaluronic acid (a proteoglycan), and chondrocyte
cells, which are responsible for cartilage production. Collagen, hyaluronic acid
and water entrapped within these organic matrix elements yield the unique elastic
properties and strength of cartilage. Chondrocytes produce both Type I and Type
II collagens. Type II collagen is not found in bone, whereas Type I collagen is
found in bone. (U.S. Pat. No. 5,686,116) It has previously been shown that the
endogenous growth factors TGF beta and BMP induce both new cartilage and bone formation.
Wozney et al. Science, 242:1528-1533 (1988) and Sporn et al. J. Cell Biol. 105:1039-1045 (1987).
In cartilage, collagen synthesis is required for repair, healing and augmentation,
as well as for the successful bonding of grafts and prosthetic devices. (U.S. Pat.
No. 5,686,116) Collagen is the major structural protein responsible for the architectural
integrity of cartilage. Thus, an adequate supply of chondrocytes is essential in
order to produce sufficient amounts of collagen for repair, healing, and augmentation
of cartilage. Other, noncollagen proteins, such as osteonectin, fibronectin and
proteoglycans are also important for cartilage repair.
Cells such as synoviocytes that are found in joint spaces adjacent to cartilage
have an important role in cartilage metabolism. Synoviocytes produce metallo-proteinases,
such as collagenases that are capable of breaking-down cartilage. TGF beta is known
to inhibit cell-release (and probably synthesis) of metallo-proteinases and to
induce chondrocytes (cartilage forming cells) to produce new matrix components
and inhibit production of cartilage destructive enzymes so as to effect cartilage
repair, healing and augmentation. Sporn et al. (1987). It has also been shown that
mice deficient in parathyroid hormone-related peptide (PTHrP) exhibit abnormal
cartilage maturation, indicating that PTHrP is an essential factor for chondrocyte
development and maturation. (U.S. Pat. No. 5,700,774)
Cartilage implants are often used in reconstructive or plastic surgery
such as rhinoplasty. There is a need in the art for methods that increase chondrocyte
proliferation and collagen synthesis, and thus inhibit cartilage destruction and
enhance cartilage repair. Such methods would increase the clinical utility of cartilage
repair including but not limited to cartilage grafts and healing of cartilage grafts.
Although some of the above methods have met with limited success, there
remains a need in the art for improved methods for enhancing bone and cartilage
repair, healing and augmentation, and for enhancing the attachment and fixation
of bone and cartilage implants.
SUMMARY OF THE INVENTION
The present invention provides methods, kits, and compositions for 1) enhancing
bone and cartilage repair; 2) bone and prosthesis implantation; 3) attachment and
fixation of cartilage to bone or other tissues; and methods, cell culture medium
and kits for the proliferation of chondrocytes; all of which comprise the administration
of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues
thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof
or AII AT
2 type 2 receptor agonists.
These aspects and other aspects of the invention become apparent in light of
the following detailed description.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a bar graph showing the effect of AII, AIII, and GSD37B (10 μg/ml)
on chondrocyte proliferation.
FIG. 2 is a bar graph showing the effect of AII, GSD36, GSD37B, GSD38B, and
GSD28 (10 μg/ml) of the invention on chondrocyte proliferation.
FIG. 3 is a bar graph showing the effect of AII, 1GD, 2GD, and 3GD (10 μg/ml)
on chondrocyte proliferation.
FIG. 4 is a bar graph showing the effect of AII, AII(1-7), GSD22A, GSD24B, and
GSD28 (10 μg/ml) of the invention on chondrocyte proliferation.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
All references, patents and patent applications are hereby incorporated by reference
in their entirety.
Within this application, unless otherwise stated, the techniques utilized
may be found in any of several well-known references such as:
Molecular Cloning:
A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory
Press),
Gene Expression Technology (Methods in Enzymology, Vol. 185, edited
by D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to Protein Purification"
in
Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.);
PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990.
