Note: Descriptions are shown in the official language in which they were submitted.
`" 94/0014~ 213~3~ PCI/US93/06254
TARGETED DELIVERY OF GROWTH FACTORS FOR BONE REGENERATION
This invention relates to specific target delivery systems; namely, delivery of
growth promoting factors to bone for its regeneration by a chelant composition system.
Compositions containing these systems and a process for making them are also part of this
5 invention.
Historically, many physical conditions and diseases exist which cause bone loss in
mammals, e.g., intentional and accidental traumatic injuries, osteoporosis and periodontal
diseases. Thus it is often desired in the medical and dental fields to provide a composition
which will stimulate and enhance bone regeneration in a mammal, e.g. a human patient.
One class of proteins which may be useful for regeneration of bone is polypeptide
growth factors (GF), which are also described as tissue growth promoting factors. Growth
factors are polypeptides which stimulate a defined population of target cells. As
multifunctional molecules, they may stimulate or inhibit cell proliferation as well as affect cell
function, depending on the type of the target cells and the presence of other signal peptides.
Examples of growth factors are platelet-derived growth factors (PDG F's), i nsul i n-l i ke growth
factors (IGF's), transforming growth factors (TGF) such as beta's (TGF-13's) and alpha (TGF-a),
epidermal growth factor (EGF), fibroblast growth factor (FGF's) including acidic fibroblast
growth factor (aFGF) and basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and
bone morphogenetic proteins (BMP's), including osteogenic and osteoinductive factors.
20 Combinations of tissue growth factors may be beneficial for promoting bone regeneration.
For example the combinations of PDGF and IGF-I or PDGF and IGF-II promote bone
regeneration and wound healing [See, for example, Lynch et al., Proc. Nat'l. Acad. Sci. (USA) 84,
7696-7700 (1987); Lynch et al., J. Clin. Invest. 84, 640-646 (1989); Lynch et al., J. Clin.
Periodontol. 16,545-588(1989); Lynchetal.,J.Periodontol.62,458-467(1991);andUSPatents
25 4,861,757 and 5,019,559]
PDGF's are polypeptides of about 28-35 kilodaltons (kD). They are found in
numerous cell types in the body. PDGF derived from human platelets contains two polypeptide
sequences, PDGF-A and PDGF-B polypeptides [See H.N. Antoniades and M. Hunkapiller Science
220, 963-965 (1983)]. PDGF-A is encoded by a gene localized in chromosome 7 [C. Betsholtz et
30 al., Nature320, 695-699(1986)], and PDGF-B isencoded bythesisoncogene [R. Doolittleetal.,
Science 221, 275- 277 (1983); Waterfield et al., Nature 304, 35-39 (1983)] localized in
chromosome 22 [R. Dalla-Favera, Science 218,686-688 (1982)].
Because the two polypeptide chains of PDGF are encoded by two different genes
localized in separate chromosomes, human PDGF occurs in three forms, a disulfide-linked
35 heterodimer of PDGF-A and PDGF-B, or two different homodimers (homodimer of PDGF-A and
homodimer of PDGF-B). The role of PDGF in bone formation is not clear. Some studies have
indicatedthatitpromotesboneresorption[Tashjianetal.,Endocrinologyl11, 118-124(1982);
Canalis et al., J. Ce/l Physiol. 140, 530-537 (1989)]. Other studies have shown that PDGF
WO 94/00145 C A 2 1 3 9 3 2 3 PCI /US93/062~4
sti mulates the prol if eration of osteoblasts in vitro and, when given via repeated su bperiosteal
injectionsinnewbornrats,newboneformationinvivo[PicheandGraves,Bone10,131-138
(1989); Joyce etal., in Clinical and ExDerimental ADproachesto Dermal and E~idermal Repair:
Normal and ChronicWounds, pp. 391-416(1991).
IGF's, or somatomedins, are polypeptides of about 7.5 kD that have a strong
homologytohumanproinsulin[Humbel,HormonalProteinsandPeptides12,57-79(1984)].
IGF-I and IGF-II share a 62% sequence homology. Their actions are mediated through two
distinct receptors. The IGF-I receptor is named type-l receptor (IGF-IR), and the IGF-II receptor is
named type-ll receptor (IGF-IIR).
IGF-I by itself has also been extensively studied for its effects on bone growth. In
vivo, the continuous local application of IGF-I inside a titanium chamber implanted into the
adult rabbit tibia did not significantly alter bone formation [Aspenberg et al., Acta Orthop.
Scand., 60,607-10 (1989)]. Continuous systemic administration of somatomedic-C (IGF-I) also
failed to promote the repair of bone wounds resulting from a femoral osteotomy in rats
[Kirkeby and Ekeland, Acta Orthop. Scand.; 61,335-38 (1990)]. A preliminary study in a small
number of animals suggested that continuous infusion of IGF-I into the arterial supply of one
hind limb for 14 days resulted in increased cortical bone formation in that limb in older, but not
young, rats. The action appeared to be the result of an increased number of osteoblasts and
decreased numberof osteoclasts ISPenceretal., Bone 12,21-26(1991)]. The local application
of IGF-I to the grovvth plate of young hypophysectomized rats resulted in a smal I but significant
effect on unilateral longitudinal bone growth [A. M. Isgaard et al., J. Physiol. 250, E367-372
(1986)]. IGF-I and PDGF have also been isolated from bone matrix [Hauschka et al., J. Biol.
Chem. 261, 665-74 (1986); and Canalis et al., Cal. Tiss. Internatl. 43,346-51 (1988)].
In vitro, there are apparently conflicting data on the effects of IGF-I on bone cells.
25 Pfeilschifter, et al., [Endocrinology 127, 69-75 (1990)] reported only a modest effect of IGF-I
alone on bone matrix apposition in cultured fetal rat calvarial. Significant effects on bone
matrix formation were seen when IGF-I was combined with PDGF-BB, TGF-~, or both PDGF-BB
and TGF-~. In contrast, McCarthy et al., [Endocrinology 124,301-7 (1989)] reported that IGF-I
and IGF-II stimulate significant DNA and collagen synthesis in bone cultures. Hock et al.,
30 [Endocrinology 122, 254-60 (1988)] found that IGF-I stimulates primarily pre-osteoblast
replication in vitro and that collagen and bone matrix synthesis is stimulated independently of
cell replication. Canalis et al., [J. Cell. Physiol. 140, 530-537 (1989)] reported that PDGF-BB
opposed the stimulatory effect of IGF-I on collagen synthesis, IGF-I prevented the PDGF effect
on collagen degradation and that PDGF-BB and IGF-I had additive effects on calvarial DNA
35 synthesis. Piche and Graves [80ne 1 O, 131 -8 (1989)] also reported that in vitro IGF-I did not
stimulate significant 3H-thymidine incorporation into bone derived cells nor did it enhance the
activity of PDGF in this regard. IGF-I in combination with PDGF, EGF and TGF-B resulted in
uptakebythebonecellsnearlyequaltothatachievedbylO%fetalbovineserum. Receptors
-2-
"'') 94/00145 C A 2 1 3 q 3 2 3 PCl/US93/06254
for IGF-I and ll have been demonstrated in osteoblast-enriched cultures from fetal rat bone
[Centrei la et al ., J. Cell Biol. (abstract) 107, 62a (1988)~ The role of IG F-l i n bone metabol ism
has been reviewed recently by Canalis et al., [J. Endocrinol. Invest. 12, 577-84 (1989)].
Another class of growth factors which have been studied for thei r effect on bone
5 growth is FGFs. In vivo, both aFGF and bFGF and their respective mRl\A's have been detected at
the site of bone fractures [Joyce et al ., (1991) ibidl . Both aFGF and bFG F have been isolated
from bone matrix [Hauschka et al., (1986) ibid]. bFGF and IGF-I have been used in combination
to promote the healing of skin wounds [Lynch et al., J. Clin. Invest. (1989) ibid].
In vitro, bFGF did not significantly alter 3H-thymidine incorporation in bone
fracture calluses [Joyce et al., (1991) ibid]. bFGF has been reported to enhance mitogenesis in
fetal calvarial bone cultures but did not simulate differentiated function of osteoblasts directly
[Canalis et al., J. Clin. Invest. 81,1572 (1988)]. aFGF has the same reported biological effects on
bone as bFGF but generally requires higher concentrations [Canalis, J. Clin. Invest. 79, 52-58
(1987)]. Both aFG F and bFG F tend to decrease matrix synthesis i n the fetal rat calvarial model
[Canalis et al., (1989) ibid]. Cultured bovine bone cells synthesize both bFGF and aFGF and store
themintheirextracellularmatrix[Globusetal.,Endocrinologyl24,1539(1989)]. bFGFhasbeen reported to enhance the capacity of bone marrow cells to form bone-like nodules in vitro
[Noff et al ., F.E. B.S. Letters 250, 619-21 (1989)]. Both aFG F and bFG F increased DNA synthesis i n
cells cultured from parietal bones while bFGF was a more potent stimulator of alpha 1 Type 1
20 procollagenmRNA[McCarthyetal.,Endocrinology125,2118-26(1989)j. Theyareboth
mitogenic and chemotactic for cells derived from the periodontal ligament and bind to
pretreated denti n slabs [Terranova et al ., J. Periodontol. 60,293-301 (1989); Terranova et al ., J.
Periodontol. 58, 247-257 (1987); Terranova, In The Bioloqical Mechanisms of Tooth Extraction
and Root Resorption, Davidovitch Z. ed.; pp. 23-34 (1989)].
The TGF-B family of proteins also appearto have potential as modulators of bone
growth. In vitro TGF-B is produced by osteoblasts and stimulates proliferation and collagen
synthesisbythesecells[Robeyetal.,J. CellBiol. 105,457-463(1987); Rosenetal.,Exper. Cell
Res. 165,127-138(1986); Hocketal.,Cal. Tissuelnt. 32,385(abstract)(1988)]. Invivoinjections
of TGF-B stimulate chondrogenesis and osteogenesis [Joyce et al., J. Cell Biol. 11 O, 2195-2207
(1990); Noda et a I ., Endocrinology 124,2991 -2996 (1989)] .
Bone inductive factors, such as bone morphogenetic proteins, osteogenin and
osteoinductive protein 1, can also stimulate bone formation. They are often strucurally similiar
toTGF-Band arecharacterized bytheirabilitytoinduceectopiccartilageand boneformation
when implanted subcutaneously or intramuscularly in mammals (US Patents 4,877,864;
4,619,989; 4,455,256; 4,596,574; and 4,563,489; Wozneyetal., Science242,1528-1534(1988)].
Tarqeted Delivery
While growth factors appear to have the ability to modulate bone growth, their
efficacy as therapeutic agents is limited by the ability to deliver them to the site of bone deficit.
-3-
WO 94/0014~ 2 ¦ 3 9 3~ 3 PCI /US93/062S4
In diseases such as osteoporosis this deficit occurs at various times throughout the entire
skeletal system. Thus it would be ideal to target the delivery of the growth factors to skeletal
tissue with preference given to the site of the bone deficit. Current methods for delivery of
proteins, such as, polymers, bone grafts and liposomes are unsatisfactory because they allow
5 only local ized del ivery or system ic distri bution without targeti ng.
Various aminophosphonic and aminocarboxylic acid complexes, having a metal
complexed to an aminophosphonic or aminocarboxylic acid ligand, are known to deliver
agents to bone [US Patents 4,508,704, 4,515,767,4,560,548,4,606,907, 4,897,254, 4,898,724,
5,059,412,5,064,633, and 5,066,478] .
Clearly,itwouldbedesirabletodeliverpreferentiallytothebonesitethedesired
growth factor(s) and retai n its activity for the i ntended use. More preferred would be the
ability to deliver the desi red growth factor(s) preferential Iy to the site of inj ured or depleted
boneandstillretainitsintendedactivity. Mostpreferredwouldbetheabilitytodeliverthe
desired growth factor(s) preferentially to the site of injured or depleted bone and allow for the
activation of the bone growth factor(s) on an "as needed" basis as determined by the natural
bone homeostasis mechanisms in the body. The present invention has the ability to met the
above objectives.
Present Invention
The present invention provides compounds for stimulating and enhancing bone
20 growth by administering bone growth promoting factors which have been modified by being
associated with a polyaminomethylenephosphonic acid ligand in a way that allows their
pre~erential localization to skeletal tissue. The compounds for this delivery system are
represented by the formula
GF-[(CL)-L-AP] (I)
wherein: GF is a growth promoting factor or combinations thereof;
CL is an acid cleavable li nker which is covalently bonded to G F;
zisO, 1 or2;
q is from 1 to the sum of the amino groups present on the native GF;
L is a linking moiety; and
AP is a polyaminomethylenephosphonic acid ligand.
Formulations for administering the compounds of Formula I to mammals, and
methods for the use of the compounds of Formula I for targeted delivery to bone, and
35 processesforpreparingthecompoundsof Formula I arealsocontemplated bythisinvention.
When the compositions or formulations containing compounds of Formula I are
used, sites of injured or depleted bone are treated, and bone regenerated. Natural and
recombinanttissue growth factors are commercially available from R&D Systems (Minneapolis,
-4-
~ )94/00145 213~23 PCl/US93/06254
MN), Collaborative Research, Inc. (Bedford, MA), Genzyme, Inc (Cambridge, MA), ICN
Biomedicals, INC., (Cleveland, OH), Peprotech, Inc. (Rocky Hill, NJ) and UBI (Lake Placid, NY).
Bone inductive proteins can be purified from bone [Celeste et al., Proc. Natl. Acad. Sci. (USA) 87,
9843-9847 (1990); Wang etal., Proc. Natl. Acad. Sci. (USA) 85, 9484-9488 (1988); US Patents
4,455,256 and 4,619,989].
In the compounds of Formula 1, although any of the growth promoti ng factors
(GF) mentioned before may be used, preferably GF is chosen from PDGF's, IGF's, FGF's, TGF's or
cartilage/bone inductive factors (BMP's).
