Note: Descriptions are shown in the official language in which they were submitted.
W092/21365 ~ glaPcr/US92/04356
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THERAPEUTIC COMPOSITIONS FOR OSTEOINDUCTION
t
TECHNICAL FlFln
The present invention relates to the field of osteoinduction
(bone growth). Specific~lly, the present invention relates to
novel therapeutic formulations comprising the administration of
bone ~ 3 tic proteins and a Vitamin D compound, resulting in
o synergistic bone growth.
B.t~ R( OF THE INVENTION
In healthy individuals bone growth generally proceeds nor-
mally and fractures heal without the need for pharmacologic
intervention. Nonetheless, in certain instances bones may be
weakened or may fail to heal properly. For example, healing may
proceed slowly in the elderly and in patients undergoing treatment
with corticosteroids, such as transplant patients and those being
treated for chronic lung disease. Another example is osteopor-
osis. Osteoporosis is an abnormal loss of bony tissue often
occurring in post r -~ al woman and elderly men. The disorder
increases the risks of small fractures occurring in the bones,
particularly the spine. At present, osteoporosis is treated
mainly by supplements of calcium, vitamin D, estrogen, or
calcitonin, a hormone which controls the body's use of calcium.
2s Unfortunately, these treatments are merely preventative against
the further loss of bone. There is a need in the art for treat-
ments that go beyond the prevention of bone loss and promote bone
formation and/or reverse bone loss.
(1989) ~Bone ~ ' . ic Proteins and Vitamin D", Nutrition
Reviews, Vol. 47, pp. 364-366 concludes that Vitamin D in the diet
prevents the loss of the osteoinductive activity of bone matrix.
Turner, R. T., J. Farley, J. J. Vandersteenhoven, S. Epstein,
N. H. Bell, and D. J. Baylink, (1988) ~Demonstration of Reduced
Mitogenic and Osteoinductive Activities in Demineralized Allo-
geneic 80ne Matrix from Vitamin D-deficient Rats", The Journal of
Clinical Investi4ation. Inc.. Vol. 82, pp. 212-217, discloses the
_ . _ _ _ . . . _ . _ .
WO 92~21365 PCr/US92/04356
211~1~1Q; - 2 -
implantation of deminer~lized bone matrix from Vitamin D-deficient
rats i nto normal rats . The demi ner~l i zed bone m~tri x from Vi t~mi n
D-deficient rats did not promote osteoinduction as effectively ~s
demineralized bone matrix from normal rats.
Sampath, T. K., S. ileintraub, and A. H. Reddi, 1l984) ~Extra-
cellular Matrix Proteins Involved in bone Induction are Vitamin D
Dependent~, Biochemical and BioDhvsical Research Communications.
Vol. 124, pp. 829-835, discloses a study involving implantation of
demineralized bone matrix from norm~1 r~ts and demineralized bone
matrix from rachitic rats wherein the rachitic bone matrix did not
induce bone growth whi1e the normal bone matrix did. The study
concluded that these results demonstrate that Vitamin D is
necessary to produce bone inductive proteins in the bone matrix of
a living rat.
lS U.S. Patent No. 4,761,471, Urist, assigned to the Regents of
the University of California. issued August 2, 1988, discloses a
bone ~ , Lic protein composition comprising BMP factor and
8MP associated protein having a molecular weight of 34,000
daltons. Use of such factors and compositions to induce bone
formation in mammals is also disclosed.
U.S. Patent Ho. 4,455,256, Urist, assigned to the Regents of
the University of California, issued June 19, 1984, discloses a
bone . .~:, tic protein h~ving a molecular weight in the range
of 1,000 to 100,000 daltons.
\larious other bone ~ ,r, - tic proteins/factors, osteoin-
ductive factors, osteogenic factors ~nd other proteins/factors
rel~ted to bone growth ~re disclosed in the following public~-
tions: U.S. Patent 4,968,590, 1~' ath and Rueger, issued
November 6, 1990; U.S. Patent 4,698,328, Neer, Potts and Slovik.
~ssued ~ctober 6, 1987; U.S. Patent 4,877,864, Wang, Wozney and
Rosen, issued October 31, 1989; U.S. Patent 4,861,757, Antoni~des,
Lynch and Williams, issued August 29, 1989; U.S. P~tent 4,810,691,
Seyedin, Thomas, Bentz. Ellingsworth ~nd Armstrong, issued M~rch
` 7, 1989; U.S. Patent 4,804,744, Sen, issued February 14, 1989;
U.S. Patent 4,795,804, Urist, issued January 3, 1989; U.S. Patent
4,789,663, 1lal1~ce, Smestad, McPherson, Piez ~nd Ross, issued
December 6, 1988; U.S. P~tent 4,789,732, Urist. issued December 6,
1988; U.S. P~tent 4,774,322, Segedin, Thom~s, Bentz, Ellingsworth
and Armstrong, ~ssued September 27, 1988; U.S. P~tent 4,698,328,
_ _ _ . _ _
WO 92/21365 2 ~ 4 ~ ~ . PCr~US92/04356
- 3 -
Heer and Slovik, issued October 6, 1987; U.S. Patent ~,6Z7,982,
Seydin and Thomas, issued December 9, 1986; U.S. Patent 4,619,989,
Urist, issued October 28, 1986; U.S. Patent 4,596,574, Urist,
issued June 24, 1986; U.S. Patent 4,563,489, Urist, issued January
7, 1986; U.S. Patent 4,563,350, Nathan, Seyedin and Bentz, issued
January 7, 1986; U.S. Patent 4,526,909, Urist, issued July 2,
1985; U.S. Patent 4,434,894, Seyedin and Thomas, issued February
23, 1984; U.S. Patent 4,294,753, Urist, issued October 13, 1981;
European Patent Application 349 048, Bab, Muhlrad, Gazit and
Shteyer, published January 3, 1990; European Patent Application
309 241, Chu, Nathan and Seyedin, published March 29, 1989;
European Patent Application 336 760, Bentz, Nathan, Rosen, Dasch
and Seyedin, published October 11, 1989; European Patent Applica-
tion 145 ISS, Sen, published July 10, 1985; World Patent Applica-
lS tion 89/10934, Roos, Burns, Guy and McKnight, publ ished November
16, 1989; llorld Patent Applications 89/09787 and 89/09788,
Oppermann, Kubersampath, Rueger and Ozkaynak, published October
19, 1989; and llorld Patent Application 88/00205, llang, liozney and
Rosen, published Janaury 14, 1988.
OBJECTS OF THE PRESENT INVENTION
It is an object of the present invention to provide a method
for generating new bone growth in a mammal.
It is a further object of the present invention to provide a
pharmaceutical composit~on which can be used to generate new bone
growth in a mammal.
SUMMARY OF THE INVENTION
The present invention relates to a method of generating new
bone growth in mammals comprising administration to a mammal a
combination of a safe and effect~ve amount of a Vitamin D
compound, and a safe and effective amount of one or more BMPs or
osteoinductive extract comprising one or more BMPs.
The present invention further relates to a composition for
generating new bone growth in ma~mals comprising a safe and
effective amount of a Vitamin D compound; a safe and effective
35 amount of a BMP or osteoinductive extract comprising one or more
BMPs; and a pharmaceutically-acceptable carrier.
DETAII Fn l)F~r~TPT10N OF THE INVENTION
The present invention comprises the administration to a
mammal of a combination of a safe and effective amount of a
.
., _ . _ _ _ . .
21 10410
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Vitamin D compound and a saFe and effective amount of one or more BMPs or
an osteoinductive extract comprising one or more BMPs. It has been
determined that treatment with a Vitamin D compound, BMP or osteoinductive
extract alone increases bone growth. Surprisingly, it has been further
determined that treatment with a Vitamin D compound in ~,UIllL~ dLiull with
osteoinductive extract or in UolllL~ill " I with at least one BMP results in a level
of new bone growth greater than that achieved through ddllli~ lLldLiull of the
BMP, osteoinductive extract or Vitamin D compound alone. Subjects in need
oF such treatment suffer from a variety of ailments which may be treated via this
procedure, including but not limited to, bone fractures (closed and open), non-
union fractions, congenital defects, as an adjunct in plastic surgery, in treating
oncological resections, all diseases classified as u~L~ Jol u~is, rheumatoid
arth ritis, U~ od l l11 11ti~,, septic arthritis, rickets, orga n ic i ncu, uu, d li~ll I of prosthetic
joints and dental implants, periodontal disease and defects, as well as
osteopenic and u~Leollldldcic conditions and disease.
As used herein, "safe and effective amount" means an amount of
compound or uu" ,~,osiL;,.I~ sufficient to signif cantly induce a positive " l~ldiriudLioll
in the condition to be treated, but low enough to avoid serious side effects (ata reasonable benefiVrisk natio), within the scope of sound medical judgment.
2û The safe and effective amount of the compound or co,,,~,u~iLiu,l will vary with
the particular condition being treated, the age and physical condition of the
patient being treated, the severity of the condition, the duration of the treatment,
the nature of concurrent therapy, the specific compound or cûl,,r~ ,
employed, the particular pharn~ce~' "y ~cceptable can-ier utilized, and like
factors within the knowledge and expertise of the attending physician.
As used herein, "fracture reduction" means the restoration of a bone
fracture by surgical or manipulative means to its normal anatomical relation.
As used herein, "BMP" means bone IllLn,ullo~ , protein.
As used herein, "q.s." means quantity sufficient.
As used herein, all p~lu~llldg~ are by weight unless otherwise
specif ed.
As used herein "regional treatment" includes treating bone fractures
(closed and open), treating non-union Fractures,
~ s
21 104~0
s
treating congenital defects, as an adjunct treatment to plastic
surgery, treating oncological resections, organic incorporation of
prosthetic joints, organic incorporation of dental implants. and
treatment of periodonta~ disease and defects.
S As used herein ~systemic treatment~ includes treating
diseases classified as osteoporosis, rheumatoid arthritis. osteo-
arthritis, septic arthritis, rickets, and osteopenic conditlons
and di seases .
As used herein, all dose ranges for systemic treatment are
recited as the dry weight of the actives per kg body weight of the
mammal .
As used herein, all dose ranges for regional treatment are
recited as the dry weight of the actives per cm2 surface area of
mineralized tissue to be treated.
IS As used herein, ~mineralized tissue~ means bone and teeth.
Vitamin O ComDounds
One component involved in the method of the invention is a
Vitamin D compound. As used herein, ~Vitamin D compound~ includes
Vitamin D, ergocalciferol (Vitamin Dz), cholecalciferol (Vitamin
D,) and their biologically active metabolites and precursorS
Preferred Vitamin D compounds include, but are not limited to,
Vitamin D2 (Sigma, St. Louis, liO), Vitamin D3 (Sigm~, St. Louis,
MO), 1-~2-hydroxy Vitamin D~ -fluoro Vitamin D3, 3-deoxy-1,25-
dihydroxy Vit~min D" 25-hydroxy-5,6-trans Vitamin D3, 25-hydroxy
Vitamin D2 . 25-hydroxy Vitamin D, (Hoffman LaRoche), 1.25-dihy-
droxy Vitamin D2, 24.25-dihydroxy Vitamin D2, 24,25-dihydroxy
V~tamin D3 (Hoffman LaRoche), and 1,25-dihydroxy Vitamin D,
(Duph~r, Veenenda~l, Holland). Preferably, the Vitamin D compound
is selected from 25-hydroxy V~t~min D2, 25-hydroxy Vitamin D3,
1,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D2, 24,25-dihy-
droxy Vitamin D3, and 1,25-dihydroxy Vitamin D3, more preferably
1,25-dihydroxy Vitamin D~. Addition~l Vitamin D compounds useful
in the present invention are well known to those skilled in the
art and include, but dre not limited to, those disclosed by the
following U.S. Patents, U.S. Patent 4,970,203, DeLuca and Kwiecinski, issued
November 13, 1990; U.S. Patent 4,927,815, DeLuca, Kutner, Perlman and Schnoes,
issued May 22, 1990; U.S. Patent 4,857,518, DeLuca,
,~
-
WO 92121365 PCr/US92~04356
0 6-
Ikekawa and Tanaka, issued August IS, 1989; U.S. Patent 4,851,401,
DeLuca, Kutner, Perlman and 5chnoes, lssued July 25, 1983; U S.
