BONE-TARGETED ALKALINE PHOSPHATASE - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 416

While the Fc region was incorporated into ENB-0040
primarily to facilitate purification, Fc is known to consider-
ably increase the half-life of short lived proteins, as dis-
cussed at length elsewhere in this topic, thus allowing us to
use lower doses and less frequent injections. The pharma-
cokinetics (PK) and tissue distribution properties of ENB-
0040 were determined in studies of adult and newborn mice
comparing different administration routes [23]. Initially,
using a single IV injection of 5mg/kg ENB-0040 into adult
wild-type (WT) mice, the authors found the circulating half-
life to be 34 h, with prolonged retention of radioactive
iodine-labeled ENB-0040 in bone, with as much as
1 m g/g of bone (wet) weight. Skeletal levels of bone-targeted
test material are apparently highly stable, as no significant
decrease in radiolabeled ENB-0040 was observed during the
course of the experiment. Conversely, no sustained accumu-
lation of sALP-FcD 10 was observed in muscle, as radio-
labeled enzyme levels in this tissue decreased in parallel
with ENB-0040 enzymatic activity in blood. Figure 28.1C
shows a histochemical staining for ALP activity in untreated
and treated Akp2 / bone sections revealing the successful
homing of ENB-0040 to bone tissue. Because Akp2 / mice
die between days 12 and 16, and IV injection is very difficult
in such small animals, PK analysis of ENB-0040 was
planned using intraperitoneal (IP) and subcutaneous (SC)
administration in newborn WT mice. However, IP injection
proved unreliable due to the high intra-abdominal pressure
seen in young animals that led to unpredictable losses of
drug solution through the injection site. Since SC injections
proved reproducible, SC injection route was used to generate
PK data used to predict circulating levels of ENB-0040
achieved after repeated daily doses. Circulating ENB-0040
reached steady state serum concentrations oscillating
between C min and C max values of 26.4 and 36.6 m g/mL,
respectively, after 5-6 daily doses at 10mg/kg. Prediction
validity was tested using 5 daily SC injections of 10mg/kg
ENB-0040. Circulating ALP activity measured 24 h after the
last injection (C min ) was in good agreement with predicted
concentrations. In WT mice, serum TNAP levels measured
in the same conditions were found to be 0.58 m g/mL. Thus,
the authors calculated that the SC injection regimen would
achieve steady-state circulating ENB-0040 concentrations

early mortality, rickets, epileptic seizures, lack of an acel-
lular cementum layer in teeth, as well as elevated concen-
trations of inorganic pyrophosphate, calcium, and
pyridoxal-5-phosphate (PLP) in plasma. As described ear-
lier in this chapter, disease severity, which is inversely
proportional to age at onset, varies from a generally fatal
perinatal form to an adult form characterized principally by
nontraumatic fractures and bone pain. ENB-0040 is cur-
rently under investigation for treatment of life-threatening
(perinatal and infantile HPP) childhood and adult HPP. As
reviewed by Drake and Koshla [32] in an Editorial that
accompanied the first report of successful treatment of HPP
mice [23], this bone-targeted form of TNAP shows great
promise for treatment of human HPP.
As with any protein, the formation of antibodies is
a potential concern. Anti-ENB-0040 antibodies were
detected in some TNAP knockout mice before and after
ENB-0040 treatment, but antibodies were not associated
with clinical symptoms or lessening of enzyme effect
over time.
Because ENB-0040 contains the human IgG1 Fc region, a
possible interaction with various immune cells was consid-
ered. The likelihood of such an interaction is low, since
monkeys have a homologous receptor for the Fc region of
human IgG1, and no clinical signs of adverse immunological
reactions were seen even at very high doses of ENB-0040 in
primate toxicology studies.
One potential concern regarding ENB-0040 treatment
is that it could induce hypocalcemia or hypophospha-
temia as bone mineralization is stimulated. This poten-
tial outcome was examined in the mouse HPP model,
and no evidence of either biochemical abnormality was
detected, either acutely or with chronic ENB-0040
administration.
Another potential concern related to ENB-0040 treatment
in HPP is vitamin B 6 toxicity and a resulting peripheral
neuropathy [33]. The primary circulating form of Vitamin
B 6 is PLP, which must be dephosphorylated to pyridoxal
(PL) to enter cells. In animal studies, ENB-0040 treatment
has not resulted in excessively high PL levels and there has
been no evidence of peripheral toxicity, even at very high
ENB-0040 doses.
The possibility that ENB-0040 could induce ectopic
calcification has been investigated in animal safety stud-
ies, as we have shown that TNAP expression and a fibrillar
scaffold is sufficient to promote mineralization of any
extracellular matrix [34] and, in particular, that TNAP
upregulation in vascular smooth muscle cells causes
medial vascular calcification [35,36]. Histological evalua-
tion of organs and blood vessels in ENB-0040 treated
mice and rats failed to demonstrate any such pathology.
Monkey studies indicated microscopic evidence of
reversible, dose-dependent calcium deposition at injection
sites.

50 times higher than normal WT TNAP concentrations.
In clinical attempts to use ERT by injections of Paget's bone
disease plasma, purified liver ALP, or purified placental
ALP, only an eightfold elevation in serum ALP activity
had been achieved [16,17,19].

28.3 APPLICATIONS AND INDICATIONS

Preclinical trials using daily SC injections of newborn
Akp2 / mice with purified ENB-0040 blocked disease
manifestations in this model of infantile HPP, including