PDGF, preferably in combination with IGF-I, has been shown to increase new bone
formationwhenappliedeitheraloneordirectlytodiseasedbone(USPatents4,861,757and
5,019,559andcopending USapplicationSerial No. 582,332,filedSeptember 13,1990, H.Antoniades and S. Lynch). PDGF contains 30 free amino groups per molecule which can
potentially be modified to increase PDGF's affinity for bone. PDG F is avai lable from R&D
SystemsandGenzyme, Inc. PDGFhasbeendescribed in USPatents4,861,757and 5,019,559.IGF-I has been shown to increase new bone formation when applied, either alone
or preferably in combination with PDGF, directly to diseased bone. IGF-I contains 4 free amino
groups per molecule which can potentially be modified to increase PDGF's affinity for bone.
IGF-I is available from R&D Systems and Genzyme, Inc. IGF-I is described by R. E. Humbel, Eur. J.
Biochem. 190, 445-462 ( 1990).
IGF-llhasbeenshowntoincreasenewboneformationwhenapplieddirectlyto
diseased bone. IGF-II contains 2 free amino groups per molecule which can potentially be
modified to increase PDGF's affinity for bone. IGF-II is available from R&D Systems and
Genzyme, Inc. IGF-II isdescribed by R. E. Humbel, Eur. J. Biochem. 190, 445-462 (1990).
bFGF has been shown to increase new bone formation when applied directly to
25 diseased bone. bFGF contains 15 free amino groups per molecule which can potentially be
modified to increase PDGF's affinity for bone. bFGF is available from R&D Systems and
Genzyme, Inc. bFGF isdescribed by Gospodarowic2 et al., Endocrinol. Rev. 8, 95-114 (1987).
aFGF is a low molecular weight homodimer polypeptide which has been shown to
increase new bone formation when applied directlyto diseased bone. aFGF contains 14 free
30 amino groups per molecule which can potentially be modified to increase PDGF's affinity for
bone. aFGF is available from R&D Systems and Genzyme, Inc. aFGF is described by
Gospodarowicz et al., Endocrinol. Rev. _, 95-114 (1987).
TGF-~, is a low molecular weight (about 25 kDa, amino acids) homodimer
polypeptide which has been shown to increase new bone formation when applied directly to
35 diseased-bone. TGF-~l contains 18 free amino groups per molecule which can potentially be
modified to increase PDGF's affinity for bone. TGF-,BI is available from R&D Systems and
Genzyme, Inc. TGF-~l is described by Sporn et al., J. Cell Biol. 105, 1039-1045 (1987).
WO 94/00145 ~ 93~ PCI`/US93/06254
BMP's have been shown to increase new bone formation when applied within
mesenchymal tissues. The BMP's have been reviewed by Celeste et al. [Proc. Natl. Acad. Sci.
(USA) 87, 9843-9847 (1990)] and found to be as follows:
BMP
No. of kDa
BMPFree AminO approximate
G r oups
2 10 12.9
3 11 14.5
4 8 13.1
11 15.6
6 8 15.7
7 9 15.7
The polyaminomethylenephosphonic acid ligands (AP of Formula 1) are either
covalently bonded to the G F of Formula I (z = 0), or have a cleavable I i nker (L) present (z = l ).
The AP ligands may be straight or branched-chain moieties, cyclic moieties, polymers (including
dense star polymers, their dendrimers and dendrons), or aryl moieties, which ligands contain at
least two, preferably three or more, nitrogen atoms. Preferably, the ligands are polyamino-
methylenephosphonic acid ligands of one of the formula
Rl R2 Rl R2 R
25 R/ N ~3 ( ) n ~ R
m
(II)
wherei n:
each Rl independently is hydrogen, Cl-C4 alkyl, phenyl, hydroxy Cl-C3 alkyl, -
CH2COOH, -CH2PO3H2 or an L moiety;
35 with the proviso that only one of R' may be an L moiety and one L moiety must be present and
with the proviso that at least one-half of the total R1's are -CH2PO3H2;
each R2 and R3 independently is hydrogen, CpC4 alkyl or L moiety;
withtheprovisothatonlyoneLmoietyispresentinFormulall;
-6-
~ '~ 94/0014~ 2 i ~ 9 ~ 2 3 PCr/US93/062~4
n is independently 2, 3 or 4;n' is independently 2, 3 or 4; and
misOtolO;or
S
R2
\ ( C ~ N J
n
R3 m
( I I I )
wherein: R', R~, R3, n and m are defined as before; or
Rl N
Rl
( IV )
35 wherein: R' is defined as before.
2~393~23
WO 94/001 4~ PCl /US93/062~4
The iinking moiety (L in Formula 1) is represented by the formula
R6
C ) G
I y
R5
(V)
wherein: G is hydrogen, NH7 or
R7
~\~4
R4 is an electrophilic group capable of being attached to protein;
Rs and R6 are independently hydrogen or -COOH;
with the proviso that when G is hydrogen, then one of Rs or R6 is COOH;
R7 is hydrogen, hydroxy or C~-C4 alkoxy; and
yisO, 1,2,30r4;
with the proviso that when y is 1, 2, 3 or 4, then only one of Rs or R6 may be COOH.
In the present invention the following terms are defined as follows. The term
"straight or branched-chain moieties" refers to an alkyl group having from 1 to about 100
30 carbon atoms which may be either a straight-chain moiety such as, for example, ethyl, propyl,
n-butyl, n-dodecane and the like, or a branched-chain moiety such as, for example, isopropyl,
tert-butyl, 2,5,7-trimethyldodecyl and the like. Both the straight and branched-chain moieties
must contain at least 2 nitrogen atoms, preferably from 3 to 50 nitrogen atoms, and more
preferably from 3 to 25. Some examples of these moieties include
-8-
~,/
" '~ 94/0014~ 2 1 ~ 9 32 3 PCI/US93/06254
HOOC _ (CH 2)3 ~ po3H2
PDTMP N \ \ N
H203P l l PO3H2
APEDTMP H2N~ N~ N P3H2
H 2O3P ¦ po3 H 2
HOOC I P3H2
H203P I N
CEDTMP N ~ PO3H2
H203P
H2N
PO3H2
ABEDTMP H2O3P N ~ PO3H2
H203P
WO 94/00145 ~ 93~ PCl`/US93/06254
po3 H2
H2N ~ N \ \ iN
APIPTMP
PO3H2
APDTM P ~ N /~/ N PO3 H 2
PO3H2
P3H2
H2N ~N P3H2
ABDTMP N I PO3H2
H203p 1 \~1
~ PO~H2
2 5 wherei n:
PDTMP = (N-propylcarboxyl)ethylenediamine-N,N',N'-trimethylenephosphonic
acid;
APEDTMP = [N-(4-aminophenyl)ethyl]ethylenediamine-N,N',N'-trimethylene-
phosphonic acid;
CEDTMP = 1-(carboxyl)ethylenediamine-N,N,N',N'-tetramethylenephosphonic
acid;
ABEDTMP = [1-(4-aminobenzyl)]ethylenediamine-N,N,N',N'-tetramethyiene-
phosphonic acid;
APIPTMP = N-(4-aminophenyl)-N,N-bis-[propyl(iminodimethylenephosphonic
35 acid)];
APDTMP = N-[(4-aminophenyl)ethyl]-N,N-bis-[ethyl(iminodimethyiene-
~hosphonic acid)]; and
-1 0-
2139323
`"O 94/0014~ PC~r/US93/06254
ABDTMP = N-[1-(4-aminobenzyl)-N,N'-ethylenediamine-N',N"-ethylenediamine-
N,l~l,N',N"-pentamethylenephosphonic acid;
with ABEDTMP and ABDTMP being preferred. The compounds are shown in the Examples.
The term "cyclic moieties" refers to aliphatic, saturated ring systems having at
least 2 nitrogen atoms, preferably from 3 to 10, and more preferrably from 3 to 8; and about
twice as many carbon atoms present as nitrogen atoms. Some examples of these moieties
include 1,4,7,10-tetraazacyclododecane,1,5,8,12-tetraazacyclotetradecane,2-[(4-amino-
benzyl)-1,4,7,10-tetraazacyclododecane~-1,4,7,10-tetramethylenephosphonic acid,
1-[(a-carboxyl)-4-amino-2-methoxybenzyl]-1,4,7,10-tetraazacyclododecane-4,7,10-tri-
methylenephosphonic acid, and 1-[(~-phosphonyl)(4-aminophenyl)ethyl]-1,4,7,10-tetra-
azacycl ododecane-4,7,10-tri methylenephosphoni c acid .
The term "aryl moieties" refers to an aromatic ring system which may have one ormore additional cyclic or aromatic rings or substitution by straight or branched-chain moieties.
The total number of atoms in the backbone of the aryl ring is from 3 to 30, preferably from 6 to
16, and more preferably from 8 to 16. The aryl moiety contai ns at least 2 nitrogen atoms,
preferably from 3 to 10, and more preferrably from 3 to 8. Some examples of these moieties
include pyrazolyl,3-methylpyrazolyl, 5-methylpyrazolyl, imidazolyl,4-methylimidazolyl, 5-
methylimidazolyl,1,4-dimethylimidazolyl,1,5-dimethylimidazolyl, pyridazinyl, pyrimidinyl,
2,4,6-trimethylpyrimidinyl, pyrazinyl, purinyl, pterdinyl,3,6,9,15-tetraazabicyclo[9.3.1]penta-
deca- 1 (15),11,13-tri ene-3,6,9-tri methyl enephosphoni c aci d (PCTM P),6-(a-ca rboxyl -4-a m i no-
benzyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,9-dimethylenephos-
phonicacid (PCAPCDMP),13-(4-aminobenzyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-
1(15),11,13-triene-3,6,9-trimethylenephosphonic acid (PCABTMP), and 6-[(a-phosphonyl-4-
aminophenyl)ethyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,9-
25 dimethylenephosphonic acid (PCAPCTMP). PCTMP, PCAPCDMP and PCAPCTMP, with PCTMP
and PCAPCTMP preferred, and are taught in Kiefer et al.'s copending US Patent Application
Serial No.805,551, filed December 10,1991 (assigned by unrecorded assignment toThe Dow
Chemical Company), the disclosure of which is hereby incorporated by reference. Other hetro
atoms such as oxygen may be present.
The term "polymers, including dense star polymers" are defined as described in
European Appln.0 271 180, published June 15,1988, the disclosure of which is hereby
i ncorporated by reference. These "dense star polymers" are also referred to as STARBU RST-~
polymers (a Trademark of Michigan Molecular Institute, Midland, Ml) or STARBURST'~
dendrimers and described in the same European publication. Preferred STARBURST'~35 dendrimers have polyaminoimine groups where the surface has the amino groups converted to
aminomethylenephosphonic acid groups and with at least one 4-aminophenyl moiety on the
surface. These STARBURSTT~ oendrimers are prepared as described in European Appln.
0 271 180, published June 15,1988, the disclosure of which is hereby incorporated by reference.
1 1
WO 94/0014~ 2~-~93~3 PCr/US93/06254
The "polymers" also include arborols (G. R. Newkome, J. Org. Chem. 50, 2004-2006 (1985), and
either linear or branched polymer containing amines (V. P. Torchilin et al., Byull. Eksp. Biol.
Med. 102,63-65 (1986).
Also included within the definition of the AP term of Formula I is
5 H2N-(CH2)~-C(PO3H)2OH
where n is from 1 -3.
Such compounds are described in US Patents 5,039,819, 5,019,651, 4,922,007, 4,621,077,
4,134,969,4,117,086, 4,108,962, and 3,962,432.
Theterm "cleavablelinker" (CLofFormulal)meansthatthelinkagebetweenthe
polyaminomethylenephosphonic acid (AP of Formula 1) [having a bifunctional chelanting agent
(BFCA) on the linking moiety (L of Formula 1)] and the protein of the GF is reversible or
cleavable under certain physiological conditions. Such linkages are taught in the art and
include linkages containg thiourea, thioether, peptide, ester, and difulfide groups [C. F. Meares
et al., Int'l. J. Cancer, Supp. 2,99-102 (1988) and references contained therein, US Patent
5,045,312]. Alsotaughtintheartarelinkagesbetweenmoleculesandproteinswhichcontainan amidine linkage. These linkages are prepared by reacting a molecule containing an
imidoester with the amine groups of the protein [O. R. Zaborsky, Immobilized Enzvmes in Food
and Microbial Processes, p187-202, A. C. Olson and C. C. Cooney, eds., Plenum, New York
(1974)]. Similarlytaught in the artare amide, diester, thioether, hydrocarbon, and disulfide
20 linkages [C. H. Paik et al., ~. Nucl. Med. Q, 1693-1701 (1989) and M. K. Haseman, Eur. J. Nucl.
Med. 12, 455-46û (1986)]. Cleavable diphosphonate and amidated diphosphonate linkers are
taught in US Patent 5,094,848. These linkers are cleaved in situ by enzymes such as
phosphodiesterase, 5'-nucleotidase, and acid phosphatase. Cleavable linkagesare alsotaught
in US Patent 5,094,849 in the form of an alkylidene hydrizide bond. These linkages are formed
25 by reacting a carbonyl containing molecule with a molecule containing a hydrazide moiety.
Additionally, acetal glycosides have been proposed as selectively cleavable linkages [L. F. Tietze,
Nachr. Chem., Tech. Lab. 36, 728-737 (1988)]. Linkers that are acid cleavable may be particularly
advantageous. Especially preferred are acid cleavable linkers wherein their rate of cleavage is
at leastten-fold greater at pH 5 compared to their rate at pH 7; both rates are determined at
30 37C. Some examples of acid cleavable linkers are disclosed in US Patents 4,542,225 and
4,618,492 and the references mentioned therein and our copending application US Serial No.
026,800, filed March 4,1993, which is hereby incorporated by reference. In this latter paending
application, the preferred acid cleavable linker is 4-isothiocyanatophthalic anhydride.
The term "group present to permit coupling to a protein" or "electrophilic
35 group(s) caDable of being attached to protein(s)" refersto an electrophilic group (R~ of the L
term of Formula 1) that can bind to an amino acid of a protein, e.g. the GF. Some examples
known to those skilled in the art of suitable groups inciude, but are not limited to, amino,
maleimido, diazo, isothiocyanato, vinylpyrido, bromoacetamido, carboxyl, and N-hydroxy-
-12-
~' ~ 94tO0145 ^ 2 1 ~ ~ 3 2 3 PC~r/~S93/06254
-
succinimido active ester When these electrophilic groups are present, the ligand (L-AP of
Formula 1, prior to attachment) is a BFCA.