Patent 4,851,400, DeLuca, Ikekawa and Tanaka, issued July 25,
1989; U.S. Patent 4,847,012, DeLuca, Kutner, Perlman, Phelps,
S Schnoes and Sicinski, issued July 11, 1989; U.S. Patent 4,816,417,
Dame, DeLuca and Pierce, issued March 28, 1989; U.S. Patent
4,769,181, DeLuca, Schnoes, Sicinski and Tanaka, issued September
6, 1988; U.S. Patent 4,755,329, DeLuca, Lee and Schnoes, issued
July S, 1988; U.S. Patent 4,719,205, DeLuca, Schnoes, Sicinski and
Tanaka, issued January 12, 1988; U.S. Patent 4,719,204, DeLuca,
Schnoes, Sicinski and Tanaka, issued January 12, 1988; U.S. Patent
4,717,721, DeLuca, Ikekawa, Ostrem and Schnoes, issued January S,
1988; U.S. Patent 4,689,180, DeLu~a, Schnoes, Sicinski and Tanaka,
issued August 25, 1987; U.S. Patent 4,619,920, DeLuca, Ikekawa,
IS Kobayashi and Tanaka, issued October 28, 1986; U.S. Patent
4,594,192, DeLuca, Ikekawa, Kobayashi and Tanaka, issued June 10,
1986; U.S. Patent 4,588,716, DeLuca and Schnoes, issued May 13,
1986; U.S. Patent 4,588,528, DeLuca, Ikekawa and Tanaka, issued
May 13, 1986; U.S. Patent 4,564,474, DeLuca, Ikekawa, Kobayashi
and Tanaka, issued January 14, 1986; U.S. Patent 4,555,364,
DeLuca, Lee, Phelps and Schnoes, issued November 26, 1985; U.S.
Patent 4,554,106, DeLuca, Lee, Phelps and Schnoes, issued November
19, 1985; U.S. Patent 4,552,698, DeLuca, Ikekawa, Kobayashi and
Tanaka, issued November 11, 1985; U.S. Patent 4,512,925, DeLuca,
Lee and Schnoes, issued April 23, 1985; U.S. Patent 4,505,906,
DeLuca, Schnoes, Sicinski and Tanaka, issued March 19, 1985; U.S.
Patent 4,502,991, DeLuca, lkekawa, Kobayashi and Tanaka, issued
March S, 1985; U.S. Patent 4,500,460, DeLuca, Ikekawa, Kobayashi
and Tanaka, issued February 19, 1985; U.S. Patent 4,481,198, Chu,
DeLuca, Kabakoff and Schnoes, issued November 6, 1984; U.S. Patent
4,461,766, DeLuca, Hart and Schnoes, issued July 24, 1984; U.S.
Patent 4,448,726, DeLuca, Paaren, Schnoes and Smith, issued 11ay
IS, 1984; U.S. Patent 4,448,721, DeLuca, Morzycki and Schnoes,
issued May IS, 1984; U.S. Patent 4,428,946, DeLuca, Jorgensen and
Schnoes, issued January 31, 1984; U.S. Patent 4,411,833, DeLuca,
Ikekawa, Kobayashi and Tanaka, issued October 25, 1983; U.s.
Patent 4,367,177, DeLuca, Schnoes and liichman, issued January 4,
1983; U.S. Patent 4,358,406, DeLuca, Ikekawa, Kobayashi and
Tanaka~ issued November 9, 1982; U.S. Patent 4,338,312, DeLuca,
.
WO 9ZJ2136S ~ PCr~US92/043~i6
- 7
Jorgensen and Schnoes, issued July 6, 1982; U.S. Patent 4,338,250,
DeLuca, Hamer, Paaren and Schnoes, issued July 6, 1982; U.S.
Patent 4,336,193, DeLuca, Fivizzani, Paaren, Schnoes and ilichmann,
issued June 2Z, 1982; U.5. Patent 4,313,942, DeLuca, Frank, Paaren
and Schnoes, issued February 2, 198Z; U.S. Patent 4,307,231,
DeLuca, Paaren, Schnoes, Tanaka and l~lichmann, issued December 22,
1981; U.5. Patent 4,307,025, DeLuca, Ikekawa, Morisaki, Oshida,
Schnoes and Tanaka, issued December 22, 1981; U.S. Patent
4,305,880, DeLuca, Ikeka~a, Kobayashi and Tanaka, issued December
15, 1981; U.S. Patent 4,297,289, DeLuca, Fivizzani, Paaren and
Schnoes, issued October 27, 1981; U.S. Patent 4,292,250, DeLuca,
Levan and Schnoes, issued September 29, 1981; U.S. Patent
4,265,822, DeLuca, Hamer, Paaren and Schnoes, issued May 5, 1981;
U.S. Patent 4,264,513, DeLuca, Fivizzani, Napoli and Schnoes,
issued April 28, 1981; U.S. Patent 4,263,214, DeLuca, Napol i,
Onisko and Schnoes, issued April 21, 1981; U.S. Patent 4,260,804,
DeLuca, Esvelt and Schnoes, issued Apri1 1, 1981; U.S. Patent
4,260,549, DeLuca, Hamer, Paaren and Schnoes, issued April 7,
1981; U.5. Patent 4,254,045, DeLuca, Ikekawa, Morisaki, Oshida and
Tanaka, issued March 3, 1981; U.S. Reissue Patent 30,538, DeLuca,
Lam and Schnoes, issued March 3, 1981; U.S. Patent 4,248,791,
DeLuca, Ikekawa, Kobayashi and Tanaka, issued February 3, 1981;
U.S. Patent 4,234,495, DeLuca, Hamer, Paaren and Schnoes, issued
November 18, 1980; U.S. Patent 4,230,627, DeLuca, Napoli, Onisko
and Schnoes, issued October 28, 1980; U.S. Patent 4,229,359,
Alper, DeLuca, Schnoes and Tanaka, issued October 21, 1980; U.S.
Patent 4,229,358, DeLuca, Napoli, Onisko and Schnoes, issued
October 21, 1980; U.S. Patent 4,229,357, DeLuca, Napoli, Onisko
and Schnoes, issued October 21, 1980; U.S. Patent 4,226,788,
DeLuca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued October 7,
1980; U.S. Patent 4,226,787, DeLuc~, Napoli, Onisko and Schnoes,
issued October 7, 1980; U.S. Patent 4,224,231, Alper, DeLuca,
- Schnoes and Tanaka, issued September 23, 1980; U.S. Patent
4,224,230, DeLuca, Napoli, Onisko and Schnoes, issued September
35 23, 1980; U.S. Patent 4,223,131, DeLuca, Schnoes and ~lichman,
issued September 16, 1980; U.S. Patent 4,217,288, DeLuca, OniskD
and Schnoes, issued August 12, 1980; U.S, Patent 4,209,634,
DeLuca, Esvelt and Schnoes, issued June 24, 1980; U.S. Patent
4,202,829, DeLuca, Hamer, Paaren and Schnoes, issued Hay 13, 1980;
~=
21 1~410
- 8 -
U.S Patent 4,201,881, DeLuca. Ikeka~a, Kobayashi, Schnoes and
Tanaka, issued Hay 6, 198û; U.S. Patent 4,196,133, Oe~uca,
Ikekawa, Kobayashi, Schnoes and Tanaka, issued April 1, 1980; U.S.
Patent 4,195,027. OeLuca, Hamer, Paaren and Schnoes, issued March
25, 1980; U.S. Patent 4,188,345, DeLuca, Hapol i, Oniski and
Schnoes, issued February 12, 1980; and U.S. Patent 3,906,014,
DeLuca, Lam and Schnoes, issued September 16, 1975. Additional
Vitamin O compounds useful in the present inYention and disclosed
by these rerérl - include, but are not limited to, hydroxylated
24-homo-vitamin 0; cyclopeneano-vitamin 0; hydroxylated 26-homo
vitamin D; 1 Q-hydro%yvitamin 0; 1-hydroxyvitamin D; 1 l-hydroxy-
vitamin 2; I Q,25-dihydroxy-22Z-d-h~J-u~iitamin 0; 26,26,26,-
27,27-pentafluoro-1 Q-hydroxy-27-methoxyvitamin 0~; 2 Q-fluoro-
vitamin D3; 1,24-dihydroxy-delta 22-vitamin D3; 23,23-difluoro-
25-hydroxy-vitamin 0~; 1-hydroxy-3.5-cyclovitamin D; 23.23-di-
fluoro-1 Q,25-dihydroxy-vitamin D3; 1,23-dihydroxyvitamin D;
hydroxyvitamin D2; 23,23-difluoro-} Q,25-dihydroxy-vitamin 03;
23,23-difluoro-25-hydroxy-Yitamin D,; 26,26,26,27,27,27-hexa-
fluoro-l Q,25-dih~u'~u~r-holesterol; 23,25-dihydroxyvitamin D3;
26,26,26,27,21.27-hexafluoro-1 ,25-dih~u,u~ ,olecalciferol; 1
Q,25-dihydroxy-2 ~-fluorovitamin 0,; 24-fluoro-25-h.~.u~ ,olecal-
ciferol; 5,6-trans-vitamin 0; 1 Q-hydroxy-25-keto-27-nor-chole-
calciferol; fluorcvitamin D; 1 Q-hydroxy-2 ~-fluorocholecalci-
ferol; 3-deoxy-1 ~-hru,u~ lecalciferol; 25-hydroxy-26,26,26,-
27.27,27-hexafluorocholecaliferol; ~-hydroxy-3,5-cyclovitamin D;
25-h~u. u~.h~lecalciferol; 24,24-difluoro-1 Q,25-dihjul u~.l,ûle-
calciferol; 25~ UluA.r~holec~lciferol; 25-l~d.u~ lecalciferol-
26,23-lactone; 24~24-difluoro-lQ~2s-dik~dluAr~llolecalciferol;
24~24-difluoro-2s-hrJlù~r~holecalciferol; 3.5-cyc~ovitamin D; and
3-deoxy-Q-l.~i û~r~hùiecalciferol . Additional Vitamin O compounds
useful in the present invention further include those disclosed in
The Handbook of Vitamins, L. J. Hachlin, Ed., Hercel Dekker, lnc.
(1984), Vitamin D cûmpormds
useful in the present invention disc]osed by this reference,
inc]ude, bur are not limited to, 1,25-dihydroxy Yitamin 0,
3-deoxy-1,25-dihydroxy Vit~min D, 27-nor-25-hydroxy Vitamin D3,
26,21-bis-nor-25-hydroxy Vitamin D3 24-nor-25-hydroxy Vitamin 03,
25-hydroxy Vitamin 0, 1,25-dihydroxy Vitamin 0, 1Q-hydroxy Vitamin
03 and 25-fluoro-1Q-hydroxy Vitamin D3.
r
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WO 92/21365 2 1 1 ~ Pcr/US92/04356
A safe and effective amount of a Vitamin D compound ls dosed
in combination with at least one ~MP or in combination with an
osteoinductive extract comprising at least one BMP.