Academic Press, San Diego, Calif.),
Culture of Animal Cells: A Manual of Basic
Technique, 2
nd Ed. (R. I. Freshney. 1987. Liss, Inc. New
York, N.Y.),
Gene Transfer and Expression Protocols, pp. 109-128, ed. E.
J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion,
Austin, Tex.).
As defined herein the phrase "enhancing bone repair" refers to increasing the
rate of new bone formation via bone remodeling, osteogenesis, osteoconduction and/or
osteoinduction. The methods for enhancing bone repair in a mammal of the invention
include those that stimulate bone formation and those that reverse bone loss. The
methods can thus be used for (1) providing a subject with an amount of a substance
sufficient to act prophylactically to prevent the development of a weakened and/or
unhealthy state; or (2) providing a subject with a sufficient amount of a substance
so as to alleviate or eliminate a disease state and/or the symptoms of a disease
state, and a weakened and/or unhealthy state.
As used herein the term "enhancing cartilage repair" comprises healing and regeneration
of cartilage injuries, tears, deformities or defects, and prophylactic use in preventing
damage to cartilaginous tissue.
The present invention fulfills the need for methods to enhance bone repair in
a mammal suffering from bone fractures, defects, and disorders which result in
weakened bones such as osteoporosis, osteoarthritis, Paget's disease, osteohalisteresis,
osteomalacia, periodontal disease, bone loss resulting from multiple myeloma and
other forms of cancer, bone loss resulting from side effects of other medical treatment
(such as steroids), and age-related loss of bone mass. In addition, bony ingrowth
into various prosthetic devices can be greatly enhanced so that such artificial
parts are firmly and permanently anchored into the surrounding skeletal tissue
through a natural osseous bridge.
The present invention further fulfills the need for methods to enhance the repair
of cartilage in a mammal, by accelerating the proliferation of chondrocytes and
thereby increasing the synthesis of collagen for use in cartilage repair. Such
methods have application in the healing of cartilage, for example articular cartilage
tears, deformities and other cartilage defects in humans and other animals. The
methods have prophylactic use in preventing damage to cartilaginous tissue, as
well as use in the improved fixation of cartilage to bone or other tissues, and
in repairing defects to cartilage tissue. De novo cartilaginous tissue formation
induced by the compounds of the present invention contributes to the repair of
congenital, trauma induced, or other cartilage defects of other origin, and is
also useful in surgery for attachment or repair of cartilage. The methods and compositions
of the invention may also be useful in the treatment of arthritis and other cartilage
defects. The methods of the present invention can also be used in other indications
wherein it is desirable to heal or regenerate cartilage tissue. Such indications
include, without limitation, regeneration or repair of injuries to the articular
cartilage. The methods of the present invention provide an environment to attract
cartilage-forming cells, stimulate growth of cartilage-forming cells or induce
differentiation of progenitors of cartilage-forming cells and chondrocytes.
The methods and kits of the present invention also provide improved chemically
defined medium for accelerating the proliferation of chondrocytes (cartilage-forming
cells). In another embodiment, the compositions and methods of the present invention
can be used to treat chondrocytic cell lines, such as articular chondrocytes, in
order to maintain chondrocytic phenotype and survival of the cells. The treated
cell populations are therefore also useful for gene therapy applications.
U.S. Pat. No. 5,015,629 to DiZerega (the entire disclosure of which is hereby
incorporated by reference) describes a method for increasing the rate of healing
of wound tissue, comprising the application to such tissue of angiotensin II (AII)
in an amount which is sufficient for said increase. The application of AII to wound
tissue significantly increases the rate of wound healing, leading to a more rapid
re-epithelialization and tissue repair. The term AI refers to an octapeptide present
in humans and other species having the sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
[SEQ ID NO:1]. The biological formation of angiotensin is initiated by the action
of renin on the plasma substrate angiotensinogen (Clouston et al.,
Genomics
2:240-248 (1988); Kageyama et al,
Biochemistry 23:3603-3609; Ohkubo
et al.,
Proc. Natl. Acad. Sci. 80:2196-2200 (1983); each reference hereby
incorporated in its entirety). The substance so formed is a decapeptide called
angiotensin I (AI) which is converted to AII by the angiotensin converting enzyme
(ACE) which removes the C-terminal His-Leu residues from AI [SEQ ID NO: 37]. AII
is a known pressor agent and is commercially available.