The "polyaminomethylenephosphonic acid moieties" (AP of Formula 1) are
represented by a wide variety of possible groups, such as the cyclic moieties, straight or
5 branched-chain moieties, aryl moieties, polymers including dense star polymers, as defined
above, which have at least one portion of the moiety containing between two nitrogen atoms
a methylene (-CH2-)n group where n is 2, 3 or 4 Ipolyaminomethylene group). More than one
such polyaminomethylene group may exist in the moiety. The moiety also contains at least two
methylenephosphonic acid group (-CH2-PO3H2) covaiently attached to the polyamino-
1 O methylene group via a nitrogen. The polyami nomethylenephosphonic acid moieties arepreferably represented by Formula 11, 111 or IV but not limited thereto. The polyaminomethyl-
enephosphonic acid moieties preferably have a group present to permit coupling to a protein
or electrophilic group(s) capable of being attached to protein(s) as described herein The
polyaminomethylenephosphonic acid moieties (represented by L-AP in Formula 1) may be
linked to the protein (GF in Formula 1) by a cleavable linker (represented by CL in Formula 1),
preferably an acid cleavable linker, as described herein.
The terms "growth factor" and "tissue grovvth promoting factor" mean any
molecule which stimulates the proliferation, differentation, metabolism or migration of
mammalian cells. The factors can be derived from natural sources or made by recombinant
20 DNA technology or chemical synthesis. Preferably the factors are purified.
Theterm "purified" asused herein referstofactorswhich, priortomixingwith
the other growth factors, are 90% or greater, by weight, of the specified protei n (i .e., is
substantially free of other proteins, lipids, and carbohydrates with which it is naturally
associated). A purified protein preparation will generally yield a single major band on a
25 polyacrylamide gel for each subunit. Most preferably, the purified factor used in the
compositions of the invention is pure as judged by amino-terminal amino acid sequence
analysis.
The ligands of this invention (AP of Formula 1) may be in the form of their
pharmaceutically acceptable salts. The term "ligand" as used herein is understood to include
30 these salts. The term npharmaceutically acceptable saltn means a cation acceptable for
pharmaceutical use. These are cations that are not substantially toxic at the dosage
administered toachievethedesired effect. Illustratively,thesesaltsincludethose of alkali
metals, such as sodium and potassium; alkaline earth metals, such as calcium and magnesium;
ammonium; light metals of Group IIIA including aluminum; and organic primary, secondary
35 and tertiary amines, such astrialkylamines, including triethylamine, procaine, dibenzylamine,
N,N'-dibenzylethylenediamine, dihydroabiethylamine, N-(C,-C )alkyipiperidine, and any other
suitable amine. Sodium and potassium salts are preferred. The term " pharmaceutically
acceptable" means suitable for administration tO warmblooded animals, e.g. mammals,
-13-
~393%3
WO94/0014~ PCT/US93/062~4
esDecially human beings, and includes being nontoxic, e.g. suitable for pharmaceutical use and
is not poisonous to the warm-blooded animai . The pharmaceutically acceptable salts of the
compounds of the present invention are prepared by conventional ion exchange processes or
by treating the ligand or compound of Formula I with an appropriate base.
5 Preparation of Polyaminomethylener~hosphonic Acids
Various processes are known to prepare the po~yaminomethylenephosphonic
acid moieties of the present invention. A few of these processes are discussed below.
A. Preparation from Amines
Much literature exists which describes the pre,~aration of
10 polyaminomethylenephosphonic acids, particularly the straight or branched-chain moieties,
from amines. For example, US Patent 2,599,807, the disclosure of which is hereby incorporated
by reference, teachesthe preparation of polyaminomethylenephosphonic acids by heating an
aqueous solution of an amine with chloromethylenephosDhonic acid in the presence of a base
such as sodium carbona~e at pH > 10. Other examples are shown by the references given below
describing the preparation of ethylenediaminetetramethylenephosphonic acid (EDTMP) from
the corresponding ethylenediamine (EDA), using a variety of phosphonomethylating agents as
shown in the table below.
PREPARATION OF EDTMP
REACTION REFERENCE
20EDA + CICH2PO3H2 A.E. Martell etal,Nature
321 (1956);
A.E. Martell et al, S. Inorg.
Nucl. Chem. 33,3353
(1971) ,
A.E. Martell et al, Inorg.
Chem. 15, 2303 (1976);
A.E. Martell et al, Inorg.
Nucl. Chem. Letters 7,1103
(1971)
EDA + CICH2PO2H, then H92CI2 US Patent 3,160,632**
EDTA* + PCI3 + H3PO4 USPatent3,959,361**
30EDA + H3PO3 + H2CO K. Moedritzer et al ., J. Org.
Chem. 31,1603 (1966)
EDA + PC13 + H2CO British Patent 1,142,294**
EDA + (-ocH2cH2o-)p(o)cl US Patent 3,832,392* *
*EDTA=ethylenediamine~etramethylenecarboxylic
acid
35 ** = the disclosure of which is hereby
incorporated by reference
14-
94/0014~ 21~ 9 3 2 3 PCr/US93/06254
Additional examples invoiving the preparation of cyclic polyaminomethylene-
phosphonic acids from the corresponding amines can be found in US Patent 4,937,333, the
disclosure of which is hereby incorporated by reference, and D W. Swinkels et al., Recl. Trav.
- Chim. Pays-BAS 110,124-128(1991).
The preparation of polyaminomethylenephosphonic acids from amines via an
intermediate aminomethylenephosphonate ester or mixed aminomethylenephosphonic acid
esterisfound inWO91/07911,publishedJune 13, l991,thedisclosureofwhichishereby
incorporated by reference. This process uses phosphonomethylating agents (e.g.
formaldehyde and dialkyl phosphite) in an aqueous solution which forms the peralkyl
0 phosphonate esters. These ester are then hydrolyzed to the aminomethylenephosphonic 3cids.
The reference also descri bes how to make al kyl or aryl su bstitutions on the carbon between the
nitrogen and phosphorous by treatment of the ester with a strong base (e.g. n-butyl lithium)
and alkylating with an alkyl or aryl halide.
B. Preparation of Polyaminomethylenephosphonic Acids from Carboxylic Acids
Polyaminomethylenecarboxylic acids can be converted into the corresponding
polyaminomethylenephosphonic acids by various known processes. The general reaction to
transform the corresponding carboxylic acid involves a reagent capable of donating a
phosphonic acid group. For example, the reaction of EDTA with PCI3 in nitrobenzene to give
EDTMP (e.g. US Patent 3,832,392, the disclosure of which is hereby incorporated by reference)
C. Preparation of Bifunctional Chelating Agents (BFCAs~ having Ami ne Groups
Capable of Conversion to Polyaminomethylenephosphonic Acids
Simon et al.'s (assigned by unrecorded assignment to The Dow Chemical
Company) copending US PatentApplication Serial No. 565,379, filed August9, 1990,the
disclosure of which is hereby incorporated by reference, teaches the process to make various
25 linearorbranchedlinearpolyaminomethylenephosphonlcacidsthatarecapableofbeing
attached to proteins. WO 84/03698, published September 27,1984, describes open chain
polyamine based bifunctional chelating agent intermediates which can be phosphonomethyl-
ated to yield BFCAs. The synthesis of linear or branched polyalkylene polyphosphonate BFCAs
is also described in US Patent 4,8û8,541, the disclosure of which is hereby incorporated by
30 reference~
US Patents 3,994,966 and 4,622,420 and various references given therein describehow to make various ethylenediamine and diethylenetriamine based BFCAs with different
groups for attachment to protein amine groups, all of which can be converted from the amine
to a polyaminomethylenephosphonic acid BFC to be attached to the amine group of the
35 protein.
Open chain polyaminomethylenephosphonic acid BFCAs containing the linkage
through an aminocarboxylic acid can be prepared from the corresponding polyamineintermediate BFCAs as described in European Appln. 0 279 307, pubiished August 24,1988, the
-15-
WO 94/0014~ 353 PCI/US93/06254
disclosure of which is hereby incorporated by reference. US Patents 4,994,560, 5,006,643 and
5,064,956 also describe how to make various open chain and cyclic amine containing BFCAs
which can be phosphonomethylated to yield the corresponding polyaminomethylenephos-
phonic acid BFCAs.
Cyclic polyamine BFCAs are taught in EP Appln. û 353 450, published February 7,
l990,anddescribeshowtopreparemanydifferentcyclicaminecontainingBFCAswhichcan
then be carboxymethylated to yield aminomethylenecarboxylates. The amine containing
intermediates of these BFCAs can alternatively be phosphonomethylated toyield the
corresponding polyaminomethylenephosphonic acid BFCAs. Similarly, US Patent 4,885,363
describesavarietyof 1,4,7,10-tetraazacyclododecanebasedaminecontainingintermediates
which can be converted by phosphonomethylation tothe corresponding polyaminomethylene-
phosphonic acid BFCAs. The cyclic BFCAs based on the 1,4,7,10-tetraazacyclododecyltetra-
methylenephosphonic acid and the open chain BFCAs based on diethylenetriaminopenta-
methylenephosphonic acid are a preferred group of polyaminomethylenephosphonic acid
BFCAs ligands.
The attachment group from the polyami nomethylenephosphonic acid BFCAs to
the protein may be substituted from the cyclic polyaminomethylenephosphonic acid itself and
are exemplified by J. P. L. Cox in J. Chem. Soc., Chem. Commun. 797 (1989), and M. K. Moi et al,
in J. Amer. Chem. Soc. 11û,6266-6267 (1988). Similar compounds are shown in WO 89/11475,
20 published November30,1989.
Cyclic amine compounds based on the 1,4,8,11-tetraazacyclotetradecane have
been described by M. K. Moi et al, in Inorg. Chem. 26, 3458-3463 (1987). These can be
converted to the tetraaminomethylenephosphonic acid counterpart compounds by processes
mentioned above. Similar compounds which may be used in the present invention are
25 described in US Patent 4,678,667. Additional examples of cyclic polyamines are described by T.
J. McMurry et al. in Bioconjugate Chem. 3(2),108-117 (1992). These intermediate bifunctional
cyclic amines can be phosphonomethylated to give the corresponding polyaminomethylene-
phosphonic acid BFCA (the L-AP portion of Formula 1).
Bicyclopolyazamacrocylocarboxylic acid BFCA intermediates are disclosed in
30 Kiefer et al .'s (assigned by unrecorded assignment to The Dow Chemical Company) copendi ng
US Patent Application Serial No. 8û5,270, filed December 10, 1991, the disclosure of which is
hereby incorporated by reference, can be phosphonomethylated to give the polyaminomethyl-
enephosphonic acid BFCAs.
Bicyclopolyazamacrocylophosphonic acid BFCAs are disclosed in Kiefer et al.'s
35 (assigned by unrecorded assignment to The Dow Chemical Company) copending US Patent
Application Serial No. 805,551, filed December 10,1991, the disclosure of which is hereby
incorporated by reference, teaches the process to make various bicyclopolyazamacrocylophos-
phonic acid BFCAs having polyaminomethylenephosphonic acid BFCAs.
-16-
`''') 94/0014~ PCl /US93/062~4
2139323
D. Preparation of Bifunctional Chelating Agents (BFCAs) having their Nitro
Groups Reduced to Am i ne Groups
When the reduction of a nitro group, especially a nitrophenyl group, to the
corresponding amino group is desired, particularly for compounds described in Paragraph C
5 above, this reduction is readily accomplished by methods known in the art. For example, the
processes listed in Survev of Orqanic Svntheses 1, 411-417 (1970), pub. John Wiley & Sons, and
references contained therein.
E. Preparation of Bifunctional Chelating Agents (BFCAs) having their Amino
Groups Converted to Electrophilic Groups
The BFCAs of the ligands of the present invention (e.g. L of Formula 1) have a
group present to permit coupling to a protein or electrophilic groups capable of being
attachedtoaprotein. Theprocesstoconverttheaminogrouptoelectrophilicgroupscapableof being attached to a protein is well known in the art. Some references that provide suitable
processare: C. F. Mearesetal.,Acc.Chem. Res.17,202-209(1984)and referencesgiventherein;
15 D. Parker, Chem. Soc. Rev. 19, 271-291 (1990) and references given therein; C. F. Meares et al., J.
of Protein Chem. 2,215-228 (1984) and references gi ven therei n; C. F . M eares " Protei n
Tailoring Food Med. Uses, Amer. Chem. Soc. SvmP., 339-352 (1985), ed. R. E. Feeney and
J. R. Whitaker, pub. Dekker, NY, NY. Examples of BFCAs have been given before. Additionally,
many reagents are available for forming an amide bond in aqueous solution which brings
20 together an amine containing molecule with a carboxylate containing molecule. ~or example,
commercially available water soluble carbodiimides have been developed for this jpurpose
[J . V. Staros, Anal. Biochem. 156,220-222 (1986)] .
Another method for linking two nucleophiles together is to convert one, such as
an amine into a Michael acceptor, by reaction with acryloyl chloride or the chemical equivalent.
25 This converts the amino group to the acrylamide group which can then react with a different
nucleophilic amine (Chem. Abst. 83:80295b).
Other methods for preparing drug-carrier conjugations are described by
M. J. Poznansky and R. L. Juliano in Pharmacol. Rev. 36, 278-336 (1984) and WO 90/14844.
F. Preparation of BFCAs by Other Methods
The molecules for attaching two nucleophilic moieties together areltermed
bifunctional crosslinking agents. If the two reactive ends of the molecule are the same, they
are termed "homobifunctional " crosslinking agents. If the two reactive ends of the molecule
are different, they are termed "heterobifunctional" crosslinking agents.
T. Kitagawa et al . i n Chem. Pharm. Bull. 29,1130- 1135 (1981) and references given
35 therein disclosed how to make heterobifunctional crosslinking agents for protein modification.
Such reagents possess two selectively reactive groups such as a maleimide group (which reacts
with thiol moieties) and N-hydroxysuccinimidyl ester (which reacts with amlne groups such as
Iysine). These reagents allowthe combination of two moleculestogether if one rholecule
-17-
~g3~3
W O 94/00145 PC~r/US93/06254
contains amine groups and the other molecule containsthiol group(s). Similar compounds
containing a maleimide group has been reported by O. Nielsen in Synthesis819-821 (1991).