A preferred dose range for administration of the Vitamin D
S compound for systemic treatment is from about I ng to about I mg,
preferably from about 10 ng to about 500 /l9, more preferably from
about 20 ng to about 10 ~9.
For purposes of regional treatment, the dose range of the
Vitamin D compound is preferably from about I ng to about I mg,
lo preferably from about 10 ng to about 500 ng, more preferably from
about 10 ng to about S0 ng, most preferably from about 20 ng to
about 30 ng.
Preferably, doses are administered over a I day to 6 month
period, more preferably from about I week to about 1 month.
Preferably doses are administered from about once per month to
about S times per day, more preferably from about once per week to
about once per day.
Bone l~u..~r. etic Proteins
In one: ' ~., t of the present invention, a Vitamin D
compound is administered in combination with one or more BMPs to
generate new bone growth in a mammal. These BMPs are preferably
selected from the group consisting of BMP-I, BMP-2, BMP-3, BMP-4,
BMP-S, BMP-6 and BMP-7.
A safe and effective amount of a BMP, preferably selected
from the group consisting of BMP-I, BMP-2, BMP-3, BMP-4, BMP-S,
BMP-6 and BMP-7, is dosed in combination with a Vitamin D
compound .
A preferred dose range for administration of the BMP for
systemic treatment is from about I pg to about 100 19, preferably
from about I ng to about 10 1l9, ~ore preferably from about 10 ng
to about 2.5 ug.
For purposes of regional treatment, a preferred dose range
for the BMP is from about I pg to about 100 119. more preferably
from about 1.5 IL9 to about 90 ug, preferably from about 1.8 ~9 to
about 75 1~9, more preferably from about 2.0 1~9 to about S0 1l9,
more preferably still from about 2.2 1~9 to about 25 ag, more
preferably from about 2.3 1~9 to about 10 119, most preferably from
about 2.5 !~9 to about 5 1~9. Preferably the dose range is at least
~bout 2 . 5 ,ug .
WO 92/21365
,, : A PCI-/US92~0435C
2~ 10-
Preferably, doses are administered over a 1 day to 6 month
period, more preferably from about I week to about I month.
Preferably doses are administered from about once per month to
about S times per day, more preferably from about once per week to
S about once per day.
As used herein, ~BMP-I~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO~
As used herein, "BMP-2~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO:2.
Io As used herein, ~BMP-3~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO:3.
As used herein, "BMP-4~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO:4.
As used herein, rBMP-S~ means a peptide encoded by a DNA
IS sequence comprising SEQ ID NO:S.
As used herein, ~BMP-6~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO: 6.
As used herein, "BMP-7~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO: 7.
As used herein, rA~, rT~, ~G~, and ~C~ refer to the nucleo-
tides containing adenine, thymine, guanine and cytosine
respectively.
Osteoinductive Extract
Another component of the invention is ~n osteoinductive
extract. As used herein, ~osteoinductive extract~ means a
chemical extract of bone, comprising one or more various bone
.~ ., tic proteins, including, but not limited to, BMP-1,
BMP-2, BMP-3, BMP-4, BMP-S, BMP-6 and BMP-7, wherein each BMP has
a molecular weight of from about 28,000 to about 40,000 daltons.
The 28,000 to 40,000 dalton molecular weight r~nge is in
reference to the BMP's dimer weight. Preferably, the molecular
weight of the dimer is from about 30,000 to about 34,000 daltons.
The BMP dimer comprises two monomers, each having a molecular
weight of from about ~4,000 to about 20,000 daltons, preferably
from about 15,000 to about 17,000 daltons.
A preferred method of obtaining the osteoinductive extract is
~s fol l ows:
Snip the skin at the ankles of a 7-8 week old Long-Evans r~t
(Charles River laboratories, ~ilmington, MA). Remove both tibiae
, ... . ...... . ..
W0 92/2i365 ~ PCr/US92/04356
and pl~ce in cold water. Rinse the bone with distilled water to
remove non-osseous t~ssue (t~ssue other than bone). Allow the
bone to air dry. Grind the bones by placing ~n an Osterizer
(Oster Co~mercial, Hilwaukee, 1~) blender with water and ice.
llith the blender set at ~liquefy~ speed, cont~nue to add bone.
Allow the blended material to settle for a few minutes. Decant
the liquid layer. Place the solid layer on a stirring plate and
add distilled water to wash. Continue washing until the distilled
water washes clear. Once the distilled water is clear, add ice
lo and stir. Add I ml of lmM of phenylmethylsulfonyl fluoride
(PM5F). ~lash for I hour adding ice frequently. Repeat with a
second water wash. Place the sample in an ice water bath on a
stirring plate. Defat with absolute ethanol, then defat twice
with ethyl ether. Spread bone material onto glass petri dishes.
IS Allow the bone chips to air dry overnight.
~eigh the bone chips following the overnight drying. Using a
sieve (U.S.A. Standard Sieve Series, Newark llire Cloth Co.,
Newark, N.J.; sieve ~40 retains particles greater than ~25 ~m and
sieve ~170 retains particles greater thàn 90 ~m), isolate the bone
particles in the 90-425um range. Grind any particles greater than
42511m in a MicroMill (Scienceware Bel-Art Products, ~
NJ) for I minute adding dry ice to the bone part~cles to keep the
material cold. Repeat the sieving and MicroMill grinding steps of
the greater than 4251Jm particles until the amount of total
recovery is greater than 2/3 of the initial weight of the bone.
Store the particles at 4C until the next step. I~eigh the
particles isolated thus far. For each gram of particles, add 25
ml of 0.6N HCl. Stir vigorously at 4-C for 2 hours. After 2
hours, stop stirring and allow the part~cles to settle. Decant
the HCl. Add fresh 0.6N HCl and stir again for 2 hours. Decant
the HCl and add fresh 0.6N HCl a third time and stir for two
hours. Decant the HCl and rinse with distilled water. Using
litmus paper, check the pH of the water for the presence of HCl.
Continue rinsing with distilled water until the pH is between
about S and S.S. Rinse the bone particles with ethanol three
times. Swirl, allow to settle, and remove the supernatant. Rinse
the bone particles with ethyl ether three ttmes as above. Dry
overnight in glass plates. The dried bone parttcles are referred
to as ~acid demineralized bone part~cles~.
. _ _ _ _ _
WO 92/21365 2 ~ 1 0 ~ ~ PCr/USs2~043s6
- 12 -
The acid demineral ized bone particles are deproteini2ed as
follows: lleigh the material following the overnight drying. For
each gram of material, add a solution of 30 ml ~H guanidine-HCl,
IOmM Tris and 1.0mM PMSF pH 6.4 to the bone materi~l in a beaker.
S Extract for 16 hours at 4- with vigorous stirring. Following the
16 hour extraction, cease stirring and allow the material to
settle. Pour off the guanidine solution and save. Extract the
material a second time for 6-7 hours using fresh guanidine-HCl
solution. Following the extraction, pour off the solution and
combine with the previously saved solution. The bone particles
are now demineralized and deproteinized.
Dialyze the saved guanidine-HCl solution against distilled
H~O at 4C using SO mm dialysis tubing (3SOO molecular weight
cutoff). Following dialysis, lyopholize the material and resolu-
ls bilize the lyophilized material in 4M Urea-O.OSH Tris-O.lM NaCl,
pH 7.4. Mix the solubilized material in a conical centrifuge tube
with Heparin-Agarose and mix overnight on a rotator at 4C. Pour
the Heparin-Agarose slurry into a column. Iiash with I column
volume 4M urea, O.OSM Tris, O.IM NaCl, pH 7.4 buffer. Collect the
fraction. Ilash ~ith 3 column volumes of 4M urea, 0.05M Tris, 0.2M
NaCl, pH 7.4 buffer. Step off the material with 3 column volumes
of 4M urea, O.OSM Tris, 0.75M NaCl, pH 7.4. Concentrate this
sample in a SO ml Amicon concentrator (Amicon Corp., Danvers, MA)
with filter (10,000 molecular weight cut off) to about 4-Sml.
Assay for protein concentration using BCA (bicinchoninic acid)
Protein Assay Reagent (Pierce, Rockford, IL) and dialyze (3500
molecular weight cutoff dialysis tubing) in 41i guanidine-O.Ol M
Tris pH 7.4. Load material on Sephacryl S-200 column and collect
fractions. The fractions containing the major protein peak are
dialyzed against IM acetic acid and assayed for activity.
Active fractions from the gel filtration ~re combined and
dialyzed against three changes of 6M urea, 25mM Ha acetate, pH
4.6. The dialysate is loaded onto a column of carboxymethyl-
sepharose (CM-Sepharose) equilibrated with the s~me buffer. The
3s column is washed with 6M urea, 25mM Na acetate, pH 4.6 and
~ctivity eluted using a O - O.SM NaCl gradient. Fractions are
analyzed for protein concentration and sodium dodecyl sulfate gel
ele_Lr~ sis. The activity located in the seven fractions
before and after the beginning of the major protein peak are
,,, _ . . . _ _ .
wo
92/21365 2 1 1 ~ 4 1 ~; PcrJUS92/04356
- 13 -
pooled for further pur1f~cation.
The pooled CM-Sepharose fractions are dialyzed three times
for 2~ hours each against 1% acetic acid. The dialysate is
- lyophilized to dryness and the protein pellet dissolved into 30 ml
s of 6M urea, 0.5M NaCl, 25mM Na phosphate, pH 7.4. The sample is
applied on a column of chelating Sepharose charged with zinc and
equilibrated with the abov~ buffer. The column is washed with the
above buffer and then eluted with a gradient from 6M urea, 0.5M
llaCl, 25mM Na phosphate, pH 7.4 to 6M urea, 0.5M NaCl, 25mM Na
acetate, pH 4.6. Aliquots of each fraction are labeled with l2'l
and analyzed by SDS gel ele~r" ' .~sis. Aliquots (lO0 pl) of
each fraction are co~bined with 400 pl of elution buffer, dialyzed
against 1% acetic acid and assayed for activity. Highly purified
molecular weight range (Mr) 25-40 kD peptides are assayed in the
bone induction assay.
A safe and effective amount of osteoinductive extract is
dosed in combination with a Vitamin D compound. For purposes of
systemic treatment, the osteoinductive extract dosed preferably
comprises ~t least one BMP in an amount from about 1 pg to about
100 pg, preferably from about 1 ng to about 10 pg, more preferably
fro~ about 10 ng to about 2.5 pg.
For purposes of regional treatment, the osteoinductive
extract dosed preferably comprises at least one BMP in an amount
from about 1 pg to about 100 pg, more preferably from about 1.5 pg
to about 90 pg, preferably from about 1.8 pg to about 75 pg, more
preferably from about 2.0 pg to about 50 pg, more preferably still
from about 2.2 pg to about 25 pg, more preferably from about 2.3
pg to about lO pg, most preferably from about 2.5 pg to about 5
1~9. Preferably the dose range is at least about 2.5 pg.
Preferably, doses ~re administered over a 1 day to 6 month
period, more preferably from about 1 week to about 1 month.
Preferably doses are administered from about once per month to
about 5 times per day, more preferably from about once per week to
~bout once per day.