Studies have shown that AII increases mitogenesis and chemotaxis in cultured
cells that are involved in wound repair, and also increases their release of growth
factors and extracellular matrices (diZerega, U.S. Pat. No. 5,015,629; Dzau et.
al.,
J. Mol. Cell. Cardiol. 21:S7 (Supp III) 1989; Berk et. al.,
Hypertension
13:305-14 (1989); Kawahara, et al.,
BBRC 150:52-9 (1988); Naftilan,
et al.,
J. Clin. Invest. 83:1419-23 (1989); Taubman et al.,
J. Biol.
Chem. 264:526-530 (1989); Nakahara, et al.,
BBRC 184:811-8 (1992); Stouffer
and Owens,
Circ. Res. 70:820 (1992); Wolf, et al.,
Am. J. Pathol. 140:95-107
(1992); Bell and Madri,
Am. J. Pathol. 137:7-12 (1990). In addition, AII
was shown to be angiogenic in rabbit corneal eye and chick chorioallantoic membrane
models (Fernandez, et al.,
J. Lab. Clin. Med. 105:141 (1985); LeNoble, et
al., Eur. J. Pharmacol. 195:305-6 (1991). Additionally, AII and angiotensin III
analogs and fragments thereof have been shown to be effective in wound healing.
(U.S. Pat. No. 5,629,292; International Application No. WO 95/08565; International
Application WO 95/08337; International Application No. WO 96/39164; all references
hereby incorporated in their entirety.)
Previous studies have suggested that angiotensin I (AI) and AII both stimulate
bone resorption in vitro by osteoclasts incubated on bone slices, but only in the
presence of osteoblastic cells, suggesting that the effect of angiotensin II was
not direct, but rather is mediated by a primary hormonal interaction on cells of
the osteoblastic lineage. (Hatton et al.,
J. Endocrinol. 152:5-10 (1997)).
AI stimulation of bone resorption was inhibited by ACE inhibitors, suggesting that
the formation of AII from AI was responsible for the stimulation of bone resorption.
Neither AI nor AII were shown to have any effect on osteoclast formation. Thus,
this study suggests that local bone destruction may be mediated by AII's stimulation
of bone resorption.
Other studies have demonstrated AII stimulation of DNA and collagen synthesis
in vitro on primary cultures of isolated, phenotypically immature osteoblasts derived
from the periosteum of fetal rat calvaraiae and human adult trabecular bone. (Lamparter
et al.,
J. Cell. Physiol. 175:89-98 (1998)) No direct AII effect was detected
on primary cell populations with a mature osteoblast phenotype, and an indirect
effect through AII-responsive osteoblastic precursor cells was proposed. Similar
in vitro studies on osteoblast-rich populations of cells demonstrated a similar
effect, while not ruling out stimulation of mature osteoblast proliferation. (Hiruma
et al.,
Biochem and Biophys. Res. Commun. 230:176-178 (1997)) Another study
suggests that AII may decelerate the differentiation and bone formation of rat
calvarial osteoblasts. (Hagiwara et al., J. of Endocrinology 156:543-550 (1998))
Based on all of the above studies, there is no expectation that the use of
angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof,
AII, AII analogues, AII fragments or analogues thereof or AII AT
2 type
2 receptor agonists would be effective in enhancing bone and cartilage repair,
or effective in accelerating chondrocyte proliferation and collagen synthesis.
Previous studies in our laboratory have demonstrated that a class of AII
and AII analogues and fragments stimulate the proliferation of mesenchymal stem
cells, which give rise to the cells that make up bone and cartilage. (U.S. patent
application Ser. No. 09/012,400, filed Jan. 23, 1998, herein incorporated by reference
in its entirety.)