Other bifuctional crosslinkers have been developed for the attachment of BFCAs
or other electrophilic molecules to proteins or other nucleophillic substrates (US Patent
4,680,338 and references given therein); dialdehyde crosslinking agents [S. Avameas et al.,
Scand. J. Immunol. _, 7-23 (1978)]; and commercially available crosslinking agents are shown in
Pierce 1989 Handbook and General Cataloq, pp 283-311 (Pierce, Rockford, IL).
Proteins and small molecules containing electrophilic groups such as amines (butalso including other similar electrophilic groups) can be modified by reaction with
10 commerciallyavailableTraut's reagent [I. Wower, Nuc. AcidRes. 9,4285-4291 (1981)] toconvert
amino groups to sulphydryl groups which can then react selectively with maleimide groups.
G . Cleavable Li nkers
Sometimes it is advantageous that the linkage between the polyaminomethyl-
enephosphonic acid BFCAs and the GF be reversible or cleavable under certain physiological
conditions. Such linkages are taught in the art and examples have been given above.
Particularly advantageous are the linkers that are acid cleavable. Some examples of acid
cleavable linkers are disclosed in US Patents 4,542,225 and 4,618,492 and the references
mentioned therein. These cleavable linkers possess a cyclic anhydride at one end of the linker
which reacts with amine groups, and a maleimido group at the other end of the linker which
20 reacts with sulphydryl groups. The linkage between the anhydride and amino groups (which
forms an amide with a carboxylic acid hereby) is easily cleaved at acidic pH. Also US Patent
4,764,368 descri bes the use of cyclohexene- 1,2-dicarboxyl ic acid anhydrides as a way to
introduce acid cleavable amide functionality between a small molecule and a large molecule.
WO90/14844describestheuseofsugarderivativesascleavablelinkersinweaklyacidic
2S conditions
H. Presence of Metal lons
Suprisingly, the addition of metal ions does not significantly inhibit the affinity of
polyaminomethylenephosphonic acids for calcific surfaces. For example, the addition of
calci um or samari um ions does not i nterfere with the affi nity of 1,4,7,10-tetraazacyclodo-
30 decanetetramethylenephosphonic acid (DOTMP) for the calcific surface of hydroxyapitite.
For example, DOTMP has been shown to go to calcific sites in vivo whencomplexed with ions of samari um, holmium, gadolinium or yttri um in US Patent 4,976,950.
Also ethylenediaminetetramethylenephosphonic acid (EDTMP) wnen complexed with calcium
or other metal ions has also been shown to go to calcific sites in vivo (European Appln.
35 0 462 787, published December 27,1991).
I. Clusters of Aminophosphonic Acids for Attachment to Proteins
Polymers having polyaminomethylenephosphonic acids, including dense star
polymers such as described in European Appln.0 272 180, published June 15,1988, are
-18-
"'') 94tO014~ 2 1 ~ 9 3 2 3 PCl/US93/06254
prepared by reacting the amine with formaldehyde and dialkyl phosphite in an aqueous
solution which forms the peralkyl phosphonate ester. This ester is then hydrolyzed'to the
aminomethylenephosphonic acid on the dense star polymer. Particularly, the dense star is in
the PAMAM form having amine groups on its surface. These amine groups are theh reacted
5 with phosphonomethylating agentstoform the polyaminomethylenephosphonic acid, which
is then conjugated to the GF. The advantages of the dense star as the bone seeking moiety are
that it can be water soluble, have a controlled size, and specific groups and quantity of groups
available for conjugation to GF.
The polymers of the L-AP of Formula I also include arborols (G. R. Nev~kome, J.
1O Org. Chem. 50, 2004-2006 (1985) which are capable of being attached to proteins, e.g. GF. The
arborols are monocascade spheres which possess a three-deminsional microenvi roment having
the outer surface covered with polar functional groups. These arborols can be con~erted into
polyamines by methods known in the art for converting esters and alcohols into arnines (e.g.
SurveyofOrqanicSynthesis 1,411-417(1970),pub.JohnWileyandreferencesgivqntherein).
The 2 -l~orol polyamines can be converted into polyami nomethylenephosphonic acids by the
met , described above. By starting the arborol cascade poiymer with the appropriate group
such as p-nitrobenzyl bromide, the arborols can contain a group capable of attaching to
proteins. Thus aborols containing polyaminomethylenephosphonic acid groups and a group
capable of attaching to a protein by either a stable covalent linkage or cleavable linkage can be
20 prepared.
Also, other polymers, either linear or branched, containing amines and a group
capable of attachment to a protein have been described [V. P. Torchilin et al., Byull. Eksp. Biol.
Med. 102, 63-65 (1986)]. Such polyamine containing polymers can be converted by methods
described above to polymers containing polyaminomethylenephosphonic acid groups.
By adjusting the stoichiometries of the protein to reactive group containing
cluster of polyaminomethylenephosphonic acid, the type of product can be altered. Also, by
adjustingthestoichiometryduringthestepthatintroducesthelinkagecontainingthereactive
group, a cluster compound can be linked to two molecules of protein which may be identical to
each other or different molecules of protein.
J . Alternate Processess
Selective attachment of the polyami nomethyleneohosphonic acid BIFCAs to
noncritical amine groups on a GF may be accomplished by first mixing the GF with a soluble or
solid support immobilized form of the receptor from cells which will form a GF-rekeptor
aggregate. This aggregate can then be reacted with an excess of the reactive form of the poly-
35 aminomethylenephosphonic acid BFCA. Since the GF is bound to the receptor, only amine
- groups not involved in the GF receptor interaction will be modified. The GF modilfied in this
fashion is then able to interact with its receptor in vivo to yield biological activity, The
aggregate between the modified GF and the receptor can then be broken down i nto the now
l g
WO94/00145 2~39~23 PCr/US93/06254
selectively modified GF and the reusable receptor, which after separation will yield fully active
modified GF that sti 11 recognizes the receptor.
The compounds of Formula I can be prepared in several different ways. For
example, the AP portion can be reacted with L to form the L-AP portion; the L-AP portion then
5 attached to the CL; with the CL-L-AP group then attached to the GF. Alternatively, GF is
reacted with CL to form the ~iF-CL portion; which GF-CL is then reacted with L-AP to form
compounds of Formula 1. The degree of modification of the GF is described by the q term of
Formula 1, which is arrived at experimentially by adjusting the concentrations, time,
temperature, pH, and stoichiometry of the reactions between GF, CL and the L-AP molecules.
1 0 Formulations
The formulations of the present invention are in the solid or liquid form. Theseformulations may be supplied as a single substance for direct use or as two or more substances
(e.g. in kit form) such that the two components are mixed at the appropriate time prior to use.
Whether premixed or as a kit, the formulations may require a pharmaceutically-acceptable
carrier or adjuvant.
The compounds of this invention may also be administered parenterally, that is,
subcutaneously, intravenously, intramuscularly, or interperitoneally, as injectable dosages of
the compound in a physiologically acceptable diluent with a pharmaceutical carrier which can
be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related
20 sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as
propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-1 ,3-dioxolane-4-
methanol, ethers such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or
glyceride, or an acetylated fatty acid glyceride with or without the addition of a
pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as
25 pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose,
or emulsifying agent and other pharmaceutical adjuvants.
Illustrative of oils which can be used in the parenteral formulations of this
invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut
oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil.
30 Suitable fatty acids include oleic acid, stearic acid, and isostearic acid. Suitable fatty acid esters
are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty alkali metal,
ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for
example, dimethyl dialkyl ammonium halides, alkyl pyridinium haiides, and alkylamines
acetates; anionic detergents, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,
35 and monoglyceride sulfates, and sulfosuccinates; nonionic detergents, for example, fatty
amine oxides, fatty acid alkanolamides, and polyoxyethylenepoly-propylene copolymers; and
amphoteric detergents, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline
-20-
21393~3
-'') 94/0014~ PCr/US93/062~4
quarternary ammonium salts, as well as mixtures. The parenteral compositions of this
inventionwilltypicallycontainfromaboutO.001~/otoaboutlO% byweightofcornpoundof
Formuia I in solution. Preservatives and buffers may also be used advan-tageously. In order to
- minimize or eliminate irritation at the site of injection, such compositions may contain a non-
5 ionicsurfactanthavinga hydrophile-lipophilebalance(HLB)offromabout 12toabout 17. The
surfactant can be a single component having the above HLB or can be a mixture of two or more
components having the desired HLB. Illustrative of surfactants used in parenteral formulations
are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate
Other possible formulations for the compounds of Formula I include; orally, using
10 known oral pharmaceutical formulations some of which may contain magnesium hydroxide in
excessive amounts [P. J.Neuvonen et al., Eur. J. Clin. Pharmacol. 35,495-501 (1988)]; trans-
dermal delivery using known proceduresfrom B. Kari, Diabetes35, 217 (1986), B. R. Meyeret
al., ain. Pharmacol. Ther. 44, 607 (1988); implants using adsorption of the compound onto
activated carbon particles which are then implanted or injected [A. Hagiware, Gan to Kagaku
Ryohol5, 1038-1042(1988)orChem.Abst. 109:156158n(1988)];andnasaldelivery,either
alone or in combination with permeation inhancers [W. A. Lee, ~ioPharm. 22-25 (Nov/Dec,
1990) or Wall Street J. B 1, (August 16,1989)]; and others by R. Langer, Sci. 249,1527- 1533 (Sept.
28,1990). The formulations may also be applied locallytothe site of the injured or depleted
bone by direct topical application to that site. The latter method may require surgery to expose
20 the site of injured or depleted bone.
Bone regeneration using the compounds of the present invention is more
effective than that achieved in the absence of treatment (i.e. without applying exogenous
agents) or by treatment with similar levels of unmodified growth promoting factprs, i .e. only
GF of Formula 1. Because of the relatively high cost of GF production and the potential ability
25 of GF to cause adverse toxic affects when delivered in high doses systemically, it i~ also
desireable to use the ligand to ensure delivery to the desired site and reduce the overall dose of
G F and its toxic effects to the mammal .
In the method of regenerating bone of a mammal, especially a human patient,
according to the invention there is administered to the mammal either by direct application to
30 the area of injured or depleted bone or by indirect application, for example, via systemic
circulation (such as following parenteral, intramuscular or subcutaneous injection) an effective
amount of a composition that i ncl udes a compound of Formula 1.
The amount of (CL) -L-AP of Formula I which is needed to aid in bor~e delivery of
the GF can be any effective amount. The amount of compound of Formula I to be administered
35 in order to treat any of the diseases desired for such a delivery system can vary widely according
to the particular dosage unit employed, the period of treatment, the age and sex of the patient
treated, the nature and extent of the disorder treated, and other factors wel l-known to those
WO 94/0014~ 2~3~3 PCr/US93/062~4
practicing the medical arts. Moreover the compounds of Formula I can be used in conjunctlon
with other agents known to be useful in the treatment of bone diseases.
The effective amount of compounds of Formula I to be administered according to
the present invention will generally range from about 0.005 to 50 mg/kg of body weight of the
5 patient and can be administered as frequently as one or more times per day. The compounds
of Formula I can be administered in a pharmaceutically acceptable formulation as described
above. The compounds of Formula I can have one or more different active compounds
administered either simultaneously or sequentially; and such compounds may be administered
with other known active agents for regenerating bone.
The i nvention wi ll be further clarified by a considerati on of the following
examples, which are intended to be purely exemplary of the present invention.
Defi nitions
The terms used in the present examples and not defined previously herein are as
follows:
BMP = bone morphogenetic protei ns
BSA = bolvine serum albumin
FCS = fetal calf sera
HPLC = high pressure liquid chromatography
HSA = human serum albumin
PBS = phosphate buffered saline
RP-HPLC = reverse phase HPLC
SCN-BDTMP = 4-isothiocyanatobenzyldiethylenetriaminopentamethylenephos-
phonic acid
All solventsand reagentswereobtained from commercial suppliersand used
25 without further purification, except as defined below.
All water was passed through a Barnstead NANOpure'~ ion exchange/carbon bed
water purification system and had a resistivity of about 18.5 megaohms
Acetic acid was glacial acetic acid from Aldrich Chemical Co. at greater than
99.99%
1 % HSA in 1.0 mL aliquots in PBS (20mM phosphate, pH 7.4,0.15M NaCI) were
received frozen from the Institute of Molecular Biology, Inc. (IMB)
12sl-PDGF was obtained from IMB (frozen in 135 IlL aliquots, containing 100 1l9 of
nonradioactive IGF and 26.5 IlCi of carrier free l251-PDGF), as well as unlabelled
purified recombinant PDGF-BB and IGF-I, all in 0.1M acetic acid
Centricon'~ membrane filters of 10,000 and 3,000 molecular weight cutoff
(Amicon Division of W. R. Grace and Co.) were used for washing and
concentrating the aqueous protein solutions
'~'') 94/0014~ ~ JL ~ a1 PCI/US93/062S4
Growth Factor Modification
General Methods
Purified recombinant human PDGF-BB and recombinant human IGF-I and their
respective radioiodinated forms were suppl ied by the Institute of Molecular Biology, Inc. The
5 identities of both PDGF-~ and IGF-I were established by amino acid composition and amine
terminal amino acid sequence. Amino acid composition was determined by gas phasehydrolysis of the protein followed by RPHPLC essentially according to the method of Eveleigh
and Winter [J. W. Eveleigh and G. D. Winter, "Amino Acid Composition Determination" Protein
Sequence Determination, pp. 91-95 (1970)]. The amine terminal amino acid sequence was
10 obtained by the Edman degradation followed by formulation and analysis of the PTH amino
acid by HPLC essentially according to the method of Hunkapiller and Hood lM. W. Hunkapiller
and L. E. Hood, Methods in Enzymology91, 486-489, (1983)]. Puritieswere determined using
scanning densitometry of Coomassie blue and silver stained SDS PAGE gels and RP-HPLC.
STARTING MATERIALS
Example A
Preparation of PDGF-BB and IGF-I
Both PDGF-BB and IGF-I were produced by standard recombinant DNA techniques
and purified by conventional chromatography. Both process are well known to those skill in
the art. (PDGF: US Patents 4,061,757 and 5,045,633; IGF-I: Y. Sato et al., J. Biochem. 101,1247-
20 1252 (1987) and Wong et al ., Gene 68,193-203 (1988) .