",~": ~licallY AcceDtable Carrier
The Vitamin D compound, osteoinductive extract, or BMP may be
~dministered vi~ a pharm~ceutically acceptable carrier. The term
'. ~ic~lly-~ccept~ble carrier~, ~s used herein, means one
WO 92~21365
~ 4l0 -14 PCI/USgz/04356
or more compattble solid or liquid filler diluents or encapsu-
lating substances which are suitable for administration to a human
or lower animal. The term ~compatible~, as used herein, means
that the components of the pharmaceutical compositions are capable
S of being commingled with the compound(s) of the subject invention,
and with each other in a manner such that there is no interaction
which would substantially reduce the pharmaceutical efficacy of
the pharmaceutical composition under ordinary usage situations.
Pharmaceutically-acceptable carriers must, of course, be of
sufficiently high purity and sufficiently low toxicity to render
them suitable for administration to human or lower animal being
treated .
Some examples of substances which can serve as pharmaceuti-
cally-acceptable carriers are sugars such as lactose, glucose and
sucrose; starches such as corn starch and potato starch; cellulose
and its derivatives, such as sodium ~u~ . thylcellulose,
ethyl cel 1 ul ose, cel 1 ul ose acetate; powdered tragacanth; mal t;
gelatin; talc; stearic acid; magnesium stearate; calcium sulfate;
vegetable oils such a peanut oil, cottonseed oil, sesame oil,
olive oil, corn oil and oil of theobroma; polyols such as
propylene glycol, glycerine, sorbitol, mannitol, and polyethylene
glycol; sugar; alginic acid; pyrogen-free water; isotonic saline;
phosphate buffer solutions; cocoa butter (suppository base);
emulsifiers, such as the Tweens; as ~ell as other non-toxic
compatible substances used in pharmaceutical formulations.
Wetting agents and lubri~ants such as sodium lauryl sulfate, as
well as coloring agents, flavoring agents, excipients, tableting
agents, stabilizers, antioxidants, and preservatives, can also be
present. Other compatible pharmaceutical additives and actives
(e.g., NSAI drugs; pain killers; muscle relaxants) may be included
in the pharmaceutically-acceptable carrier for use in the
compositions of the present invention. For example, art-known
local anesthetics may be included in the pharmaceutically-accept-
able carrier (e.g., benzyl alcohol; Novocaine~; lidocaine).
Additional examples of carriers include collagen, deminer-
alized bone particles, ceramic and metallic implant materials,
collagen membrane and bone grafts (isogenic or allogenic)~
The choice of a pharmaceutically-acceptable carrier to be
used in conjunction with the compounds of the present invention is
_ _ _ _ _ ,
21 10410
- 15 -
determined by the way the compound is to be ~dministered. The
, preferred modes of administering the compounds of the present
invention are by injection, oral administration, topical-oral
administration, and nasopharyngeal administration or a combination
S of modes ~i.e., osteoinductive extract via injection and Vitamin
compound via oral administration). If the compound is to be
injected, the preferred pharmaceutically-acceptable carrier is
sterile, physiological s~line. Suitable pharmaceutically-
acceptable carriers for oral administration include those suited
for tablets, and capsules. Suitable pharmaceutically-acceptable
carriers for topical-oral administration include those suited for
pastes, gels, and liquids. Suitable pharmaceutically-acceptable
carriers for nasopharyngeal administration include those suited
for drcps, sprays, mists and pawders.
A separate pharmaceutically-acceptable carrier may be used in
conjunction with each active component of the present invention or
a single pharmaceutically-acceptable carrier may be employed in
conjunction with a mixture of the active components of the present
invention. In either case, the pharmaceutically-acceptable
carrier is used at a concentration sufficient to provide a
practical size to dosage relationship. The pharmaceutically-
acceptable carriers, in tot~l, may comprise from about 0.1% to
about gg.g9959% by weight of the pharmaceutical compositions of
the present invention, preferably from about 50X to about gg.gg9X,
and most preferably from ~bout 75% to about g9.9%.
Specific oral and inject~ble carriers useful in this
invention are described in the following U.S. Patents: U.S. Patent No. 4,401,663,
Buckwalter, et al, issued August 30, 1983; U.S. Patent No. 4,424,205, LaHann, et
al, issued January 31, 1984; U.S. Patent No. 4,443,473, Buckwalter, et al, issued
April 12, 1984; U.S. Patent No. 4,493,848, LaHann, et al, issued January 15, 1984.
RCl!l c:: cllLLLi yc ~ of the present invention are provided in
the Examples hereinafter.
Pl,~ ly-acceptable carriers suitable for the preparation of unit dosage
forms for oral a.~ ;."., topical-oral ad,.1i11i~L~iu1~, nasopharyngeal
A~11~1;1~;~11,11;~ 11l and injection are well-known in the art. Their selection will depend
on secondary ~ like taste cost, and/or shelf stability,
-'
21 10410
- 16 -
which are not eritical for the purposes of the present invention, and can be made
without difficulty by a person skilled in the art. Pl~ liy-acceptab~e
earriers useful in the rnmrn~itl~mC of the present invention are deseribed more
S fully hereinafter.
A. Oral Dose ~ormc
Preferably, the vitamin D eompound is adl.lilu~,clc~ via an oral dose
form. Various oral dosage forms can be used, including such solid forms as
tablets, capsules, grarlules, bulk powders and ulicLu~a~J~ulc~ of the drug. These
oral forms comprise a safe and effective amount, usually ât least about .5%, andpreferab~y from about 1% to about 10% of tbe compound of the present
invention. Tablets can be compressed, enteric-coated, sugar-coated or film-
coated containing suitable binders, lubricants, surfactants, diluents, ~ lr~ g~
âgents, colormg agents, f~avoring agents, ~JIc~lYali~, flow-inducing agents,
and melting agents. Liquid oral dosage forms include aqueous and
solutions, emulsions, ~U*)~ iol~ solutions and/or cll~r~n~inne
from non-crrt~ granules, containing suitable solvents, IJlC:~ClV~
clL~Ul~iryillg agents, suspending agents, diluentc, sweeteners, melting agents,
coloring agents, and flavoring agents. Preferred earriers for oral a~lluli~lLa~ion
inelude gelatin and propylene glyeol. Speeifie examples of ~,1.", 1.,;.,~,.1;. ~lly-
acceptable carriers and e%cipients that may be used in r.""",l~l;"~ oral dosage
forms eontaining compounds of the present invention are described in U.S.
Patent 3,903,297, Robert, issued September 2, 1975. Techniques and
cnmrocition~ for making solid oral dosage forms are described in Marshall,
"Solid Oral Dosage Forms," Modern E~ C Vol. 7. (Banker arld
Rhodes, editors), 359-427 (1979). Techniques and eompositions for making
tablets (~UlllplC;~,d, formulas amd molded), capsules (hard and soft gelatin) and
pillsaredescribedinRemin~ton'sPl,..",~ irs~lSciences(ArthurOsol,editor),
1553-1593 (1980).
B. Topieal-oral I:~ose Forms
"Topical-oral carrier", as used herein, denotes a carrier for the
component of interest which results in a composition which is administered
topically to the oral cavity, held therein for a period of time, and then is largely
c~lJc.,~ d rather than being
.~
..... ..
W092/2136~ 211i~D Pcr/us92Jo4356
- 17
swallowed. Such compositions include toothpastes, tooth gels,
tooth powders, mouthwashes, mouthsprays, prophyl axi s pastes,
dental treatment solutions, biogels or other sustained release
- products, and the like.
S Components of the topical-oral carrier are suitable for
administration to the oral cavity of a human or lower animal and
are compatible with one another and the other components, espe-
cially the Vitamin D compound and oste6inductive extract or BMP,
used in an oral composition of the subject invention. Preferred
topical-oral carriers thus provide the desired characteristics for
toothpastes, tooth gels, tooth powders, mouthwashes, mouthsprays,
prophylaxis pastes, dental treatment solutions, and the like. The
topical-oral carriers of the subject invention comprise components
typically used in such compositions which are well known to a
lS skilled practitioner. Such components include, but are not
limited to anticaries agents, antiplaque agents, anticalculus
agents, dental abrasives, surfactants, flavoring agents,
sweetening agents, binders, humectants, thickening agents,
buffering agents, preservatives, coloring agents and pigments,
ethanol, and water.
Preferred compositions of the subject invention are in the
form of toothpastes. Components of such toothpastes generally
include a dental abrasive (from about 10% to about 50%), a sur-
factant (from about O.SX to about IOX), a thickening agent (from
about O.IX to about 5X) a humectant (from about IOX to about 55%),
a flavoring agent (from about 0.04X to about 2X), a sweetening
agent (from about O.lX to about 3X), a coloring agent (from about
0.01% to about 0.5X) and water (from about 2X to about 45X). Such
toothpastes may also include one or more of an anticaries agent
(from about 0.05% to about 0.3% as fluoride ion), an anticalculus
agent (from about O.lX to about 13%), and an antiplaque agent
(from about 0.1% to about SX).
Other preferred compositions of the subject invention are
mouthwashes and mouthsprays. Components of such mouthwashes and
mouthsprays include water (from about 45% to about 95X), ethanol
(from about 0% to about 25%), a humectant (from about OX to about
50X), a surfactant agent (from about 0.01% to about 7%), a
flavoring agent (from about 0.04% to about 2X), a sweetening agent
-(from about 0.1% to about 3%), and a coloring agent (from about
2~ ~4~0
- 18 -
0.001% to about 0.5%). Such mouthwashes and mouthsprays may a1so
include one cr more of an anticaries ~gent (from about O.QS% to
about 0.3X as fluoride ion), an anticalculus agent (from about
O.OIX to about 3%), and an antiplaque agent (from about 0.1% tc
S about SX).
Other preferred compositicns of the subiect inventicn are
dental soluticns. Components cf such dental soluticns generally
include water (from about 90% to about 99X), preserYative (frcm
about 0.01% tc abcut 0.5%), thickening agent (from about OX to
about 5%), flavoring agent (from about 0.04% to about 2%),
sweetening agent (from about 0.1% to about 3%), and surfactant
(from OX to about SX).
"Topical-oral carrier~ as used herein, also denotes fibers,
strips or tubes which can be impregnated with the active
components of the present invention and inserted or implanted into
a periodontal pocket. Such compositions of the subject invention
can readily be achieved by one of ordinary skill in the art using
the teachings disclosed hereinbefore, the following references.
and related well-known technol-
ogies: U.S. Patent No. ~,666,897 issued to Golub, McNamara
lhy on May 19, 1987; European Patent Application No.
244,118 Al in the name of Baker. published on November 4, 1987;
European Patent Appl ication No. 286,802 A2 in the name cf
Kametaka, Miyazaki, Hayashi, Handa ~ I(ameda, published Octcber 19,
1988; Addy, M., L. Rawle, R. Handley, H. Newman ~ J. Ccventry,
~The development and in vitro ev~luation cf acryl ic strips and
dialysis tubing fcr local drug delivery~, Jcurnal of Periodon-
toloqY, Vol. 53 (1982), pp. 693-698; Goodson, J.M., A.D. Haffajee
~ S.S. Socransky, ~Periodontal therapy by local delivery cf
tetracycl ine, Journal of Cl inical PeriodontoloaY, Vol . 6 (1979),
pp. 83-92; Goodson, J., D. Holborow, R. Dunn, P. Hogan ~ S.
Dunham, ~Monolithic tetracycline containing fibers for controlled
delivery tc periodontal pockets~, Journal of PeriodontclcqY, Vcl.
54 (1983), pp. 575-579; Dunn, R., J. Gibson, B. Perkins, J.
Goodson ~ L. ~aufe, ~Fibrous delivery systems fcr antimicrobial
agents~, PolYmer Science and Technoloav. Vol. 32 (1985), pp.