A peptide agonist selective for the AT2 receptor (AII has 100 times higher affinity
for AT2 than AT1) has been identified. This peptide is p-aminophenylalanine 6-AII
["(p-NH
2-Phe)6-AII)"], Asp-Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36]
wherein Xaa is p-NH
2-Phe (Speth and Kim, BBRC 169:997-1006 (1990). This
peptide gave binding characteristics comparable to AT2 antagonists in the experimental
models tested (Catalioto, et al.,
Eur. J. Pharmacol. 256:93-97 (1994); Bryson,
et al.,
Eur. J. Pharmacol. 225:119-127 (1992).
The effects of AII receptor and AII receptor antagonists have been examined in
two experimental models of vascular injury and repair which suggest that both AII
receptor subtypes (AT1 and AT2) play a role in wound healing (Janiak et al.,
Hypertension
20:737-45 (1992); Prescott, et al.,
Am. J. Pathol. 139:1291-1296 (1991);
Kauffman, et al.,
Life Sci. 49:223-228 (1991); Viswanathan, et al.,
Peptides
13:783-786 (1992); Kimura, et al.,
BBRC 187:1083-1090 (1992).
Many studies have focused upon AII(1-7) (AII residues 1-7) or other fragments
of AII to evaluate their activity. AII(1-7) elicits some, but not the fill range
of effects elicited by AII. Pfeilschifter, et al.,
Eur. J. Pharmacol. 225:57-62
(1992); Jaiswal, et al.,
Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards
and Stack,
J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al.,
J. Pharmacol. Exper. Ther. 265:664-673 (1991); Jaiswal, et al.,
Hypertension
17:1115-1120 (1991); Portsi, et al.,
Br. J. Pharmacol. 111:652-654 (1994).
As hereinafter defined, a preferred class of AT2 agonists for use in accordance
with the present invention comprises angiotensinogen, angiotensin I (AI), AI analogues,
AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues
thereof or AII AT
2 type 2 receptor agonists having p-NH-Phe in a position
corresponding to a position 6 of AII. In addition to peptide agents, various nonpeptidic
agents (e.g., peptidomimetics) having the requisite AT2 agonist activity are further
contemplated for use in accordance with the present invention.
The active AII analogues, fragments of AII and analogues thereof of particular
interest in accordance with the present invention comprise a sequence consisting
of at least three contiguous amino acids of groups R
1-R
8 in
the sequence of general formula I
in which R
1 and R
2 together form a group of formula
- wherein
- X is H or a one to three peptide group, or is absent,
- RA is suitably selected from H, Asp, Glu, Asn, Acpc (1-aminocyclopentane
carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu(NH2), Gly, Asp(NH2)
and Suc,
- RB is suitably selected from Arg, Lys, Ala, Orn, Citron,
Ser(Ac), Sar, D-Arg and D-Lys;
- R3 is selected from the group consisting of Val, Ala, Leu,
norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;
- R4 is selected from the group consisting of Tyr, Tyr(PO3)2,
Thr, Ala, Ser, homoSer and azaTyr;
- R5 is selected from the group consisting of Ile, Ala, Leu,
norLeu, Val and Gly;
- R6 is His, Arg or 6-NH2-Phe;
- R7 is Pro or Ala; and
- R8 is selected from the group consisting of Phe, Phe(Br),
Ile and Tyr, excluding sequences including R4 as a terminal Tyr group,
or is absent.
Compounds falling within the category of AT2 agonists useful in the practice
of the invention include the AII analogues set forth above subject to the restriction
that R
6 is p-NH
2-Phe.
