˘
BANSAL, GITTENS, AND ULUDAG
2798
efficiency (2.7 per protein). Our unpublished
results indicated that BSA with &6 aminoBPs
per protein was not targeted to bone [BSA delivery
to tibia and femur: 0.10 ꢃ 0.02% and 0.15 ꢃ 0.06%
is the first report on the feasibility of targeting
proteins as large as antibodies (&150 kDa), which
is expected to facilitate the evaluation of antibody-
based therapeutic agents for bone diseases. The
synthesized di(bisphosphonic acid) allows attach-
ment of multiple copies of BPs per protein site, in
addition to giving a minimal distance between the
protein attachment site and the BP moieties.
Further studies are underway to prepare dendri-
tic BPs in which a controlled number of BPs can
be conjugated onto a single protein site.
(
n ¼ 3), respectively; BSA-BP delivery to tibia and
femur: 0.02 ꢃ 0.01% and 0.02 ꢃ 0.01% (n ¼ 3),
respectively]. In this regard, the newly synthe-
sized di(bisphosphonic acid) seems beneficial over
the previously used conjugation approaches by
enabling successful protein targeting to bone with
less protein modification.
We are currently pursuing more in-depth
studies probing further correlation(s) between
conjugate properties (protein size, overall charge,
and the conjugation efficiency) and bone targeting.
Based on our previous structure-property studies
ACKNOWLEDGMENTS
This study was supported by operating grants
from Canadian Institutes of Health Research
(CIHR) and the Whitaker Foundation. Infrastruc-
ture support was provided by Canadian Founda-
tion for Innovation (CFI) and Alberta Heritage
Foundation for Medical Research (AHFMR). We
thank C. Kucharski for technical help with the
animal studies and Dr. V. Somayaji for NMR
studies.
1
2–16
on BP-protein conjugates,
the BP prepared
for this study (6) is expected to be beneficial in two
aspects: (i) because of its ability to load a higher
number of BPs per protein attachment site (2 vs.
1), and (ii) because of the introduction of a minimal
tether length to link the BPs to the protein. It will
be important to probe both of these aspects of the
new BP so as to elucidate the relative contributions
of each aspect. We also noted that the in vitro
binding for the conjugates, especially in high
phosphate and serum-containing media, was
exceedingly high compared with the unmodified
proteins (>10-fold difference at times). In rats,
however, the difference in bone targeting was not
as much: 1.5- to 3.7-fold differences at the 2-day
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CONCLUSIONS
In conclusion, a COOH-containing di(bisphospho-
nic acid) (6) was synthesized and conjugated to
model proteins. The conjugation efficiency was
controlled by the reagent concentrations in the
reaction medium. The conjugates exhibited an
in vitro HA affinity that was proportional to the
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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 11, NOVEMBER 2004