47-S9; Dunn, R., J. Gibson, B. Perkins, J. Goodson ~ L. Laufe,
~Fibrous delivery systems for antimicrobial agents~, Polvmer
Material Science Enqineerinq, Vol. Sl (1984), pp. 28-31; Olanoff,
21 10410
- 19 -
L. ~ J. Anderson, 'Controlled rslease of tetr~cycline ~ A
physiological ph~,",~-G~in~tic model of the pregnant r~t-, ~Qy~
of Pharmacokinetics and BioDharmaceutics. Yol. 8 (1980), pp.
599-620; Elkayam, R., H. Friedman, A. Stabhol2, A. Soskolne, H.
Sela ~ L. Golub, ~Sustained release deYice containing minocycline
for local treatment of pericdontal disease~, Journal of Ccntrolled
~, Vol. 7 (1988), pp. 231-236; and Goodson, J r 'Multi-
center evaluation of tetr~cycline fiber therapy. I. Experimental
Design~, Journal of Oent~l Research, Vol. 68 (1989), p. 197; and
r~F~I .. ~es cited therein.
C. Iniectable Oose Forms:
The actiYe components of the present invention are also
useful when injected. The dosage of the active components of the
present invention which is both safe and effective to provide bone
growth activity will vary ~ith the particular condition being
treated, the severity of the condition, the duration cf treatment,
the specific mixture of compounds employed and its usage concen-
tration, and like factors within the specific knowledge and
expertise of the ~ttending physician and ~ ~te with a
reasonable benefit/risk ratio associated with the use of any drug
compound. In addition, lower dosages will be utilized when only
local or minor bone growth is desired, whereas higher dosages will
be utilized when general or major bone gro~th is desired.
Hethods and m~terials for manufacturing injectables can be
found in Reminqton' s Ph.""~ tical Sciences. 17ed., 1985, Ch~pter
85, p. 1518. Preferably, the injectable compo-
sition is an aqueous solution.
The aqueous solutions preferably consist of water (preferably
from about 80% to about 99.999X), ~ suitable solubilizer, various
types of acids, and an antimicrobial agent. Several solubilizers
are known . Exampl es of such sol ubi l i zers are as fol l ows: urea
compounds (e.g., urea; urethan); surfactants (e.g., Tweens; Spansi
sodium deoxycholate and Pluronics); cellulosic agents (e.g.,
Cdr~G~j thylcellulose); L.,bu~ tes (e.g., sorbitol; mannitol~;
~ vitamins (e.g., nicotin~mide); xanthine derivatives; and ~lco-
hols (e.g., benzyl alcohol). Examples of acids to be used include
the following: glucuronic; galacturonic; fumaric; gentisic;
~cetic; citric and l~ctobionic. Types of antimicrobial agents
~' ,
_
W092/21365 , ~ 4ln PCI~/US92~04356
_ - 20 -
that c~n be used are the following: phenylmercur~c n~trate;
th~merosal; benzethon~um chlor~de; benzalkonium chloride; phenol;
cresol; and chlorobutanol . An art-known local anesthet~c (e.g.
benzyl alcohol; ~lovoca~ne~; lidocaine) may also be included.
Preferably the osteo~nductive extract and the BMP s are
adm~nistered v~a an ~njectable dose form.
The follow~ng examples further descr~be and demonstrate the
preferred . - I im l,s ~l~th~n the scope of the present ~nvent~on.
The examples are g~ven solely for the purpose of ~llustration and
are not to be construed as lim~tat~ons of the present ~nvent~on
s~nce many variat~ons thereof are poss~ble without depart~ng from
~ts sp~r~t and scope.
EXAMPLE I
An ~njectable composit~on compr~s~ng the osteo~nduct~ve
lS extract and an oral composit~on compr~s~ng 1 25-dihydroxy Vitamin
D3 for bone fracture repair ~s prepared by comb~n~ng the follow~ng
components ut~l~z~ng conventional mix~ng techn~ques.
BMP comDosit~on
Percent by ~le~ght
Z0ComDonent of ComDosition
BMP - I 0 . 04
NaCl o . 90
Ster~le water a.s.
100 . 00
1.25-d~hvdroxY Vjtamin D comDosit~on
Percent by ~e~ght
ComDonent of ComDosit~on
1 25-d~hydroxy Vitam~n D 0.01
30Corn starch 18 . 49
Lactose 63.00
Tal c 18 . 00
Stear~c ac~d O.S0
100 . 00
0.1 cc of the BMP composit~on ~s ~njected ~nto the fracture
site at the time of fracture reduction and once daily thereafter.
100 119 of the 1 25-dihydroxY Vitam~n D3 composit~on is orally
adm~n~stered 2~ hours before fracture reduction and once daily
- _ thereafter. The BMP and 1 25-dihydroxy Vitamin D, are admin~s-
, ~_
.
.
W092/21365 2llaA ~ a Pcr~US92/04356
tered until desired repair is ~chieYed, perferably over a seven
day period.
EXAHPLE 1 1
An injectable composition for bone fracture repair is
s prepared by combining the following components ut~lizing conven-
t i onal mi xi ng techn i ques .
Percent by Weight
ComDonent of CQmpQs i t i on
BMP - 2 0 . 04
lo25-hydroxy Vitamin D2 .l
NaCl 0.09
Sterile water for injection o.s.
100 . 00
0.1 cc of the composition is injected into the fracture site
Is at the time of fracture reduction and once daily thereafter until
desired repair is achieved.
EXAMPLE I 11
A composition for inducing bone growth following reconstruc-
tive surgery is prepared by combining the following components
utilizing conventional mixing techniques.
Percent by ~leight
ComDonent of ComDos i t i on
BMP-3 0.04
1,25-dihydroxy Vitamin D2 0.01
25NaCl O . 90
Steri 1 e water o . s .
100 .00
0.1 cc of the composition per cm2 of surface area of
surgically r, ~ru~ed bone is deposited directly onto the bone
30surface.
EXAMPI F IV
A composition for accelerating the healing and providing a
stronger bond between natural bone and an artificial prosthesis is
prepared by combining the following components utilizing conven-
3stional mixing techniques.
Percent by l~leight
ComDonent of ComDosition
BMP- 1 0 . 04
BHP- 2 0 . 04
WO 92~21365 PCI`/US92/04356
- 22 -
BMP-~ 0.01
24,25-dihydroxy Yitamin D3 O.OI
NaCl 0 . 90
Steri 1 e water q . s .
100.00
0.1 cc of the composition per cm2 surface area of natural
bone proximate to the prosthesis is deposited directly onto the
natural bone.
EXAMPLE V
A topical oral carrier composition for periodontal therapy is
prepared by combining the following components utilizing conven-
tional mixing techniques.
Percent by ileight
ComDonent of ComDosition
SBMP-2 0.04
NaCl O . 90
Steri 1 e water a . s .
100 .00
After the patient is prepared using conventional periodontal
surgical therapy O.l cc of the composition per exposed tooth is
deposited into the surgery site. Soft tissue flaps are then
sutured to close the surgical site. This treatment is useful for
restoring alveolar and supporting bone in the periodontium lost by
di sease .
EXAMPLE Vl
An injectable composition comprising the BMPs 2, 3, 4 and S
and an oral composition comprising 1,25-dihydroxy Vitamin D3 for
treatment of osteoporosis is prepared by combining the following
components utilizing conventional mixing techniques.
osteoinductive extract comDosition
Percent by ~leight
ComDQnent of ComDos i t i on
BMP-2 O.OOl
BMP-3 O.OOl
35BMP-4 O.OOl
BMP - S 0 . 001
NaCl O . 9O0
Steri 1 e water a . s .
100.000
WO 92/21365 PCI`/US92/0435
2110~10 6
1.25-dihYdroxY Vitamin D~ comDosit~on
Percent by Weiqht
CompQnent of ComDosit~on
1, 25 -di hydroxy Vi tami n D, 0 . 01
Corn starch 18.49
Lactose 63 . 00
Tal c 18 . 00
Stearic acid 0.50
100 .00
1 cc of the BMP composition is injected intravenously once
per day~ 50 mq of the 1,25-dihydroxy Vitamin D3 composition is
orally administered within one hour of the osteoinductive extract
injection and once daily thereafter. The osteoinductive extract
and 1,25-dihydroxy Vitamin D3 are administered over a 7-day
period.
EXAMPLE VII
A composition for inducing bone growth of a non-union
fracture is prepared by combining the following components
ut~lizing conventional mixing techniques. As used herein,
~non-union fracture~ means a fracture that has failed to heal
normal ly.
Percent by Weight
ComDonent of ComDosition
8MP - 4 0 . 004
251,25-dihydroxy vitamin D3 0.01
Ac~d dem~neral~zed bone particles go.ooo
NaCl 0.900
Sterile water for ~njection ~.s.
100 . 000
At the time of fracture reduct~on, a sufficient quant~ty of
the above composit~on ~s deposited d~rectly into the non-union
site thereby filling in ~ny bone deficit.
WO 92/2136~
PCI /US92/043~i6
211~ 410 SEQUENCE LISTING
(1) 6ENERAL INFORMATION:
(i) APPLICANT: STONE, ROGER L.
(ii) TITLE OF INVENTION: THERAPEUTIC FORMULAS FOR O5TEOINDUCTION
(iii) NUMBER OF SEQUENCE5: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: The Procter ~ Gamble Company
(B) STREET: 11810 East Miami River Road
(C) CITY: Cincinnati
(D) STATE: Ohio
( E) COUNTRY: U . S .A .
(F) ZIP: 45239-8707
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFT~IARE: Patentln Release ~1.0, Version ~1.25
(vi ) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
( v i i i ) ATTORNEY/AGENT INFORMAT ION:
(A) NAME: Corstanje, Brahm J.