Particularly preferred combinations for R
A and R
B are
Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys. Particularly preferred embodiments of this
class include the following: AII, AIII or AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe
[SEQ ID NO:2]; AII(3-8), also known as des1-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe
[SEQ ID NO:3]; AII(1-7), Asp-Arg-Val-Tyr-Ile-His-Pro {SEQ ID NO:4]; AII(2-7). Arg-Val-Tyr-Ile-His-Pro
[SEQ ID NO:5]; AII(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8), Ile-His-Pro-Phe
[SEQ ID NO:7]; AII(1-6), Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8]; AII(1-5), Asp-Arg-Val-Tyr-Ile
[SEQ ID NO:9]; AII(1-4), Asp-Arg-Val-Tyr [SEQ ID NO:10]; and AII(1-3), Asp-Arg-Val
[SEQ ID NO:11]. Other preferred embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe
[SEQ ID NO:12] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO:13]. Still another
preferred embodiment encompassed within the scope of the invention is a peptide
having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31]. AII(6-8), His-Pro-Phe
[SEQ ID NO:14] and AII(4-8), Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested
and found not to be effective.
In a particularly preferred embodiment of the methods for chondrocyte proliferation,
collagen synthesis, cartilage repair, and attachment and fixation of cartilage
to bone or other tissues, the active compounds of the present invention are selected
from those comprising the following general formula:
wherein
- R1 is selected from the group consisting of Hydrogen, Gly, and Asp;
- R2 is selected from the group consisting of Arg, Citron, or Ornithine;
- R3 is selected from the group consisting of Val, Ile, Ala, Leu, and
norLeu, or Pro;
- R4 is selected from Tyr, Tyr(PO3)2, and Ala;
- R5 is selected from the group consisting of Ile, Ala, Val, Leu, and
norLeu; and
- R6 is Phe, Ile, or is absent.
Most particularly preferred embodiments of this class of compounds are selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:
13, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:12, SEQ ID NO:24, SEQ ID NO:26, SEQ
ID NO: 32, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,
SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45.
In a particularly preferred embodiment of the methods for bone repair and bone
and prosthesis implantation, the active compounds of the present invention are
selected from those comprising the following general formula:
wherein
- R1 is selected from the group consisting of Ile, Pro, Ala, Val, Leu,
and norLeu;
- R2 is selected from Tyr and Tyr(PO3)2; and
- R3 is Phe, or is absent.
Most particularly preferred embodiments of this class of compounds are selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:24, SEQ ID NO:31,
SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO:41, and SEQ ID NO: 45.
Another class of compounds of particular interest in accordance with the
present invention are those of the general formula II
in which
-
- R2 is selected from the group consisting of H, Arg, Lys,
Ala, Orn, Citron, Ser(Ac), Sar, D-Arg and D-Lys;
- R3 is selected from the group consisting of Val, Ala, Leu,
norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;
- R4 is selected from the group consisting of Tyr, Tyr(PO3)2,
Thr, Ser, homoSer, Ala, and azaTyr;
- R5 is selected from the group consisting of Ile, Ala, Leu,
norLeu, Val and Gly;
- R6 is His, Arg or 6-NH2-Phe;
- R7 is Pro or Ala; and
- R8 is selected from the group consisting of Phe, Phe(Br),
Ile and Tyr.
A particularly preferred subclass of the compounds of general formula II has
the formula
wherein R
2, R
3 and R
5 are as previously
defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe
[SEQ ID NO:2]. Other preferred compounds include peptides having the structures
Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO:17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ
ID NO:18]. The fragment AII(4-8) was ineffective in repeated tests; this is believed
to be due to the exposed tyrosine on the N-terminus.
In the above formulas, the standard three-letter abbreviations for amino acid
residues are employed. In the absence of an indication to the contrary, the L-form
of the amino acid is intended. Other residues are abbreviated as follows:
| TABLE 1 |
| |
| Abbreviation for Amino Acids |
| |
| |
| Me2Gly |
N,N-dimethylglycyl |
| Bet |
1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt |
| |
(betaine) |
| Suc |
Succinyl |
| Phe(Br) |
p-bromo-L-phenylalanyl |
| azaTyr |
aza-α'-homo-L-tyrosyl |
| Acpc |
1-aminocyclopentane carboxylic acid |
| Aib |
2-aminoisobutyric acid |
| Sar |
N-methylglycyl (sarcosine) |
| Orn |
Ornithine |
| |
It has been suggested that AII and its analogues adopt either a gamma or a beta
turn (Regoli, et al.,
Pharmacological Reviews 26:69 (1974). In general,
it is believed that neutral side chains in position R
3, R
5 and
R
7 may be involved in maintaining the appropriate distance between active
groups in positions R
4, R
6 and R
8 primarily responsible
for binding to receptors and/or intrinsic activity. Hydrophobic side chains in
positions R
3, R
5 and R
8 may also play an important
role in the whole conformation of the peptide and/or contribute to the formation
of a hypothetical hydrophobic pocket.