Analysis of the amine terminal amino acid residues 1 through 20 of the PDGF-BB
(provided by IMB) gave a single, intact amine terminus and was identical to those residues
predicted by the cDNA sequence.
Sequence analysis of the amine terminal residues 1 through 53 of the human IGF-I25 (produced by IMB) yielded a single intact amine terminus identical to those residues predicted
by the cDNA sequence. The amino acid compositions were found to be consistent with that
predicted by the integrated human IGF-I gene. Both growth factors were greater than 95%
pure.
Exam~le B
30 Biological Activity of PDGF-BB and IGF-I
Potency or bioactivity of each factor was measured by cell culture mitogenic assay
in which the effective dose at half-maximal stimulation (EDso) is defined as one unit. Both
bioassays are based on the measurement of the incorporation of ~3H~-Thymidine into DNA of
BALB/c 3T3 mouse fibroblasts. The 3T3 cells are seeded, in DMEM supplemented with 10% FCS,
35 into 96 well assay plates at 2500 cells per well. Assay plates are incubated (37C) for seven days
prior to use. This allows for the deoletion of serum components and induces quiescence m the
cells. In the PDGF-BB bioassay standards and test samples are added in triplicate on the
afternoonoftheseventhdayandareallowedtoincubateovernightforl8hours. Thecellsare
-23-
WO94/0014~ ~i39323 PCr/US93/06254
then exposed to 1.0 }lCi of 3H-thymidine for 6 hours, which is incorporated according to the
biological activity of the PDGF.
In the bioassay for IGF-I, the cells are preincubated with PDGF-BB, prior to theadditionoftestsamplesandstandards. ThepreincubationenablesthecellstorespondtolGF-I
5 when the IGF-I is added in the presence of Epidermal Growth Factor (EGF). After an overnight
incubation period the cells are exposed to 1.0 ~Ci of 3H-thymidine which is incorporated
according to the biological activity of IGF-I.
In both the PDGF-BB and IGF-I assays, following extensive washing, the cells areIysed and the samples are quantitated on a liquid scintillation counter. The resulting data are
10 plotted to generate a dose response curve from which unit activity determinations can be
made.
A unit is defined that concentration of GF which induces 50% of the maximum
cellular response in the appropriate assay (EDso value). Maximum response in the PDGF asnd
IGF-I assays is defined as the cellular response to a 5% Fetal Calf Sera (FCS) standard. The
specific acti vity was calcu I ated by d ivi d i ng the nu mber of u nits based on the m itogeni c assay by
the mass as determined by amino acid analysis. The potency for both PDGF-BB and IGF-I prior
to protein modification was 1 to 3 ng/mL (see Figure 3 for PDGF-BB; Figure 4 for IGF-I).
Typically,thespecificactivitieswereintherangeof3tolOxlO5unitspermgofprotein.
Example C
20 Radiolabel led ~ 251-PDGF
The "cold" PDGFdescribed in ExampleAwaslabelled bythe [125l]-Bolton Hunter
method which principally labels the primary amines of accessible Iysine and N-terminal
residues. '251-PDGF is also commercially available from NEN. NEN normally supplies the 1 251-
PDGF in a sodium citrate buffered salt solution containing 1% BSA as a carrier protein. Prior to
25 use this BSA must be removed from the commercially available ~251-PDGF to allow efficient
modificationofthePDGF. Inordertoproducethecarrierfreeformof1251-PDGF,antibody
affinity chromatography was used. The immobilized anti-PDGF antibody (supplied by IMB)
exhibits specific binding to the AB and BB PDGF isoforms, and no binding to the AA
homodimer. 42.6 ~lCi 1251-PDGF in a neutral pH buffer was applied to a 200 ~lL anti-PDGF
30 column. TheBSAwasexhaustivelyflushedfromthecolumnwithPBSandthe1251-PDGFelutedinO.lMaceticacid. Basedonaspecificactivityof35~1Ci/~gthisparticularlotofl251-PDGFis
estimated to have, on average, one [1251] attached for every 1.8 PDGF dimers.
ExamDle D
Radiolabelled 1251-lGF
The "cold" IGF described above was labelled with [1251] using the lactoperoxidase
or chloramine T method which iodinatestyrosine residues forming iodotyrosyl products. 1251-
lGF-liscommerciallyavailablefromNEN. 1251-lGF-lwassuppliedcarrierfreeandfreezedried
~ 94/0014~ 2 1 3 ~ 3 2~ PCl/US93/06254
from 100 IlL of 1 OOmM sodium citrate, pH 4.5. Based on a specific activity of 208 IlCil~g, this lot
of 1251-lGF-I is estimated to have on average, one [125lj attached for every 1.4 IGF-I protein
chains.
ExamDle E
5 Preparation of two PDGF solutions
A vial containing radioactive PDGF was placed in the well of a Capintec dose
calibratorwhich was adjusted for 1251 A vial containing 50 1l9 of PDGF was rinsed with 200 ~lL
of 0.1 M acetic acid and added to a vial containing 100 1l9 of cold IGF and 1 2s~-PDG F i n 135 ~lL of
0.1Maceticacid. ThecoldPDGFwasvialwasrinsedwith7more200~LportionsofO.lMacetic
10 acid, which rinses were combined to provide one sample containing radioactive and
nonradioactive PDGF and nonradioactive IGF. The combined sample vial was shaken to mix
and two 750 ~,L aliquots of the solution were removed Each aliquot was placed on a separate
membrane of a centricon 10, labelled 1 and 2. Appropriate volume and gamma readings were
taken which verified the volume and radioactivity before and after dividing of the sample. The
two aliquot samples were spun in a centrifuge and placed in new tared filtrate cups.
Each membrane filter (1 and 2) was then treated with 1.0 mL of 3.0M sodium
bicarbonate buffer and recentrifuged. The two cups were removed and replaced with new
tared filtrate cups. Both membrane retained samples read 10.5 ~Ci. The two membrane filters
were then treated with an additional 1.0 mL of 3.0M sodium bicarbonate buffer and
20 recentrifuged. Both filtrate cups were removed and replaced with new tared filtrate cups.
Each of the two samples read 10.5 ~lCi on the membrane. The washing procedure described
above was done to remove the carrier nonradioactive IGF which should come through the
membrane. The remaining material on the membrane was then ready for conjugation.Example F
25 Preparation of H2N-BDO3TMP
Thefreebase, 1-(a-carboxyl-2-methoxy-5-nitrobenzyl)-1,4,7,10-
tetraazacyclododecane, 513 9 (1.3 mmol) was added to a stirred slurry of 776 mg (4.7 mmol) of
triethyl phosphite and 141 mg (4.7 mmol) of paraformaldehyde. The resulting slurry was
heated with stirring to 95C for 2 hours, cooled to room temperature and concentrated in
30 vacuoto give a viscous oil. The oil was chromatographed using a basic alumina column and
eluted with chloroform and, after evaporation of the solvent, to yield (55%), as a light yellow
viscous oi l, of the ester, 1 -(a-carboxyl-2-methoxy-5-nitrobenzyl)- 1,4,7,1 O-tetraazacycl o-
dodecane-4,7,10-trimethylenephosphonic acid hexaethyl ester.
The ester, 250 mg (0.3 mmol) was hydrolyzed by stirring with 3 mL of
35 concentrated HCI at 100C for 18 hours. The agueous soiution was freeze-dried to give (80%~,
asa cream colored solid, 1-(a-carboxyl-2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclodo-
decane-4,7, 1 O-trimethylenephosphonic acid (Ozl~l-BDO3TMP).
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W094/0014~ ~l393~ PCr/US93/06254
A 100 mg portion of O2N-8DO3TMP was dissolved in 20 mL of water. After
purging the system with nitrogen,120 mg of 10% Pd/C was added and the suspension placed
under an atmosphere of hydrogen with constant vigerous stirring. After 3 hours the catalyst
was removed via filtration and the filtrate Iyphilized to give 89.1 mg of H2N-BDO3TMP as a
chocolate colored solid. A 23.4mM solution of this ligand was prepared by dissolving 89.1 mg
of H2N-BDO3TMP in 4.26 mL of water.
Example G
Preparation of SCN-BDTMP
(1.) A 100 mL three necked, flask was loaded with 2.0 9 (5.76 mmol~ of 1 -(4-
10 nitrobenzyl)diethylenetriaminetrihydrochloride [prepared bythe proceduredescribed by
M. W. Brechbiel et al. in Inorg. Chem. 25,2772-2781 (1986)]. A separate solution of 10.93 9
(0.108 mol) of concentrated HCI and 6.91 9 (0.086 mol) of phosphorous acid was prepared and
added to the reaction flask. The flask was fited with a therometer, reflux condenser and a stir
bar. With constant stirring the reaction solution was brought to reflux. An addition funnel was
loadedwith 12.0g(0.144mol)of37% bywtformaldehydesolution,attachedtothereaction
flask, and dripped into the heated mixture at a rate of about 1 mUmin. The reaction was held
at reflux for an additional 16 hours, then reduced in volume under vacuum to produce an
amber semisolid. The solid was taken up in about 2 mL of water and added dropwise with
vigorous stirring to about 800 mL of methanol. The resulting white percipitate was removed by
20 filtration and dried at 45C to yield, as an off white crude solid, 2.32 9 (57%) of 1-(4-
nitrobenzyl)diethylenetriaminepentamethylenephosphonic acid (O2N-BDTMP).
(2.) A 500 mg portion of crude 1-(4-nitrobenzyl)diethylenetriaminepenta-
methylenephosphonic acid was purified on a 1.5 cm Q-Sepharose (Pharmacia) anion exchange
column eluting with 0 to 1 M ammonium acetate gredient. The desired product,1 -(4-nitro-
25 benzyl)diethylenetriaminepentamethylenephosphonic acid, is the most retained peak at UV280 nm. Collection of the last el uti ng peak resulted in about 43 m L of solution containi ng
purified product. The volume was reduced to about 26 mL and treated with 200 mg of 10%
Pd/C. The suspension was put under a balloon of hydrogen gas as stirred vigerously for about
2.5 hours. HPLC (anion exchange, eluting with 0 to 1 M ammonium acetate over 30 minute
30 gradenta2mUmin)indicatedaslightshifttoashorterretentiontimeforthereducedproduct.
The suspension was then filtered, and the filtrate Iyophilized to give 259 mg of white glassy
solid. HPLC indicated a purity of about 92% . The product was further purified by preparative
anion exchange chromatrography as described above to give, as a white solid, 144 mg of the
ammonium salt of 1-(4-aminobenzyl)diethylenetriaminepentamethylenephosphonic acid, with
35 a purity by anion exchange HPLC of >95% . The product was further characterized by as
follows.
~.vo 94/0014~ 2 I ~ 9 3 2 3 PCT/I~S93/06254
Decoupled P-31 NMR showed 3 singlets in the expected 2:2: 1 ratio.
'H NMR(D20)
2.59-3.67 (m,17H), 4.13 (b. 2H), 6.91 (d,2H),7.21 (d,2H).
5 13C NMR(D2)
52.0, 52.6, 53.3, 53.4, 54.1, 54.9, 55.8, 57.1, 57.1, 58.3, 58.3,65.2,120.5,131.1,133.5,146.8.
(3a.) A O.O9M solution of the ammonium salt of 1-(4-aminobenzyl)diethylenetri-
aminepentamethylenephosphonic acid in water was prepared by dissolving 146 mg of the
phosphonicacidin l.913mLofwater. A328}1Lportionofthissolution(containingabout25
10 mg of the phosphonic acid) was placed i n a 10 mL vial contai ni ng 1 mL of water and 1 mL of
chloroform. To the emulsion that formed was added 100 IlL of thiophosgene with vigerous
stirring for about 1 hour. The chloroform layer was removed and the aqueous portion
extracted with water using four 1 mL portions of chloroform. The washed aqueous solution of
the product was frozen in a dry ice/acetone bath and Iyophilized to give 26.5 mg of puffy white
powder. A 0.5 mg portion was dissolved in 200 ~lL of water. A 611L portion of this solutlon was
diluted with 3.8 mL of water and the UV spectrum obtained showing absorption maxima at 270
and 283 nm confirming the product,1-(4-isothiocyanatobenzyl)diethylenetriaminepenta-
methylenephosphonic acid (SCN-BDTMP).
(3b.) A 43 mg (51 ~lM) portion of 1-(4-aminobenzyl)diethylenetriaminepenta-
20 methylenephosphonic acid was dissolved in 2 mL of water and mixed with 1 mL of chloroform.To the stirred solution was added 150 ~lL of thiophosgene in one portion. After vigerous
stirring for 2.5 hours the chloroform layer was removed and the aqueous portion extracted
with water using three 3 mL portions of chloroform. The washed aqueous solution of the
product was frozen in a dry ice/acetone bath and Iyophilized to give 45.4 mg of puffy white
25 powder. A 2.4 mg portion was dissolved in 960 IlL of water. A 611L portion of this sol ution was
diluted with 3.8 mL of water and the U\l spectrum obtained showing peaks at 272 and 282 nm
confirming the product,1-(4-isothiocyanatobenzyl)diethylenetriaminepentamethyiene-
phosphonic acid.
(3c.) Preparation of 3.47mM and 34.7mM solutions of SCN-BDTMP
AlOOmgportion(1.389x10-5mol)ofSCN-BDTMPwasdissolvedin400~LofO.3M
sodium bicarbonate buffer. The resulting solution had a r~H of 7.51 which was adjusted to a pH
of 9.48 by the addition of 5 IlL of 50% (wt) sodium hydroxide. The final solution was 34.7mM
of SCN-BDTMP
A 511L portion of the above prepared 34.7mM SCNBDTMP solution was added to
35 45 ~L of 0.1 M sodium bicarbonate and mixed thoroughly. The final solution was 3.47mM of
SCN-B DTM P.