(B) REGISTRATION NUMBER: 34,804
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 513-245-2B58
(B) TELEFAX: 513-7~1-3012
WO 92/2
1365 ~ 4 1 o Pcr/usg2/o4356
- Z5 -
(2) INFORMATION FOR SEQ ID NO:I:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2487 base pairs
S (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
(D) TOPOLO6Y: l inear
( i i ) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:I:
lSGCC6CTTCCC TCGCCGCCGC CCCGCCAGCA TGCCCGGCGT GGCCCGCCTG CCGCTGCTGC 60
TbbGGI,~b~,l GCTGCTCCCG CGI~CCGGCC GGCCGCTGGA CTTGGCCGAC TACACCTATG 120
ACCTGGCGGA r~qr~ CAC TCGGAGCCCC TCMCTACM AGACCCCTGC MGGCGGCTG 180
C~ I1,I IbG GGACATTGCC CTGGACGMG AGGACCTGAG GGCCTTCCAG GTACAGCAG6 240
CTGTGGATCT CAGACGGCAC ACAGCTCGTA AGTCCTCCAT CAMGCTGCA GTTCCAGGAA 300
25ACACTTCTAC CCCCAGCTGC CAGAGCACCA ACGGGCAGCC TCAGAGGGGA GCCTGTGGGA 360
GATGGAGAGG TAGATCCCGT AGCCG6CGGG CGGCGACGTC CCGACCAGAG CGTGTGTGGC 420
CCGATGGGGT CATCCCCTTT GTCATTGGGG GMMCTTCAC TGGTAGCCAG AGGGCAGTCT 480
TCCGGCAGGC CATGAGGCAC TGGGAGMGC ACACCTGTGT CACCTTCCTG GAGCGCACTG 540
ACGAGGACAG CTATATTGTG TTCACCTATC GACCTTGCGG GTGCTGCTCC TACGTGGGTC 600
35 G~CGCr'~ r~'CCCCAG GCCATCTCCA TCGGCMGM CTGTGACMG TTCGGCATTG 660
TGGTCCACGA GCTGGGCCAC 6lbbll~b6~l TCTGGCACGA ACACACTCGG ccA~ cccct`~ 720
ACCGCCACGT TTCCATCGTT CGTGAGMCA TCCAGCCAGG GCAGGAGTAT MCTTCCTGA 780
WO 92/21365 PCl'/US92/04356
21ila4lo -26-
AGAT6GAGCC TCA66A66T6 GAGTCCCT66 666A6ACCTA T6ACTTC6AC A6CATCAT6C 8~0
ATTAC6CTCG 6MCACATTC TCCA6666CA TCTTCCTGGA TACCATT6TC CCCM6TAT6 900
S A66T6MCGG GGTGMMCCT CCCATT66CC AA/`rC~r4C6 6CTCA6CM6 66G6ACATT6 960
CCCA!\rCCCG CM6CTTTAC M6T6CCCA6 CCTGTG6A6A 6ACCCT6CM 6ACA6CACA6 1020
6CMCTTCTC CTCCCCT6M TACCCCMT6 6CTACTCT6C TCACAT6CAC T6C6T6T66C ~080
GCATCTCTGT CACACCCGGG GAGMGATCA TCCTGMCTT CACGTCCCTG GACCTGTACC 1140
GCAGCC6CCT GTGCTGGTAC GACTATGTGG AGGTCCGAGA TGGCTTCTGG ArCAArrCGC 1200
' ~ CCCTCCGAGG CCGCTTCTGC GGGTCCMMC TCCCTGAGCC TATCGTCTCC ACTGACA6CC 1260
GCCTCTGGGT TGMTTCCGC AGCAGCAGCA ATT6GGTTGG MMGGGCTTC TTTGCAGTCT 1320
ACGM6CCAT l.lbCG66Gbl 6ATGTGMAA A66ACTATG6 CCACATTCM TC6CCCMCT 1380
ACCCAGACGA TTACCGGCCC AGCMMGTCT GCATCTGGCG 6ATCCA6GTG TCT6A6G6CT 1440
TCCAC6T66G CCTCACATTC CAGTCCTTT6 A6ATTGA6C6 CCAC6ACA6C T6T6CCTACG 1500
25ACTATCT6GA G6T6CGCGAC G66CACA6TG AGAGCAGCAC CCTCATCGGG CGCTACT6TG 1560
GCTAT6AGM GCCTGATGAC ATCMGA6CA CGTCCAGCCG Cl, 1~ 1 bG(, I 1, MGTTCGTCT 1620
CT6AC666TC CATTMCMA GCG6GCTTTG CC6TCMCTT TTTCMAGAG GT66AC6A6T 1680
6CTCTC66CC CMCC6C666 66CT6T6A6C A6CG6T6CCT CMCACCCT6 6GCA6CTACA 1740
AGT6CA6CT6 T6ACCCC666 TAC6A6CT6G CCCC.4r'\r~A 6CbbCG~lbl 6A66CT6CTT 1800
356T66C6GATT CCTCACCM6 CTCMC66CT CCATCACCA6 CCC6661.1b6 CCCM66AGT 1860
A~CCCCCCAA CM6MCT6C ATCT66CA6C r6blb6CCCC CACCCA6TAC C6CATCTCCC 1920
T6CA6TTT6A CTTCm6AG A~'~r'~G'rr4 ATGATGTGTG CAA6TAC6AC TTC6TGGAGG 1980
WO 92~
21365 PCI~/US92/04356
22il~
TGCGCAGTGG ACTCACAGCT GACTCCMGC TGCATGGCAA li1 l~lbl~bl TCTGAGMGC Z040
CCGAGGTCAT CACCTCCCAG TACMCMCA TGCGCGTGGA GTTCMGTCC GACMCACCG 2100
s TGTCCAAAM GGGCTTCMG GCCCACTTCT TCTCAGMM GAGGCCAGCT CTGCAGCCCC 2160
CTCGGGGACG CCCCCACCAG CTCMMTTCC GAGTGCAGM MGMMCCGG ACCCCCCAGT 2220
GAGGCCTGCC AGGC~1~CCG GACCCCTTGT TACTCAGGM CCTCACCTTG GACGGMTGG 2280
GATGGGGGCT TCGGTGCCCA CCMCCCCCC ACCTCCACTC TGCCATTCCG GCCCACCTCC 2340
GCCGG ACAGMCTGG Tl.u11,1[,1 1C TCCCCACTGT GCCC61~GC GGACCGGGGA 2400
lS CCCTTCCCCG TGCCCTACCC CCTCCCAm TGATGGTGTC TGTGACATTT CCTGTTGTGA 2460
AGTAAAAGAG GGACCCCTGC GTCCTGC 2487
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(AJ LENGTH: 1547 base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
( i i ) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGGGACTTCT TGMCTTGCA GGGAGMTM CTTGCGCACC CCACTTTGCG CC61,1i,~11 60
TGCCCCAGCG GAGCCTGCTT CGCCATCTCC GAGCCCCACC GCCCCTCCAC ~C~1G66C~1 120
TGCCCGACAC TGAGACGCTG TTCCCAGCGT rNVU~ C ACTGCGCGGC cr~cA~ccrr~ 180
CA'`~'\r'`~CC l\C~r~r~. Mî~^~.Cr'`~ CATTCGGTCC TTGCGCCAGG TCCTTTGACC 240
WO 92/21365 ~ 28 - PCI/US92/04356
AGAGTTTTTC CATGTG6ACG CTCTTTCMT GGACGTGTCC CCGCI, ~bl, 11 CTTAGACGGA 300
CTGCGGTCTC CTMMGGTCG ACCATGGTGG CC~ ACCCG CTGTCTTCTA GCGTTGCTGC 360
S TTCCCCAGGT C~ l b~ l b6GC GGCGCGGCTG GCCTCGTTCC GGAGCTGGGC CGCAGGMGT ~20
TCGCGGCGGC GTCIllbG6GC CGCCCCTCAT CCCAGCCtTC TGACGAGGTC CTGAGCGAGT 480
TCGAGTTGCG GCTGCTCAGC ATGTTCGGCC TGMMCAGAG ACCCACCCCC AGCAGGGACG 5~0
CCGTGGTGCC CCCCTACATG CTAGACCTGT ATCGCAGGCA CTCAGGTCAG CCGGGCTCAC 600
CCGCCCCAGA CCACCGGTTG GAGAGGGCAG CCAGCCGAGC CMCACTGTG CGCAGCTTCC 660
15 ACCATGMGA ATCmGGM GMCTACCAG MMCGAGTGG GMMCMCC CGGAGATTCT 720
TCTTTMTTT MGTTCTATC Crr~rG'''\CC AGTTTATCAC CTCAGCAGAG CTTCAGGTTT 780
TCCGAGMCA GATGCMGAT GCTTTAGGM ACMTAGCAG TTTCCATCAC CGMTTMTA 840
mATGMMT CATMMCCT GCMCAGCCA ACTCGMATT CCCCGTGACC AGACTTTTGG 900
ACACCAGGTT GGTGMTCAG MTGCMGCA GGTGGGMMG TTTTGATGTC ACCCCCGCTG 960
25 TGATGCGGTG GACTGCACAG GGACACGCCA ACCATGGATT CGTGGTGGM GTGGCCCACT 1020
TGGAGGAGM ACMGGTGTC TCCMGAGAC ATGTTAGGAT MGCAGGTCT TTGCACCMG 1080
ATGMCACAG CTGGTCACAG ATMGGCCAT TGCTAGTMC TTTTGGCCAT GATGGMMG 11~0
GGCATCCTCT CCACAAMGA GMAMCGTC MGCCMMCA CMMCAGCGG MMCGCCTTA 1200
AGTCCAGCTG TAAGAGACAC CCTTTGTACG TGGACTTCAG TGACGTGGGG TGGMTGACT 1260
35 GGATTGTGGC ICCCCCGGGG TATCACGCCT mACTGCCA CGGAGMTGC CCTTTTCCTC 1320
TGGCTGATCA TCTGAACTCC ACTMTCATG CCATTGTTCA GACGTTGGTC MCTCTGTTA 1380
ACTCTMGAT TCCTMGGCA TGCTGTGTCC CGACAGMCT CAGTGCTATC TCGATGCTGT lU0
WO 92~21365 2 ~ Pcr/US92~043~;6
ACCTTGACGA GMTGMAAG GTTGTATTM AGMCTATCA GGACATGGTT 6TGGA6GGTT IS00
bTGGbIbl~6 CTAGTACAGC AAMTTMMT ACATMATAT ATATATA I547
S(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: I 774 base pai rs
(B) TYPE: nucleic ~cid
I0 (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
( i i ) MOLECULE TYPE: cDNA
'S
(xi) SEQUENCE DESCRIPTION: SEQ rD NO:3:
AGATCTTGM MCACCCGGG CCACACACGC CGCGACCTAC AGCTCTTTCT CAGCGTTGGA 60
GTGGAGACGG C'''CC~CAGC GCCblbCGCG GGT6AG6TCC GCGCAGCTGC TGGGGMGAG 120
CCCACCTGTC AGGCTGCGCT GGGTCAGCGC AGCMGTGGG G~,lbGCCG~,I ATCTCGCTGC I80
25ACCCGGCCGC G I CCCGGGu I CCb ~bbGCCC TCGCCCCAGC TGGTTTGGAG TTCMCCCTC 240
66CII,C6CC6 CCGGCTCCTT GC6CI,llb66 AGTGTCCCGC ACrCACG~G rrq~CC~\~6 300
C~ Ç~GCGÇ GTACCTAGCC ATGGCTGGGG CGAGCAGGCT 6l.~bllll.1b I6G~,IbG6~,l 360
6~11elbCGI GAGCCTGGCG CA''''~ GACCGMGCC ACCTTTCCCG GAGCTCCGCA ~20
MGCTGTGCC AGGTGACCGC ACGGCAGGTG GTGGCCCGGA CTCCGAGCTG CAGCCGCMG 480
35ACMGGTCTC TGMCACATG ~, I bC6GI. 11,1 ATGACAGGTA CAGCACGGTC CArrrrrrcc S~o