Appropriate side chains on the amino acid in position R
2 may
contribute to affinity of the compounds for target receptors and/or play an important
role in the conformation of the peptide. For this reason, Arg and Lys are particularly
preferred as R
2.
For purposes of the present invention, it is believed that R
3 may
be involved in the formation of linear or nonlinear hydrogen bonds with R
5
(in the gamma turn model) or R
6 (in the beta turn model). R
3
would also participate in the first turn in a beta antiparallel structure
(which has also been proposed as a possible structure). In contrast to other positions
in general formula I, it appears that beta and gamma branching are equally effective
in this position. Moreover, a single hydrogen bond may be sufficient to maintain
a relatively stable conformation. Accordingly, R
3 may suitably be selected
from Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr.
With respect to R
4, conformational analyses have suggested that the
side chain in this position (as well as in R
3 and R
5) contribute
to a hydrophobic cluster believed to be essential for occupation and stimulation
of receptors. Thus, R
4 is preferably selected from Tyr, Thr, Tyr (PO
3)
2,
homoSer, Ser and azaTyr. In this position, Tyr is particularly preferred as it
may form a hydrogen bond with the receptor site capable of accepting a hydrogen
from the phenolic hydroxyl (Regoli, et al. (1974), supra). R
3 may also
be suitably Ala.
In position R
5, an amino acid with a β aliphatic or alicyclic
chain is particularly desirable. Therefore, while Gly is suitable in position R
5,
it is preferred that the amino acid in this position be selected from Ile, Ala,
Leu, norLeu, Gly and Val.
In the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof,
AII analogues, fragments and analogues of fragments of particular interest in accordance
with the present invention, R
6 is His, Arg or 6-NH
2-Phe.
The unique properties of the imidazole ring of histidine (e.g., ionization at physiological
pH, ability to act as proton donor or acceptor, aromatic character) are believed
to contribute to its particular utility as R
6. For example, conformational
models suggest that His may participate in hydrogen bond formation (in the beta
model) or in the second turn of the antiparallel structure by influencing the orientation
of R
7. Similarly, it is presently considered that R
7 should
be Pro in order to provide the most desirable orientation of R
8. In
position R
8, both a hydrophobic ring and an anionic carboxyl terminal
appear to be particularly useful in binding of the analogues of interest to receptors;
therefore, Tyr and especially Phe are preferred for purposes of the present invention.
Analogues of particular interest include the following:
| TABLE 2 |
| |
| Angiotensin II Analogues |
| AII |
|
|
| Analogue |
|
Sequence |
| Name |
Amino Acid Sequence |
Identifier |
| |
| Analogue 1 |
Asp-Arg-Val-Tyr-Val-His-Pro-Phe |
SEQ ID NO: 19 |
| Analogue 2 |
Asn-Arg-Val-Tyr-Val-His-Pro-Phe |
SEQ ID NO: 20 |
| Analogue 3 |
Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His- |
SEQ ID NO: 21 |
| |
Pro-Phe |
| Analogue 4 |
Glu-Arg-Val-Tyr-Ile-His-Pro-Phe |
SEQ ID NO: 22 |
| Analogue 5 |
Asp-Lys-Val-Tyr-Ile-His-Pro-Phe |
SEQ ID NO: 23 |
| Analogue 6 |
Asp-Arg-Ala-Tyr-Ile-His-Pro-Phe |
SEQ ID NO: 24 |
| Analogue 7 |
Asp-Arg-Val-Thr-Ile-His-Pro-Phe |
SEQ ID NO: 25 |
| Analogue 8 |
Asp-Arg-Val-Tyr-Leu-His-Pro-Phe |
SEQ ID NO: 26 |
| Analogue 9 |
Asp-Arg-Val-Tyr-Ile-Arg-Pro-Phe |
SEQ ID NO: 27 |
| Analogue 10 |
Asp-Arg-Val-Tyr-Ile-His-Ala-Phe |
SEQ ID NO: 28 |
| Analogue 11 |
Asp-Arg-Val-Tyr-Ile-His-Pro-Tyr |
SEQ ID NO: 29 |
| Analogue 12 |
Pro-Arg-Val-Tyr-Ile-His-Pro-Phe |
SEQ ID NO: 30 |
| Analogue 13 |
Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe |
SEQ ID NO: 31 |
| Analogue 14 |
Asp-Arg-Val-Tyr(PO3)2-Ile-His- |
SEQ ID NO: 32 |
| |
Pro-Phe |
| Analogue 15 |
Asp-Arg-norLeu-Tyr-Ile-His-Pro-Phe |
SEQ ID NO: 33 |
| Analogue 16 |
Asp-Arg-Val-Tyr-norLeu-His-Pro-Phe |
SEQ ID NO: 34 |
| Analogue 17 |
Asp-Arg-Val-homoSer-Tyr-Ile-His- |
SEQ ID NO: 35 |
| |
Pro-Phe |
| |
The polypeptides of the instant invention may be synthesized by any conventional
method, including, but not limited to, those set forth in J. M. Stewart and J.
D. Young,
Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford,
Ill. (1984) and J. Meienhofer,
Hormonal Proteins and Peptides, Vol. 2, Academic
Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke,
The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis.
The disclosures of the foregoing treatises are incorporated by reference herein.
In general, these methods involve the sequential addition of protected amino
acids
to a growing peptide chain (U.S. Pat. No. 5,693,616, herein incorporated by reference
in its entirety). Normally, either the amino or carboxyl group of the first amino
acid and any reactive side chain group are protected. This protected amino acid
is then either attached to an inert solid support, or utilized in solution, and
the next amino acid in the sequence, also suitably protected, is added under conditions
amenable to formation of the amide linkage. After all the desired amino acids have
been linked in the proper sequence, protecting groups and any solid support are
removed to afford the crude polypeptide. The polypeptide is desalted and purified,
preferably chromatographically, to yield the final product.
Preferably, peptides are synthesized according to standard solid-phase
methodologies, such as may be performed on an Applied Biosystems Model 430A peptide
synthesizer (Applied Biosystems, Foster City, Calif.), according to manufacturer's
instructions. Other methods of synthesizing peptides or peptidomimetics, either
by solid phase methodologies or in liquid phase, are well known to those skilled
in the art.
In one aspect, the present invention provides methods and kits for enhancing
bone
and cartilage repair, implantation, and augmentation in a mammal comprising the
administration of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments
and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues
thereof or AII AT
2 type 2 receptor agonists (hereinafter referred to
as "active agents"). The compounds can be administered alone or in combination
with other compounds that enahnce bone and/or cartilage repair, implantation, and
augmentation, including but not limited to bone morphogenic protein-2, bone morphogenic
protein-4, bone morphogenic protein-6, bone morphogenic protein-7, transforming
growth factor-beta, insulin-like growth factor, and parathyroid hormone.
The active agents may be administered by any suitable route, including orally,
parentally, by inhalation spray, rectally, or topically in dosage unit formulations
containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
Such vehicles may include a tantalum or hydroxyapatite scaffold as a vehicle with
the compounds of the invention embedded therein. Alternatively, polymeric substrates
can be used for compound delivery to the bone or cartilage such as the polymeric
substrates disclosed in U.S. Pat. Nos. 5,443,515; 5,171,273; 5,607,474; 4,916,207;
and 5,324,775; all references hereby incorporated in their entirety. The term parenteral
as used herein includes topical (i.e.: placement in to the bone), subcutaneous,
intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal,
intracranial, intrathoracic, infusion techniques or intraperitoneally.
The active agents of t