WO 94/0014~ 2 ~3 9 ~ 23 PCr/lJS93/06254
Examole H
Preparation of four IGF Solutions
Vials of 100 ~L of Iyophilized radiolabelled i251-lGF-I (from NEN) or Iyophilized
recombinant IGF-I (from IMB) were each reconstituted with 100 ~L of water to give solutions of
5 1 mg/mL of IGF containing 0.5M of sodium chloride in 20mM of Tris-hydrochloric acid with
0.25% BSA as a carrier protei n. The recombinant IG F vial's contents were dissolved i n 1 Om M of
acetic acid and transferred into the glass serum vial containing iodinated IGF dissolved in 535
mg of 10mM acetic acid. This combined solution of IGF containing tracer '2sl-lGF-I was dialysed
(Spectra/Por7"A membrane from Spectrum Medical Industries) againstthree 1 L portions of
10 10mM acetic acid. The contents of the dialysis bag were then divided into 4 equal volumes and
placed onto the membranes of 4 separate Centricon"A microconcentrator membrane filters
(3000 molecular weight cutoff). The Centricons"A were then spun to concentrate the sol utions
and the contents of the membrane washed with one 1.6 mL of 0.3M sodium bicarbonate buffer
at pH = 9.48. This method gave 4 Centricon"A membrane units containing the amounts of IGF
shown below:
IGF
CentriConry membrane
119
141.2
241.7
345.7
447.7
* approximate
25 Example I
Preparation of two IGF solutions
Frozen radiolabelled '2sl-lGF as a 150 llL aliqout containing 300 1l9 Of IGF-I and
23.6 ~uCi of carrier free 12sl-lGF in 0.1 M acetic acid was allowed to thaw and then diluted to a
finalvolumeofabout1.5mLusingO.1Maceticacid. A70011Laliqoutofthissolutionwas
30 placed on each of 2 Centricon"A microconcentrator membrane filters (3000 molecular weight
cuttoff). The Centricons"A were then spun to concentrate the solutions and the contents of the
membrane washed with two consecutive 1 mL portions of 0.3M sodium bicarbonate buffer at
pH = 9.48. The calculated amount of IGF (with tracer 12sl-lGF) in membrane no. 1 was 130 ~g
(1.7 x 10-8 mol) and in membrane no. 2 was 127 1l9.
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`- ') 94/0014~ 2 1 3 9 3 2 ~ PCI /US93/06254
Example J
Preparation of 1,3-propanediamine-N-(carboxy-propyl)-N,N',N'-trimethylene-phosphonic acid
(PDTMP)
The structure of the compound PDTMP is shown by the following formula:
H00C -- ( CH2 ) 3 CH2-P03H2
N CH2 CH2 CH2 N
CH2-P03H2
o H203P-CH2
Commercial grade 3-aminopropyl-2-pyrrolidinone (5.0 9) was added slowly to a
solution of phosphorous acid (9.7 g) in 6M hydrochloric acid (50 mL). The yellow solution was
heated to reflux and treated dropwise with formalin (9.23 mL of 37% solution) over at two
15 hour period. After refluxing an additional 18 hours, the solution was cooled and the solvent
removed to give a viscous oil. Dripping the oil into 200 mL of methanol gave 5.15 9 of PDTMP
after filtering and drying. This compound showed the expected two singlets in the decoupled
3~P-NMR spectrum at 8.2 ppm and 7.5 ppm (relative to H3PO~) in a 2: 1 ratio.
This compound may be used as a iigand AP in Formula I where the L is through the20 COOH group.
Example K
Preparation of Ethylenediamine-N-(4-amino-phenethyl)-N,N',N'-trimethylenephosphonic Acid
(APE DTM P)
The structure of the compound APEDTMP is shown in the following formula:
H2N ~CH2CH2 CH2-P03H2
N CH2 CH2 N
H203P-CH2 CH2-P03H2
Three250mLErlenmeyerflaskswereloadedwith 120mLofethylenediamine
(EDA) and equipped with a stirring ~ar and placed on a stirrer. Three 10 9 samples of p-
nitrophenethylbromide were weighed and added slowly to the stirring flasks containing EDA
over a thirty minute period. Following tne addition of p-nitrophenethyl bromide the flasks
were ailowed to stir overnight (sixteen hours) at room temperature. The contents of the flasks
WO 94/00145 2~93~3 PCI/US93/06254
were transferred to a 500 mL round bottomed flask and attached to a simple distillation
apparatus. The excess EDA was distilled from the desired product at a temperature of 28 to
32C under vacuum. Following the removal of most of the starting EDA the viscous oil was
dissolved in water and extracted three times with 75 mL of methylene chloride (CH2CI2). The
CH2CI2 layers were combined and concentrated in a rotoevaporator to yield 29.34 grams of a
viscous dark oily liquid.
All of this sample was added to a 500 mL round bottomed flask along with 100 mL
of l .SM HCI. The aqueous layer was treated with activated carbon heated to 90C, then filtered
through a paper filter. The aqueous layer was stripped under vaccum and dried in a vacuum
dessicator overnight.
The resulting dry solid was washed with cold methanol and filtered to give 15.60g of N-(p-nitro-phenethyl)ethylenediamine as the hydrochloride salt. An additional 6.77 9 of
product was obtained by evaporating the filtrate and washing with methanol to give a
combined yield of 72%.
A 250 mL three-necked, round bottomed flask was loaded with 20.0 9 (0.063
moles) of N-(p-nitro-phenethyl)ethylenediamine trihydrochloride salt. To this flask was added
9.0 9 of deionized water and 22.79 9 (0.219 moles) of concentrated hydrochloric acid in a
sol ution with 17.16 9 (0.209 moles) of phosphorous acid .
The flask was attached to a reflux condenser, equipped with a stir bar, and set on
20 astirrer. A 10 mLsyringewasfilledwith 17.74g(0.219moles)of37% formaldehydesolution
and attached to a syringe pump pre-calibrated to deliver at a flow rate of 0.1 mUmin. The
reaction solution was brought up to reflux temperature, then with constant stirring, the
formaldehyde was slowly added tothe flask over a three hour period.
Following the addition of the formaldehyde solution, the reaction was allowed to25 reflux and stir for an additional three hours. The reaction was allowed to cool and the water
was removed under vaccum to give a viscous, dark solid. This was slowly added to methanol
resulting in a light brown solid. This precipitate was filtered and dried to give 20 9 (65% yield)
of N-(p-nitrophenethyl)-ethylenediarnine-N,N',N'-trimethylene phosphonic acid. A 10 9 sample
of this triphosphonic acid was dissolved in 20 mL of water and neutralized with 2 mL of
30 concentrated ammonium hydroxide. This crude ammonium salt was then purified using
reverse phase preparative HPLC eluting with water. A sample of N-(p-
nitrophenethyl)ethylenediamine-N,N',N'-trimethylene phosphonic acid (710 mg, l.21 ~moles)
purified in this manner was dissolved in 50 mL of water and placed in a hydrogenation bottle
with a 235 mL volume capacity. A blanket of nitrogen was gently blown into the bottle to
35 displace the ai r Approximately 50 mg of 10% Pd on carbon was added as a catalyst and any
catalyst on the vessel sides was rinsed down with water. Once all the catalyst was under water,
the nitrogen padded bottle was placed on a Parr hydrogenation shaker apparatus. After
removal of the nitrogen by means of a vacuum line, an atmosphere of hydrogen was
-30-
~vo 94/0014~ 2 1 ~ 9 3 2 ~ PCl /US93/062S4
introduced. This flushing was repeated to ensure that all oxygen had been removed. The final
charge of hydrogen gave a pressure of 35 psi. The hydrogenation was started by shaking the
bottle and continued until the hydrogen uptake ceased (2.5 hours). This pressure drop and the
known volume of hydrogen can be used to calculate the moles of hydrogen used which i n this
5 case was 99.2% of theoretical. The reaction mixture wasthen carefully vacuum filtered (after
removal from the depressurized hydrogenation apparatus) through a glass frit and the catalyst
thus separated waswashed with four 25 mL portions of water. The combined aqueous filtrate
was then frozen and Iyophilized overnight to yield 580 mg (86% yield) of N-(4-aminophenyl)-
ethylethylenediamine-N,N',N '-trimethylenephosphonic acid as the ammoni um salt (APEDTMP).
The decoupled 31P-NMR Spectrum of this compound showed a singlet at 8.0 ppm
(rel.- - ve to H3PO~) and a singlet at 16.9 ppm in the expected ratio of 1 :2, respectively.
This compound may be used as a ligand AP in Formula I where the L is through theNH2 group.
ExamDle L
Preparation of 1 -(Carboxy)ethylenediaminetetramethylenephosphonic Acid (CEDTMP) The structure of the compound CEDTMP is shown below:
H203P CH2 COOH CH2P03H2
I
N CH CH2 -- N
CH2P03H2
H203p -- CH2
To a solution of phosphorous acid (12.9 g) in 6M hydrochloric acid (50 mL) was
added 5.0 9 of 2,3-diaminoproprionic acid dihydrochloride (Aldrich Chemical Co., Milwaukee,
Wl). The solution was heated to reflux and treated dropwise with formalin (37% formaldehyde
in water,12.3 mL) over a ,oeriod of 45 minutes. After an additional eighteen hours of reflux,
the solvent was removed and the oil dissolved in methanol (20 mL). This solution was then
30 added with stirring to absolute ethanol (200 rnL) to yield a fine precipitate. This solid was then
filtered and dried, yielding 7.37 9 of CEDTMP. An aiiquot of this solution was diluted ten-fold
and analyzed by high performance liquid chromatography (HPLC). HPLC was performed on a
Dionex'~ 2010i lon Supression Chromatography System (Sunnyvale, CA) using an AS-7 anion
exchange column (8mm x 25cm) and eluting with 0.03M nitric acid at a flow rate of 0.6 mUmin
35 with ultraviolet detection monitored at 330 nanometers. Thls compound had a retention time
of 10.11 minutes.
WO 94/00145 2~.3~3~ PCI/US93/06254
This compound may be used as a ligand AP in Formula I where the L is through theCOOH group.
Example M
Preparation of 1-(4-aminobenzyl)ethlene-diaminetetramethyleneDhosphonic acid (ABEDTMP)
The structure of the compound ABEDTMP is shown in the formula:
NH2
11
H203p -- CH2 fH2 CH2P03H2
I
N CH CH2 N
CH2P03H2
H203P CH2
To a 50 mL round bottomed flask was added 0.6 9 (0.2256 moles) of 1-(4-
20 nitrobenzyl)ethylenediamine, concentrated HCI (1.24 9, 0.0118 mole) and then phosphorous
acid (0 0925 9, .0113 mole). The solution was brought to reflux and then treated with 0.956 9
(0.0118 mole) of 30% formaldehyde dropwise over a ninety minute period. The reaction was
then kept at reflux overnight, cooled to room temperature and then added dropwise to 100 mL
of cold methanol. The resulting precipitate was filtered and dried to yield 1-(4-nitrobenzyl)-
25 ethylenediaminetetramethylenephosphonic acid, which was dissolved in 60 mL of water in ahydrogenation flask. A 50 mg sample of 5% Pd on carbon was added and the flask connected
to a Parr hydrogenation apparatus and shaken until hydrogen uptake ceased. The solution was
then filtered to remove the catalyst and the resulting clear filtrate was frozen and freeze-dried
to yield 1.28 gm (86%) of 1-(4-aminobenzyl)-ethylenediaminetetramethylenephosphonic acid.
30 This compound gave the expected decoupled 3IP-NMR spectrum.
This compound may be used as a ligand AP in Formula I where the L is through theNH} group.
Example N
Preparation of N~-(p-aminophenyl)-norspermidine
[Bis(cyanoethyl)](p-aminophenyl)amine was prepared as described in US Patenl
2,809,985. A 9.0 9 (42 mmol) sample of the bisnitrile was dissolved in 90 mL of ethanol and 10
mL of 50% NaOH and treated with 6 9 of Raney nickel (w-2 grade) and placed under an
atmospnere of hydrogen with shaking. Shaking was continued until the hydrogen uptake
-32-
'`'O 94/0014~ 2 I 3 9 3 2 ~ P~r/US93/06254
ceased at which time the catalyst was removed by filtration The filtrate was reduced in volume
under vacuum to a viscous oil, dissolved in 40 mL of 50% NaOH, and extracted with three 75 mL
portions of chloroform The combined chloroform layers were dried over sodium sulfate,
decanted and rotoevaporated under vacuum to yield 7.2 gm (77% yield) of the title compound.
5 The product was characterized by proton NMR.
This compound may be used as a ligand AP in Formula I where the L is through theNH2 group.
Example O
Preparation of N"(4-aminophenyl)-dipropylenetriamine-N',N',N"',N"'-
0 tetramethylenephosphonicAcid (APIPMP)
The structure of the compound APIPMP is shown by the formula
NH2
H203P-CH2 ~3 CH2-P03H2
~ -(CH2)3 N (CH2)3-N ~
H203P-CH2 CH2-P03~2
N4-(p-aminophenyl)norspermidine, prepared in Example N (3.65 9,16.4 ~lmoles)
was added to 25 mL of water and treated with a solution of 6.2 9 (75.6 mMoles) phosphorous
acid in 13 mL (163 mMoles) of concentrated HCL. The resulting dark blue solution was heated
to 100C and treated dropvvise with 5.9 9 (73 mMoles) of 37 weight percent formaldenyde i n
25 water over a 2.75 hour period. The solution was kept at reflux for an additional 15 hours, then
cooled to room temperature and dripped i nto ethyl alcohol. The resulti ng brown precipitate
was vacuum filtered and dried under vacuum to give 3.84 9 (40% yield) of the title compound
(APIPMP) as a brown solid. The product thus obtained gave a retention time of 5.50 minutes
(minor peak) and 10.50 (major peak) when analyzed on the anion exchange HPLC system
30 described in Example L.
This compound may be used as a ligand AP in Formula I where the L is through theNH2 group.
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WO 94/0014~ 3 PCI /US93/06254
ExamDle P
Preparation of N"-(4-aminophenethyl)-diethylenetriamine-N',N',N"',N"'-tetra-
methylenephosphonic acid, tetrasodium salt (APDTMP)
The structure of the compound APDTMP is shown by the formu la
'ld ~
CH2
H203P-CH
2 ~H2 CH2-P03H2
N-(CH2)2 N (cH2)2-N\
15H203P-CH2 CH2-P03H2
Diethylenetriamine (20 9,0.045 mole) was dissolved in 200 mL of toluene and
treated over five minutes with a solution of p-nitrophenethyl bromide (10 9, 0.89 mole) in 150
mL of toluene. After three hours of stirring the supernatent was decanted from the gummy
sol id and extracted three ti mes with 100 m L portions of water. The combined water layers
were reduced under vacuum to a low volume and back extracted with 100 mL chloroform. The
chloroform was then evaporated under vacuum to yield 9.32 9 (83%) of N"-(p-nitrophenethyl)-
diethylenetriamine.