CC'1'4CC~C' CTCCCTGGAG GGAGGCTCGC AGCCulbGCG CCu~(,GG~,Il, CTGCGCGMG 600
GCMCACGGT TCGCAGCTTT Cl`''CGrr4' CA~'`4'1MC TCTTGAMGA AAAGGACTGT 660
2 ~ 30 PCl'/US92/04356
ATATCTTCM TCTGACATCG CTMCCMGT CTGMMCAT 1 1 ~bTGll,CC ACACTGTATT 720
TCTGTATTGG AGAGCTAGGA MCATCAGCC TGAGTTGTCC AGTGTCTGGA G6ATGCTCCC 780
5ATCATGCTCA GAGGMMCAC ATTCAGATTG ATCTTTCTGC ATGGACCCTC MMTTCAGCA 840
GMMCCMMG TCMCTCCTT GGCCATCTGT CAGTGGATAT GGCCMMTCT CATCGAGATA 900
TTATGTCCTG GCTGTCTMM GATATCACTC MTTCTTGAG C~ CC~^~ GMAATGMG 960
AGTTCCTCAT AGGATTTMC ATTACGTCCA AGGGACGCCA GCTGCCMMG AG6AGGTTAC 1020
CTTTTCCAGA GCCTTATATC TTGGTATATG CCMTGATGC CGCCATTTCT GAGCCAGMM 1080
15GTGTGGTATC MGCTTACAG GGACACCGGA ATTTTCCCAC TGGMCTGTT CCCAAATGGG 1140
ATAGCCACAT CAGAGCTGCC CTTTCCATTG A~r'`qrr^.^. GMGCGCTCT ACTGGGGTCT 1200
TGCTGCCTCT GCAGMCMC GAGCTTCCTG GGGCA6MTA CCAGTATMM MGGATGAGG 1260
TGTGGGAGGA GAGMMGCCT TACMGACCC TTCAG6CTCA GGCCCCTGM MGAGTMGA 1320
ATMAMGM ACAGAGMMG GGGCCTCATC C~CM~^~CCCA GACGCTCCM TTTGATGAGC 1380
25 AGACCCTGM MMGGCMGG AGMMGCAGT GGATTGMCC TCGGMTTGC GCCAGGAGAT 14~0
ACCTCMGGT AGACTTTGCA GATATT6GCT GGAGTGMTG GATTATCTCC CCCMGTCCT 1500
TTGATGCCTA TTATTGCTCT GGAGCATGCC AGTTCCCCAT GCCMMGTCT TTGAAGCCAT 1560
CMMTCATGC TACCATCCAG AGTATAGTGA GAGCT6TGGG GGTCGTTCCT GGGATTCCTG 1620
AGCCTTGCTG TGTACCAGM MGATGTCCT CACTCAGTAT IIIAII~.III GATGMMTA 1680
35 AGMTGTAGT GCTTMMGTA TACCCTMCA TGACAGTAGA Glt,l I~CGCl TGCA6ATMC 17~0
CTGGCMMGA ACTCATTTGA ATGCTTMTT CMT 1774
(2) INFORMATION FOR SEQ ID NO~4:
WO 92/21365 2 1~ PCI~/US92~04356
- 31 -
(i) SEQUENCE CHARACTERISTICS:
(A) LEH6TH: 1751 base pairs
(B) TYPE: nucleic ~cid
(C) STRANDEDHESS: double
S (D) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
G6CAGAG6A6 6~rr~qrrrq CCCMrt~Gr ~rqr'CCG ~CCrr~CC TA66T6A6TG 60
lS T66CATCCGA GCT6A666AC 6CGAGCCTGA 6AC6CC6CTG ~ILCI~GGC TGAGTATCTA 120
6CTT6TCTCC CC6AT6GGAT TCCC6TCCM 6CTATCTC6A 6CCT6CA6C6 CCACA6TCCC 180
CGGCCC1~6C CCAGGTTCAC TGCMCCGTT CAGA66TCCC CAG6AGCT6C T6CTG6C6A6 240
CCC6CTACTG CA666ACCTA T66AGCCATT CCGTAGT6CC ATCCCGAGCA ACGCACTGCT 300
GCAGCTTCCC T&AGCCTTTC CAGCMGTTT GTTCMGATT G6CTGTCMG MTCATG6AC 360
T6TTATTATA lbCu~ TCT6TCM6A CACCAT6ATT CCTGGTMCC GMTGCTGAT 420
66TCGTTTTA TTATGCCMG TCCTGCTAGG Arrt~r~ACt CATGCTA6TT TGATACCTGA 480
GACCrrr~AE MMMGTCG CCGAGATTCA trrCCACrrG 6rqtrqC~rC GCTCAGG6CA 540
6AGCCATGA6 CTCCT6CGGG ACTTCGAGGC GACACTTCT6 CA6ATGm6 GG~I~C6CCG 600
CC~CC~:C46 CCTAGCMGA 6TGCCGTCAT TCC6GACTAC ATGC66GATC mACCGGCT 660
TCAGTCT66G CArr'~rrlrr MGAGCAGAT CCACAGCACT GGTCTTGAGT ATCCTGAGCG 720
CCCl`rrCACC CrrrtCA~'4 CCGTGAGGA6 CTTCCACCAC GMGMCATC TG6A6MCAT 780
CC~4rrr'~CC AGTGMMCT ~ 1 1 1 11,6 1 1 11,u1 L I I I MCCTCA6CA GCATCCCT6A 840
WO 92/21365 ~ PCr/US92/04356
21104~ 32
GMCGAGGTG ATCTCCTCTG CAGAGCTTCG G~, l b l l cC6G GAGCAGGTGG A~CA'`rr~C 90O
TGATTGGGM AGGGGCTTCC ACCGTATMM CATTTATGAG GTTATGMGC CCCCAGCAGA 960
5 AGTGGTGCCT GGGCACCTCA TCACACGACT ACTGGACACG AGACTGGTCC ACCACMTGT 10ZO
GACACGGTGG GMMCTTTTG ATGTGAGCCC TGCGGTCCTT CGCTGGACCC GGGAGMGCA 1080
GCCMMCTAT GGGCTAGCCA TTGAGGTGAC TCACCTCCAT CAGACTCGGA CCCACCAGGG 1140
CCAGCATGTC AGGATTAGCC GATCGTTACC TCMGGGAGT GGGMTTGGG CCCAGCTCCG 1200
6Ccc~ 6 GTCACCTTTG GCCATGATGG CCGGGGCCAT GCCTTGACCC GACGCCGGAG 1260
GGCCMGCGT AGCCCTMGC ATCACTCACA GCGI GrCACC MGMGMTA AGMCTGCCG 1320
GCGCCACTCG CTCTATGTGG ACTTCAGCGA TGTGGGCTGG MTGACTGGA TTGTGGCCCC 1380
ACCAGGCTAC CAGGCCTTCT ACTGCCATGG GGACTGCCCC TTTCCACTGG CTGACCACCT 1440
CMCTCMCC MCCATGCCA TTGTGCAGAC CCTGGTCMT TCTGTCMTT CCAGTATCCC 1500
CMMGCCTGT TGTGTGCCCA CTGMCTGAG TGCCATCTCC ATGCTGTACC TGGATGAGTA 1560
TGATMGGTG GTACTGMM ATTATCAGGA GATGGTAGTA GAGGGATGTG 6blbCC61~1G 1620
AGATCAGGCA GTCCTTGAGG ATAGACAGAT ATACACACCA CACACACACA CCACATACAC 1680
CACACACACA CGTTCCCATC CACTCACCCA CACACTACAC AGACTGCTTC CTTATAGCTG 1740
GACTTTTATT T 1751
(2) INFORMATION FOR SEQ ID NO:S:
(i) SEQUENCE CHARACTERISTICS: t
(A) LENGTH: 2153 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
WO 92/21365 2 1 1 ~ 4 ~ o PCr/us92/o4356
( i i ) MOLECULE TYPE: cDNA
S (xi) SEqUENCE DESCRIPTION: SEQ ID HO:5:
CTGGTATATT TGTGCCTGCT GGAGGTGGM TTMCAGTM t:M('''~r'\M GGGATTGMT 60
GGACTTACAG GMGGATTTC M6TMMTTC A~`rl;~lMrl~C ATTTACTTGA ATAGTACMt 120
CTAGAGTATT ATTTTACACT MGACGACAC MMGATGTT MMGTTATCA CCMGCTGCC 180
GGACAGATAT ATATTCCMC ACCMGGTGC AGATCAGCAT AGATCTGTGA TTCAGMMTC 240
AGGATTTGTT TTGGMMGAG CTCMGGGTT GAGMGMCT CMMGCMG TGMGATTAC 300
TTTGGGMCT ACAGTTTATC AGMGATCM CTTTTGCTM TTCMMTACC MMGGCCTGA 360
TTATCATMM TTCATATAGG MTGCATAGG TCATCTGATC MMTMTATT AGCCGTCTTC 420
TGCTACATCA ATGCAGCMM MCTCTTMC MCTGTGGAT MTTGGMMT CTGAGTTTCA 480
GCmCTTAG MMTMCTAC TCTTGACATA TTCCMMTA TTTMMTAG GACAGGMAA 540
TCGGTGAGGA TGTTGTGCTC AGMMTGTCA CTGTCATGM MMTAGGTM ATTTGTTTTT 600
TCAGCTACTG GGMMCTGTA CCTCCTAGM CCTTAGGTTT l l l l l l l l l l MGAGGACM 660
GMGGACTM MATATCMC l l l I bl, l 111 GGACMAMT GCATCTGACT GTATTTTTAC 720
TTMGGGTAT I b I b6G I I l l; CTCTGGAGCT 6b 1 bbb I I ( I AGTGGGTTAT GCMA~lrr ~C~ 780
GTTTGGGAGA CMTCATGTT CACTCCAGTT TTATTTATAG MGACTACGG MCCACGMM 840
GACGGGMMT ACAMrrrAA ATTCTCTCTA ~bl lbGbl I I GCCTCACAGA CCCAGACCAT 900
mCACCTGG AAMCMGCG TCCTCTGCAC CTCTCTTTAT GCTGGATCTC TACMTGCCA 960
TGACCMTGA AGAAMTCCT GMGAGTCGG AGTACTCAGT MGGGCATCC TTGGCAGMG 1020
WO 92t21365 PCltUS92tO43~6
2110~lp 34
AGACCAGAG6 ~ Ar~AA'` GGATACCCAG CCTCTCCCM TGGGTATCCT CGTCGCATAC 1080
AGTTATCTCG GACGACTCCT CTGACCACCC AGAGTCCTCC TCTAGCCAGC CTCCATGATA 1140
S CCMCTTTCT GMTGATGCT GACATGGTCA TGAGCTTTGT CMCTTAGTT CAl\~CAr'\'~ 1200
AGGATTTTTC TCACCAGCGA AGGCATTACA MGMTTTCG ATTTGATCTT ACCCMMTTC 1260
CTCATGGAGA GGCAGTGACA GCAGCTGMT TCCGGATATA CMGGACCGG AGCMCMCC 1320
GATTTGMM TGMMCMTT MGATTAGCA TATATCMMT CATCMGGM TACACMATA 1380
GGGATGCAGA I~IGI li;l IG TTAGACACM GMMGGCCCA AGCTTTAGAT GTGGGTTGGC 1440
S TTGTCTTTGA TATCACTGTG ACCAGCMTC ATTGGGTGAT TMTCCCCAG MTMTTTGG 15OO
GCTTACAGCT CTGTGCAGM ACAGGGGATG GACGCAGTAT CMCGTMM TCTGCTGGTC 1560
TTGTGGGMG ACAGGGACCT CAGTCMMC MCCATTCAT GGTGGCCTTC TTCMGGCGA 1620
GTGAGGTACT TCTTCGATCC GTGAGAGCAG CCMCMMCG MMMTCM MCCGCMTA 1680
MTCCAGCTC TCATCAGGAC TCCTCCAGM TGTCCAGTGT TGGAGATTAT MCACMGTG 1740
25 AGCMAMCA AGCCTGTMG MGCACGMC TCTATGTGAG CTTCCGGGAT CTGGGATGGC 1800
AGGACTGGAT TATAGCACCA GMGGATACG CTGCATTTTA TTGTGATGGA GMTGTTCTT 1860
TTCCACTTM CGCCCATATG MTGCCACCA ACCACGCTAT AGTTCAGACT CTGGTTCATC 1920