A 5 9 sample of this amine was then dissolved in 3N HCI to yield a pH less than 2.
The resulting solution was ooured into excess methanol. The resulting precipitate was filtered
25 and dried under vacuum to yield 3 g of the corresponding hydrochloride salt. A 1.3 9 (.004
mole) portion of this salt was dissolved in 20 mL of water and treated with phosphorous acid
(1.5 9, 0.018 mole) and concentrated hydrochloric acid (2.0 9; 0.019 moles). The solutlon was
brought to refiux and 37% formaldehyde (1.5 9,0.019 mole) was added in a dropwise fashion.
After two add iti onal hou rs of refl ux the sol ution was cooled to room tem peratu re and
evaporated under vacuum to give a viscous oil. This oil was added dropwise to 150 mL of
methanol wlth vigorous stirri ng. The resulting white preci pitate was fi Itered and dried under
vacuum to give 1.65 9 (66% yieid) of N"-(4-nitrophenethyl)diethylenetriamine-N',N',N"',N"'-
tetra-methylenephosphonic acid . in the HPLC method descri bed for Example L this product
exhibited a single peak with retention time of 16.9 m m utes.
A 700 mg (1.1 mMoie) sample of this compound was dissolved in 44 mL (4.4
mMole) of 0.1 N NaOH. To this solution was added 100 mg of 10~0 Pd on carbon suspenbed in
56 mL water and the whole reaction mixture was placed under hydrogen with vigorous
2 1 ~ 3
~vo 94/0014~ PCr/US93/06254
shaking. After hydrogen uptake ceased, the catalyst was filtered and the filtrate evaporated
under vacuum to give 0.80 9 (100% yield) of N"-(4-aminophenethyl)diethylenetri-amine-
N',N',N"', N"'-tetramethylenephosphonic acid tetrasocdium salt (APDTMP) as a hygroscopic
yellow solid. The title compound exhibited a single peak with a retention time of 5.95 minutes
5 on the HPLC system described for Example L.
This compound may be used as a ligand AP in Formula I where the L is through theNH2 group.
Example Q
Preparation of 4-lsothiocyanatophthalic Anhydride
10 (ACL-3, an acid cleavable linker)
o
SCN ~~~
To a slurry of 4-aminophthalic acid (2.7183 grams(g),15.00 mmol) and anhydrous
potassium carbonate (8.75 9, 63.3 mmol) in 70 mL of tetrahydrofuran (THF) was added
thiophosgene (2.30 mL, 30.18 mmol). The reaction mixture was stirred 10 minutes at ambient
20 temperature, then heated to reflux for one hour. After cooling to room temperature, the
reaction product solution was filtered through celite and then concentrated to dryness under a
stream of dry nitrogen and under an efficient fume hood to avoid exposure to thiophosgene.
The crude 4-isothiocyanatophthal ic acid was thus recovered; 1 H NM R (300 M Hz, acetone-d6) d
10.22(brs,2H),7.81 (brs, lH)7.61 (s,lH),7.48(brs, lH); 13CNMR(75MHz,acetone-d6)
25 d 167.8,167.7,138.3, 135.8,134.4,131.7, 131.7,128.6,126.5.
The 4-isothiocyanatophthalic acid thus obtained was immediately heated to
reflux in a mixture of trifluoroacetic anhydride and methylene chloride for 2 hours under a
nitrogen atmosphere. After cooling to room temperature, the reaction mixture wasconcentrated under reduced pressure. Recrystallization of the resultant solid from 30 mL of
30 carbon tetrachloride afforded 4-isothiocyanatophthalic anhydride as brownish-purple crystals
in a yield of 2.3778 9, (77 percent of theoretical). The title compound melted at 106-108C.; 1 H
NMR (300 MHz, acetone-d6) d 8.15 (d, J = 8 1 Hz,1 H), 8.06 (d, J = 1.7 Hz.1 H),7.97 (dd, J = 8.1,1.7
Hz, lH); 13CNMR(75MHz,acetone-d6)d 163.3,162.6, 139.8,134.8,134.6,130.6,128.2, 123.5,
111.6; IR(CHCI3)2010(br),1840,1740cm-1; MSm/e205,161,133(base),74.
WO 94/0014~ 3~ PCI /~S93/06254
Fl NAL PRODU CTS
Exampie 1 and Comparative A
Conjugation of PDGF with SCN-BDTMP
A 400 IlL portion of 34.7mM SCNBDTMP solution (1.389 x 10 5 moles) (prepared in
5 Example G) was added to aliquot 1 from Example E. This steoichometry gives a 794: 1 molar
ratio of isothiocyanate group per amino group on the protein (Sample 1).
A 400 ~L portion of the 0.3M sodium bicarbonate buffer was added to aliquot 2
from Example E as a control (Comparative A).
Both samples 1 and 2 were mixed well. The samples, still on the membrane, were
10 allowedtosetatroomtemperatureforl4.5hours. Thesampleswerethencentrifugedatfull
speed to concentrate the samples for one hour. Following centrifugation each sample read
10.0 ~lCi on the membrane. To each membrane was added 1.0 mL of 0.1 M sodium phosphate
buffer. After swirling to mix, the samples were again centrifuged at full speed for 40 min. The
fi Itrate cups were removed and replaced with new tared fi Itrate cups. Each sample read 9.9 ~lCi
on the membrane.
To each sample was added 740 }lL portion of HSA solution to bring the volume on
the membrane to 80011L. Each solution was mixed and transferred into new tared filtrate cups.
Each membrane unit was washed with 500 ~lL of 1 % HSA. The CentriconT~ units were then
capped, inverted, and centrifuged for about 10 min. at full speed to remove the membrane
20 retentate into the cap. The wash solutions were pipetted into the corresponding filtrate cup.
The Centricon'~ caps were also washed with 200 luL portion of HSA solution which was then
transferredtothefiltratecups. Theserespectivefinaisolutionscontainedabout18.5~g(1)and
19.0~1g(ComparativeA)in 1.42mLofHSAandO.15Msodiumchloride.
Example 2
25 Conjugation of one SCN-BDTMP per IGF
A 1011L (3.47 x 10 8 mole) portion of 3.47mM SCN-BDTMP solution (prepared in
Example G) was added to Centricon "^ no.1 of Example H (containi ng approximately 41.2 1l9 of
IGF). An additional 390 ~lL of 0.1 M sodium bicarbonate buffer at pH = 9.35 was added. The
sol ution residing on the Centricon '~ membrane was then mixed wel I and allowed to set for 14
30 hours at room temperature. The unit was then reverse spun to remove the membrane's
contents. A 158 ~L aliquot of 0.1 M sodium bicarbonate was added to bring the final volume to
about 600 !lL.
Example 3
Conjugation of two SCN-BDTMP per IGF
A 20 ~lL (6.94 x 10 5 moie) portion of 3.47 mM SCN-BDTMP solution (prepared in
Example G) was added to Centricon'~ no. 2 of Example H (containing approximately 41.7 1l9 of
IGF). An additional 390 ~L of 0.1 M sodi um bicarbonate buffer at pH = 9.35 was added . The
solution residing on the Centricon'~ membranewasthen mixed well and allowed tosetfor 14
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213~3
"'O 94/0014~ PCI/US93/062~4
hours at room temperature. The unit was then reverse spun to remove the membrane's
contents. A8611LaliquotofO.lMsodiumbicarbonatewasaddedtobringthefinalvolumeto
about 60011L.
Example 4
5 Conjugation of four SCN-BDTMP per IGF
A 400 IlL (1.39 x 10-5 mole) portion of 34.7 mM SCN-BDTMP solution (prepared in
Example G) was added to Centricon'~ no. 3 of Example H (containing approximately 45.7 ~9 of
IGF). The solution residing on the Centricon"' membrane was then mixed well and allowed to
set for 14 hours at room temperature. The unit was then reverse spun to remove the
10 membrane'scontents. A20811LaliquotofO.lMsodiumbicarbonatewasaddedtobringthe
final volume to about 600 IlL.
Comparative B
Control for Examples 2, 3 and 4
A 40Q ~L portion of 0.1 M sodium bicarbonate buffer at pH = 9.35 was added to
Centricon'~ no. 4 of Example H (containing approximately 47.7 1l9 of IGF). An additional 390 IlL
of 0.1 M sodium bicarbonate buffer at pH = 9.35 was added. The solution residing on the
Centricon'~membranewasthenmixedwellandallowedtosetforl4hoursatroom
temperature. The unit was then reverse spun to remove the membrane's contents. A 23 ~L
aliquot of 0.1 M sodium bicarbonate was added to bring the final volume to about 600 ~L.
20 EXample 5
Conjugation of four SCN-BDTMP per IGF
A 400 ~lL (1.39 x 105 mole) portion of 34.7mM SCN-BDTMP solution (prepared in
Example G) was added to Centricon'~ no. 1 of Example I (containing approximately 130 lug of
IGF). The solution residing on the Centricon'~ membrane was then mixed well and allowed to
25 set for 13 hours at room temperature. The unit was then centrifuged to concentrate the
protein of the solution. The residual on the membrane was then washed with 0.5 mL of 0.1 M
sodium phosphate buffer (pH = 7.4) by adding buffer and then centrifuging for one hour. The
residual on the Centricon'~ membrane was then removed by adding 572 IlL of a 1 percent HSA
solution, swirling and pipetting. The membrane was washed with an additional 0.5 mL of 1
30 percent HSAto completely remove the membrane's contents. The final volume of combined
recovered solution was about 1.S mL containing an estimated 9.5 ~9/l OO ~L of modified IGF in
about 1 percent HSA.
Comparative C
Control for Example 5
A 400 ~L Portion of 0.3M sodium bicarbonate buffer at pH = 9.48 was added to
- Centricon'~ no. 2 of Example I (containing approximately 127 ~9 ofIGF). The solution residing
on the Centricon'~ membrane was then mixed well and allowed to set for 13 hours at room
temperature. The unit was then spun to concentrate the protein of the solution. The residual
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7,~g~
WO 94/0014~ PCl'/I~S93/06254
on the membrane was then washed with 0.5 mL of 0.1 M sodium phosphate buffer (pH = 7.4) by
adding buffer and then spi nning on a centrifuge for one hour The residual on the Centricon '~
membranewasthenremovedbyadding855,uLofal%HSAsolution,swirrlingandpipetting.
The membrane was washed with an additional 0.5 mL of 1 % HSA to completely remve the
5 membrane's contents The final volume of combined recovered solution was about 1.4 mL
containing an estimated 9.5 ~9/100 ~L in about 1 % HSA.
Example 6
IGF modified with BDTMP using ACL-3
A175uLportionofACL-3(12.1 mgACL-3in200uLoftrifluoroethanol,prepared
in Example Q) was added to a Centricon'~ containing approximately 100 1l9 of IGF in about 5ûO
IlL of 0.3M bicarbonate buffer (pH = 9.5). The resulting pH was 8.85. After 15 minutes at room
temperature,theCentricon'~ unitwasthencentrifugeduntilthevolumehaddroppedto
about 243 ~uL (243 mg weight) which took about 2 hours. To this concentrated solution was
added 300 IlL of ABDTMP solution (7.1 mg,10.5 ~uMoles in 300 IlL of bicarbonate buffer at
15 pH = 9.S). The resulting pH was 9.27. This solution was allowed to stand at room temperature
for 46 hours. The pH was found to be 9.78 and the volume was about 0.485 ~uL. This was put on
a centrifuge (Clay-Adams) for 6 hours and the volume was reduced to 117 IlL. A 2 mL aliquot of
0.3M sodium bicarbonate buffer was added and the solution again centrifuged. After 10 hours
of centrifuging the volume was about 49 IlL. An additional SOO }lL of 0.3M sodium bicarbonate
20 was added and the Centricon'~ unit centrifuged for 4 hours to give a final volume of 52 ~L. A
547~LportionofO.lMsodiumphosphatebuffer(pH=7.02)wasaddedandmixedwell. The
total weight in the Centricon'~ unitwas 599 mg or about 60011L in volume. The estimated
concentrationinthissampleis1001~91GFper600,uLofsolution(16.7~g/10011L).
Comparative D
25 Control for Example 6
A 175 ~L of trifluoroethanol was added to a Centricon'~ containing
approxi mately 100119 of IGF i n about SOO ,uL of 0.3M bicarbonate buffer (pH = 9.5). The
resultingpHwas9.50. Afterl5minutesatroomtemperature,theCentricon'~unitwasthen
centrifuged until the volume had dropped to about 230 !lL (230 mg weight) which took about
30 2 hours. To this concentrated solution was added 30011L of 0.3M sodium bicarbonate buffer.
The resulting pH was 9.46. This was allowed to set at room temperature for 46 hours. The pH
was found to be 9.79 and the volume was about 397 ~L. This was put on a centrifuge (Clay-
Adams) for 6 hours and the volume was reduced to 27 ~L. A 2 mL aiiquot of 0.3M sodium
bicarbonate buffer was added and the solution again centrifuged . After 10 hours of
35 centrifuging the volume was about 21 IlL. An additional 500 IlL of 0.3M sodium bicarbonate
was added and the Centricon'~ unit centrifuged for 4 hours to give a final volume of 27 ~L. A
573 ~L portion of 0.1 M sodium phosphate buffer (pH = 7.02) was addea and mixed wel,. The
-38-
~vo 94/0014~ 21 3 9 ~ ~ 3 PCI`/US93/06254
total weight in the Centricon'~ unit was 603 mg or about 600 IlL in volume. The estimated IGF
concentration in this sample is 100119 IGF per 600 ~uL of solution (16.7 ~g/10011L).
Exam~le 7
IGF modified with BDTMP using ACL-3
A210luLaliquotoflGFsolution(lOOIlgoflGFcontainingtraceramountsofl2sl
labeled IGF in buffer solution) was put on a membrane of a Centricon'~ membrane filtration
devices. A 500 ~1L aliquot of 0.3M sodium bicarbonate was added to both CentriconT~ unit and
it was then centrifuged to less than 50 mg remaining on the membrane. The contents on the
membrane (washed IGF) was taken up in SOO ~uL more of 0.3M sodium bicarbonate and was
10 treated with the 19.4 mg of ACL-3 dissolved i n 300 IlL of warm trifl uoroethanol . The sample
was allowed to stand for 15 minutes then centrifuged for 2 hours. At the end of this time a
30011L volume of ABHDP (35711Moles) was added to the Centricon '~ unit containing the
modified IGF and the solution then allowed to stand for about 16 hours. The pH remained
about 10duringthistime. TheCentricon'~ wasthencentrifugedforabout4hourstoreduce
the volume. The membrane retained material (modified IGF) was then diluted with 500 ~L of
sodium bicarbonate buffer and reconcentrated by centrifuging. The residual membrane
retainedvolumewa5221 mg. A1.279mLvolumeofO.lMsodiumphosphate(pH=7.02)was
added to bring the final volume up to 1.5 total. The Centricon '~ was then reverse spun to
remove the membrane retained material. This material was used in biodistribution studies in
20 rats shown in Example V.
Comparative E
Control for Example 7
The same procedure was followed as in Example 7 except that the addition of
warm trifluoroethanol did not contain the ACL-3 linking compound.
25 ExamPle 8
PDGF modified with BDTMP using ACL-3
A 100 ~L al iquot of PDG F sol ution was put on the membrane of a Centri con '~
membrane fi Itration device. The PDGF sol ution contained 10 IlCi of 1251 labeled PDG F (22
~lCj/~g) per 200 luL of 0.1 M sodium phosphate, 0.01 M sodium citrate, 0.5M NaCI and 1 % Bovine
30 Serum Albumin at pH = 4 6. A 500 ~L aliquot of 0.3M sodium bicarbonate was added to the
Centricon'~ unit and it was then centrifuged until 50 mg remained on the membrane. The
contents on the membrane (washed PDGF) were taken up in 500 ~L more of 0.3M sodium
bicarbonate. The membrane retained material was then treated with the 36.7 mg of ACL-3
dissolved in 300 IlL of warm trifluoroethanol, allowed to stand for 15 minutes, and then
35 centrifuged for 2 hours. At the end of this time a 300 IlL volume of ABHDP (357 IlMoles) was
added to the Centricon'~ unit containing the modified PDGF and the solution then allowed to
stand for about 16 hours. The pH remained about 10 during this time. The Centricon ~ was
then centrifuged for about 4 hours to reduce the volume. The membrane retained material
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WO 94/0014~ 3 PCr/US93/062~4
(modified PDGF) wasthen diluted with 500 ~L of sodium bicarbonate buffer and
reconcentratedbycentrifuging. Theresidualmembraneretainedvolumewas329mg. A1.171
mL volume of 0.1 M sodium phosphate (pH = 7.02) was added to the membrane of theCentricon'~tobringthefinalvolumeuptol.5mLtotal. TheCentricon"'wasthenreversespun5 to remove the membrane retained material . This material was used i n biodistribution studies
in rats as shown in Example Vl.
Comparative E
Control for Example 8
The same procedure was followed as in Example 8 except that the addition of
10 warm trifluoroethanol did not contain the ACL-3 linking compound.
BIOLOGICAL EXAMPLES: BIODISTRIBUTION
ExamDle I
l-PDG F-(SCN-BDTMP)
Spraque-Dawley rats weighing in the 175-230 g weight range were acclimated for
5 days prior to injection. The rats were injected via the tail vein with 50 ~L of both samples
from Example 1 (Samples 1 and Comparative A) (about 200,000 cpm). After 2, 6 and 18 hours,
the rats were sacrificed by cervical dislocation, tissues taken, weighed and the amount of
radioactivity in each tissue determined by counting for 5 min. in a scintillation counter
equipped with a Nal crystal coupled to a multichannel analyzer. The counts in each tissue were
20 compared to the counts in 50 llL standards in order to determine the percentage of injected
dose in each tissue. The counts in the tail were subtracted from the standards to give the
percent injected dose adjusted for the amount found in the tail . Background counts were
obtained and subtracted from the tissue counts. The percent dose in bone was estimated by
multiplying the percent dose in the femur by 25. The muscle and blood numbers were
25 obtai ned assum i ng that muscle is 43% and blood is 6.5% of the rat body weight. These
adjustments of the percent dose for the various body parts are accepted values for the rat
model. ~W.F. Goeckeler et al., J of Nucl. Med. 28(4),49S-504 (1987)]. The results are given in
Table I where each data point represents the average of five rats unless noted othervvise.
-40 -
`~094/0014~ 9 ~ 2 3 PCT/US93/06254
Table I
~ Injected Dose
Adjusted for Amount of Dose Found in Tail
2HR. 6HR. 18HR.
ORGAN
Ex.l* A-** Ex.1* A- Ex.l** A-
BONE 22.8 2.8 26.8 2.020.9 0.7
LIVER 14.7 12.2 8.1 2.1 2.0 0.5
KIDNEY 16.0 4.3 5.1 1.2 1.6 0.4
SPLEEN 0.7 0.2 0.6 0.1 0.5 0.0
MUSCLE 10.2 5.5 6.6 6.1 8.6 2.3
BLOOD 19.6 2.6 11.7 4.0 4.0 1.0
~average of 3 rats
**average of 4 rats
is Comparative A
It is readily apparent that conjugation of SCN-BDTMP with PDGF results in
targetingofthePDGFtobone. PDGFconjugatedtoSCN-BDTMPwas8timesmoreeffectivein
going to bone than native, unmodified PDGF. In addition, the residence time in the bone was
increased by about 30 fold.
ExamDle 11
20 l2sl-lGF-(SCN-BDTMP)
Spraque-Dawley rats weighing in the 175-230 9 weight range were acclimated for
5 days prior to injection. The rats were injected via the tail vein with 50 IlL of one of the four
samples from Examples 2, 3, 4 or Comparative B. After 30 mins. the rats were sacrificed by
cervical dislocation, tissues taken, weighed and the amount of radioactivity in each tissue
25 determined by counting for 5 min. in a scintillation counter equipped with a Nal crystal coupled
to a multichannel analyzer. The counts in each tissue were compared to the counts in 50 IlL
standards in order to determine the percentage of injected dose in each tissue. The counts in
the tail were subtracted from the standards to give the percent injected dose adjusted for the
amount found i n the tail . Background counts were obtai ned and subtracted from the tissue
30 counts. The percent dose in bone was estimated by multiplying the percent dose in the femur
by 25. The muscle and blood numbers were obtained assuming that muscle is 43/0 and blood is
6.5%oftheratbodyweight. TheresultsaregiveninTablellwhereeachdatapointrepresents
the average of five rats.
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WO94/0014~ PCT/US93/06254
Table II
% Injected ~ose
Adj-sted for Anount of Do e Found in Tail
ORGAN Ex.2 Ex 3 Ex 4 B*
BONE 9.161 9.688 13.323 6.956
LIVER 5.367 5.318 4.847 3.661
KIDNEY 8.410 11.447 12.756 7.070
SPLEEN 0.380 0.366 0.295 0.350
MUSCLE 19.614 23.440 18.379 26.078
lo BLOOD 23.909 25.006 19.521 23.169
Comparative B
The dergee of modification of the IGF with the number of bone seeking groups
added via AP of Formula I was based upon stoichiometry of reactants. The results are
summarized in Table 111 where each data point represents the average of five rats.
Table III
Degree of E I % Injected % Increase in
Modification xamp e Dose Bone uptake
l group 2 8.5 26
2 groups 3 9.2 36
4groups 4 11.7 74
none B* 6.8 0
~ Comparative
Clearly the results demonstrate that targeted delivery of IGF-I to bone can be
25 achievedbytheconjugationofSCN-BDTMPtolGF-I. AsshowninTablelll,thedegreeof
targeting is apparently dire~tly related to the degree of modification.
Example 111
'2sl-lGF-(SCN-BDTMP)
Spraque-Dawley rats weighing in the 175-230 9 weight range were acciimated for
30 5dayspriortoinjection. Theratswereinjectedviathetailveinwith50~Lofthesamplesfrom
Examples 5 or C. After 2, 6 and 18 hours the rats were sacrificed by cervical dislocation, tissues
taken, weighed and the amount of radioactivity in each tissue determined by counting for 5
min. in a scintillation counter equipped with a Nal crystal coupled to a multichannel analyzer.
The counts in each tissue were compared to the counts in 50 IlL standards in order to determine
35 the percentage of injected dose in each tissue. The counts in the taii were subtracted from the
standardstogivethepercentinjecteddoseadjustedfortheamountfoundinthetail.
Backgrounacountswereobtalnedandsubtractedfromthetissuecounts. Thepercentdosein
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~vo 94/00145 2 1 3 9 3 2 3 PCT/US93/06254
bone was estimated by multiplying the percent dose in the femur by 25. The muscle and blood
numbers were obtained assuming that muscle is 43% and blood is 6.5% of the rat body weight
The results are given in Table IV where each data point represents the average of four rats.
Table IV
~ Injected Dose
5 Adjusted for Amount of Dose Found in Tail
2 HR. 6 HR. 18 HR.
ORGAN
Ex.5 C* Ex.5 C* Ex.5 C*
BONE 16.6 11.1 14.6 9.9 13.4 6.3
û LIVER 2.8 5.4 1.4 1.8 0.7 0.9
KIDNEY 4.2 8.4 2.3 2.3 1.1
SPLEEN 0.2 0.4 0.2 0.2 0 0.1
MUSCLE 16.9 24.1 11.3 16.5 3.9 3.5
BLOOD 14.3 33.7 7.1 8.7 1.4 1.9
* Comparative '
Exam~le IV
Biological Activity of IGF-I-(SCN-BDTMP)
Various samples from the prior examples were run on an IGF-I mitogenic activity
20 assayto determine their potency. The protocoi is based on a competence-progression model in
which the assay cell line responds to varyi ng levels of IG F-l i n a dose dependent manner. Cel Is
from a mouse fibroblast line are seeded in 96 well assay plates and grown to confluence to
induce quiescence. Prior to addition of samples and standards the cells are preincubated with
PDGF-BB, which enablesthem to respond to IGF-I when it is added in the presence of EGF.
25 After an overnight incubation period the cells are exposed to 1.0 luCi of 3H-thymidine which is
incorporated at varying levels depending on the amount of IGF-I added to the assay well.
Following extensive washing, the cells are Iysed and the samples are quantitated in a
scintillation counter equipped with a Nal crystal coupled to a multichannel analyzer. The
resulting data are plotted to generate a dose response curve from which unit activity
30 determinations can be made.
A unit is defined as that concentration of IGF-I which induces 50% of the
maximum cellular response in that assay (ED;o value). Maximum response is defined as cellular
response to a 5/0 FCS standard. Maximum response due to exogenous IGF-I is usually 3-5 times
basel i ne.
- Priortodilutionfortheassay,theconcentrationoflGF-lwasdeterminedviaan
12sl-lGF-I tracer which had been spiked into the stock sample prior to manipulation. Counts
were made on a 25 IlL aliquot of the stock sample and 25 ~L of the two samples. The ratio of
counts per min (CPM)/~lg of IGF-I in the stock sample was used to determine the concentration
-43-
WO 94/00145 . PCr/US93/062~4
of iGF-I in the two samples for the bioassy. The average of the stock sample aliquots was 5,135
CPM/}lg. The values for each sample was determined by CPM per IlL and dividing the number
bythestocksample aliquotof 5,135CPM/}Ig. These measurmentsand calculationsresulted in
the foliowing values: Example 5 samplewas 107.2 ~g/mL; and Example Cwas 110.811g/mL. The
5 results are shown in table V.
Table V
Example ESDamPlveg Control %scronnptlreoEDD5o % Control
2 3.6 2.2 60 111
3 3.0 2.4 79 1 46
4 4.8 1 .7 3 5 65
B* 4.3 2.3 54 NA
5.6 2.2 39 48
C* * 3.8 3.1 82 NA
* Comparative B;
** ComparativeC
Exam~le V
l2sl-lGF-ACL-3-BDTMP)
Doses (150 ~L) of the material from Example 7 and Comparative Example E were
drawn up into 500 IlL syringes equipped with 28 gauge needles and injected into the tail veins
of 5 rats. After 6 hours the rats were sacrificed and dissected to obtain the organ distribution
of the radiolabelled material. Because of the low amount of radioactivity involved, the samples
were counted for 10 minutes. The final number of counts (less background) found in the
25 femurs of rats injected with the modified IGF material of Example 7 were 3.8 times the number
of counts (less background) found in the femurs of rats injected with the control material of
Comparative E as seen in the following Table Vl.
-44-
~094/0014~ 2l3a~ PCT/US93/06254
Table VI
Rat Example 7 Comparative E
1 673 376
2 1714 383
3 1250 281
4 1409 264
1408 375
Average 1291 336
0 Ratio 3.8
Example Vl
l2sl-PDGF-ACL-3-BDTMP)
Doses (100uL) of this material were drawn up into 500 uL syringes equipped with
28 gauge needies and i njected i nto the tail veins of 5 rats for each of Centricon l (Experi mental)
and Centricon 2 (Control). After 6 hours the rats were Aced and dissected to obtai n the organ
distribution of the radiolabelled material. Because of the low amount of radioactivity involved,
the samples were counted for l O minutes. The final number of counts (less background) found
in the femurs of rats injected with Centricon l (experimental) material were 3.8 times the
20 number of counts (less background) found in the femurs of rats injected with Centricon 2
(control) material as seen in the following Table Vll.
Table VII
Rat Example 8Comparative F
153S 683
2 750 807
3 1708 859
4 1559 525
S 1543 639
30Average 1419 703
Ratio 2
The data thus demonstrates that modification of GF's using techniques described
in the present invention results in targeted delivery of biologically active GF's to bone.
Although the invention has been described with reference to its preferred
embodiments, those of ordinary skil I i n the art may, upon reading and understanding this
disclosure, appreciate changes and modifications which may be made which do not depart
from the scope and spirit of the invention as described above or claimed hereafter.
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