TGATGTTTCC TGACCACGTA CCMMGCCTT GTTGTGCTCC MCCMMTTA MTGCCATCT 1980
CTGTTCTGTA CTTTGATGAC AGCTCCMTG TCATTTTGM MMTATAGA MTATGGTAG 2040
35 TACGCTCATG TGGCTGCCAC TMTATTMM TMTATT6AT MTMCMM AGATCTGTAT 2~00
TMGGTTTAT GGCTGCMTA MMGCATAC TTTCAGACM ACAGMMM MM 2153
(2) INFORMATION FOR SEQ ID HO:6:
Wo 92/21365 2 ~ ) Pr~/us92/o43s6
- 35
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2923 base pairs
(B) TYPE: nucleic acid
( C ) STRANDEDNESS: doubl e
S (D) TOPOLOGY: line~r
( i i ) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CGACCATGAG AGATMGGAC TGAGGGCCAG CMrrrC'~AC CGAGCCCGCC GAGAGGTGGC 60
IS GGGGACTGCT CACGCCMGG GCCACAGCGG CCGC6~1~CG GCCTCGCTCC GCCGCTCCAC 120
^~ICGCGGG ATCCGCGGGG GCAGCCCGGC CCCCCCCrr~ TGCCGG66~T GGr~rrrr~rC I80
GCGCAGTGGC TGTGCTGGTG blb66Gbl,lb CTGTGCA6CT GCTGCGGGCC CCbGCC6~1G 240
CGGCC6CC~1 T6~CC6~1bC rG~GG(~G~ 6~G~C~''C GGCAGCTGCT rrCCC~rG6C 300
CCC.~CCCCG GCCGCACGGA rCAGCrGCC6 CCbl-bCC6C AGTCCTCCTC GGGCTTCCTG 360
25 TACCGGCGGC TCMGACGCA ~r'~r'U\rrrr 6AGATGCAGA AGGAGATCTT GTCGGTGCTG 420
GGG~ CbC ArCr~rrCCCG GCCCCTGCAC GGCCTCCMC .4rrCGCACCC CC~G6C6~1~ 480
cr~r~c4cc~cc /~rr'~rC4rr4 rC4rrq~r4G CAGCTGCCTC r~rr~9r~rCc CC~ Cb66 540
CGACTGMGT CC6C6CCC~1 CTTCATGCTG GATCTGTACA ACGCCCTGTC CGCCGACMC 600
GACGAGGACG GGGCGTCGGA Grrrr~CACr CAGCAGTCCT GGCCCCACGA ArCAGCC9rr 660
35 TCGTCCCAGC GTCGGCAGCC Gr~ r r~r~GCGCACC CGCTCMCCG CMGAGCCTT 720
Clb6CCCCCG GATCTG6CAG ~C"~,rGrG TCCCCACTGA CCAr'Çrr`r~ rr~rcGcc 780
TTCCTCMCG ACGCGGACAT GGTCAT6AGC mGTGMCC TGGTGGAGTA CCAr4,^,r~C 840
WO 92/21365 PCr/US92/04356
2110~ 36-
I I L I Ll,l,l, I L GTCA6CGACA CCAr4M'`~ TTCAAGTTCA ACTTATCCCA 6ATTCCTGA6 goo
6GTGAGGTGG T6AC66CT6C A6MTTCC6C ATCTACM66 ACT6T6TTAT 66G6A6TTTT 960
5MAMCCMM CTTTTCTTAT CA6CATTTAT CM6TCTTAC A6GA6CATCA 6CACA6A6AC 1020
TCT6ACCT6T TTTT6TTGGA CACCC6T6TA 6TAT666CCT CA6M6M66 CT66CT66M 1080
m6ACATCA C66CCACTA6 CMTCT6T66 6TT6T6ACTC CACA6CATM CAT6666CTT 1140
CA6CT6A6CG T66T6ACM6 66AT66A6TC CAC6TCCACC CCCGAGCC6C A66CCT66T6 1200
66CA6AGAC6 6CCCTTAC6A TM6CA6CCC TTCAT66T66 CTTTCTTCM A6T6AGTGAG 1260
156TCCAC6T6C r,~4~CACCA6 6TCA6CCTCC Arr~rr-rrrC 6ACMCA6A6 TCGTMTCGC 1320
TCTACCCA6T CCCA66AC6T GGCGCGGblL TCCA6T6CTT CA6ATTACM CA6CA6T6M 1380
TTGMMCA6 CCT6CAG6M 6CAT6A6CT6 TAT6T6A6TT TCCM6ACCT 666AT66CA6 1440
6ACT66ATCA TT6CACCCM 666CTAT6CT 6CCMTTACT 6T6AT6GA6A AT6CTCCTTC 1500
CCACTCMC6 CACACATGM T6CMCCMC CAC6CGATTG T6CA6ACCTT 66TTCACCTT 1560
25AT6MCCCC6 A6TAT6TCCC CMMCC6T6C TGTGC6CCM CTM6CTMM TGCCATCTC6 1620
6TTCTTTACT TT6AT6ACM CTCCMT6TC ATTCTGMM MTACA6GM TATG6TT6TA 1680
A6A6CTT6T6 6AT6CCACTA ACTC6MMCC A6AT6CT666 6ACACACATT CT6CCTT66A 1740
TTCCTA6ATT ACATCTGCCT TMMMMCA C66M6CACA 6TT66A66TG GGAC6ATGA6 1800
ACmGMMC TATCTCATGC CAGT6CCTTA TTACCCA66A A6ATTTTMM G6ACCTCATT 1860
35MTMTTT6C TCACTT6GTA MT6AC6T6A GTA6TT6TT6 6TCT6TA6CA AECT6A6TTT 1920
66ATGTCTGT AGCATMG6T CTG6TMCT6 CA6MMCATA ACC6T6M6C TCTTCCTACC 1980
LrLl,lLCCcc ANVIACCCAC CMMTTA6T TTTA6CT6TA 6ATCM6CTA 11 IbG6blGl 2040
WO92/2136S 9~lDt ff4 1 0
31 -
TTGTTAGTM ATAGGGMAA TMTCTCAAA GGAGTTMMT GTATTCTTGG CTMMGGATC 2100
AGCTGGTTCA GTACTGTCTA TCMMGGTAG ATTTTACAGA GMCAGMMT CGGGGMGTG 2160
GGGGGMCGC CTCTGTTCAG TTCATTCCCA GMGTCCACA GGACGCACAG CCCAGGCCAC 2220
AGCCAGGGCT CCACGGGGCG CCC1 IG1~r~ AGTCATTGCT GTTGTATGTT CGTGCTGGAG 2280
TTTTGTTGGT GTGMAATAC ACTTATTTCA GCCMAACAT ACCATTTCTA CACCTCMTC 2340
CTCCATTTGC TGTACTCTTT GCTAGTACCA MMGTAGACT GATTACACTG AGGTGAGGCT 2400
ACMGGGGTG TGTMCCGTG TMCACGT6A AGGCAGTGCT CACCTCTTCT TTACCAGMC 2460
GGTTCTTTGA CCAGCACATT MCTTCTGGA ~1bCC66~1C TAGTACCTTT TCAGTMMGT 2520
GGTTCTCTGC CTmTACTA TACAGCATAC CACGCCACA~ GGTTAGMCC A/\~qAr \M 2580
ATMMTGAG GGTGCCCAGC TTATAAGMT GGTGTTAGGG GGATGAGCAT GCTGTTTATG 2640
MCGGMMTC ATGATTTCCC TGTAGAAAGT GAGGCTCAGA TTMMTmA GMTATTTTC 2700
TMATGTCTT mCACAATC ATGTGACTGG GMGGCMTT TCATACTMA CTGATTMMT 2760
25 MTACATTTA TMTCTACAA CTGTTTGCAC TTACAGCTTT mmGTMMT ATMMCTATA 2820
ATTTATTGTC TATTTTATAT [,IGI 11 Ibbr GlbGCbl Ib6 ''''' '''~C G6Gbl I l Ibb 2880
GG666666br 1 IGI I IbG6G GblGll.blbG IG~bGGCGG6 CGG 2923
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1448 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: doub1e
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
WO 92/2136~; PCr/US92/043~i6
2ii0~1~ 38- ~
(xi) SEQUENCE OESCRIPTION: SEQ ID NO:7:
GT6ACCGA6C GGCGCGGACG 6CCGC~16CC CCCTCTGCCA C1,1bGGGCGG lbCG6GCCCG 60
5GAGCCCGGAG CCCGGGTAGC GCGTAGAGCC GGCGCGATGC ACGTGCGCTC ACTGCGAGCT 120
G~rrCG'CCG~ ACAGCTTCGT GGCGCTCTGG GCACCCCTGT 1C~ .CG CTCCGCCCTG 180
GCCGACTTCA GCCTGGACM CGAGGTGCAC TCGAGCTTCA TCCACCGGCG CCTCCGCAGC 240
CA6GAGCGGC GGGAGATGCA GCGCGAGATC CTCTCCAm TGGGCTTGCC CCACCGCCCG 300
CGCCCGCACC TCCAGGGCM GCACMCTCG GCACCCATGT TCATGCTGGA CCTGTACMC 360
IS GCCATGGCGG TGGAGGAGGG ~rLrGGGrCC GGCGGCCAGG GCTTCTCCTA CCCCTACMG 420
~CGTCTTCA GTACCCAGGG CCCCCCTCTG GCCAGCCTGC MGATAGCCA TTTCCTCACC 480
GACGCCGACA TGGTCATGAG CTTCGTCMC CTCGTGGMC ATGACMGGA ATTCTTCCAC 540
CCACGCTACC ACCATCGAGA Gl 1i,CGI,l 1 I GATCTTTCCA AGATCCCAGA AGGGGMGCT 600
GTCACGGCAG CCGMTTCCG GATCTACMG GACTACATCC GGGMCGCTT CGACMTGAG 660
25 ACGTTCCGGA TCAGCGTTTA TCAGGTGCTC CAGGAGCACT TGGGCAGGGA ATCGGATCTC 720
1 11,1,1b~.1t,G ACAGCCGTAC C~1~1b~.bCC TCGGAGGAGG GCTGGCTGGT GmGACATC 780
ACAGCCACCA GCMCCACTG GGTGGTCMT CCGCGGCACA ACCTGGGCCT GCAGCTCTCG 840
GTGGAGACGC TGGATGGGCA GAGCATCMC CCCMGTTGG CGGGCCTGAT TGGGCGGCAC 900
rrr~(~CCCACA ACMGCAGCC CTTCATGGTG GCTTTCTTCA Ar~rCACrr'\ GGTCCACTTC 960
35 CGCAGCATCC GGTCCACGGG CA~r~rqC CGCAGCCAGA ACCGCTCCM GACGCCCMG I020
MrC,qGr~ '` CCCTGCGGAT GGCCMCGTG GCAGAGMCA GCArCACCGA CC4r'lCrC~C 1080
GCCTGTMGA AGCACGAGCT GTATGTCAGC TTCCGAGACC TGGGCTGGCA GGACTGGATC 1140
-
WO 92/2136S Pc'r/ /04356
~ 2 1 1 ~ US92
- 39 -
ATCGCGCCTG AAGGCTACGC CGCCTACTAC TGTGAGGGGG AGTGTGCCTT CCCTCTGMC IZ00
TCCTACATGA A~':fCA~CM CCACGCCATC GTGCAGACGC TGGTCCACTT CATCMCCCG 1260
S GMMCGGT6C CCM6CCCTG l.lGlbC6CCC AC6CAGCTCA AT6CCATCTC CGTCCTCTAC 1320
TTCGAT6ACA GCTCCMC6T CATCCT6MG MMTACAGM ACATGGTGGT CCG66C~,llil 1380
GGCTGCCACT AGCTCCTCCG AGMTTCAGA CCI, I I I b66G CCM6TmT CTGGATCCTC 1440
CATTGCTC 1448
The invention has been described herein with reference to
certain preferred embodiments ~nd examples. Obvious variations may
Is appear to those skilled in the art. Therefore, the invention is not
to be considered limited thereto but only by the cl~ims which follow.
~IHAT IS CLAIHED IS:
