BONE-TARGETED ALKALINE PHOSPHATASE - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 417

28.4 PREC LINICA L AND CLINIC AL RESEA RCH A subsequent prec linical stud y defined the dose/re sponse

relations hip betwee n increas ing amou nts of cGM P (curren t
good manufac turing practi ce)-produced ENB-0 040 using
bolus SC inj ections and the therape utic response after 43
days, in anticipati on of clinical trials [28] . Endpoi nts were
survival, body weight, bone length of the tibiae and femora,
and bone miner alization defects, as assessed using radi o-
graphs of the feet, rib cage, and lower limbs . To date , these
parame ters have served as accurat e indicator s of correct ion
of the HPP phenoty pe in this animal mode l. Also, radi o-
graphic man ifestations of HPP are read ily observed in
affected infant s and are thus an important endpoi nt in
preclinic al proof-of -concept stud ies to extrapolat e from
mice to humans. In addition, we used m CT (Figu re 28 .3)
and histomorp home tric analysi s to evaluate improvement in
Akp2 / bone miner alization status for represe ntative age
groups undergoing treat ment. We document ed a clear rela-
tionship betwee n daily ENB-0 040 dose and the percent age
of mice with normal bony str uctures of the foot, rib cage, and
lower limbs. We focused on establ ishing an effective
dose in 80% of the mice (ED 80 ). In mice, that dose was

Akp2 / mice, which were created via homologous
recombination by insertion of the Neo cassette into exon 6
of the mouse gene encoding TNAP (Akp2) [12], show no
detectable TNAP mRNA or protein. Phenotypically,
Akp2 / knockout mice closely mimic infantile HPP [14].
Like HPP patients, Akp2 / mice exhibit global deficiency in
TNAP activity, endogenous accumulation of the ALP sub-
strates PP i and PLP, and postnatally acquire impaired miner-
alization of skeletal matrix leading to rickets or osteomalacia.
Akp2 / mice manifest stunted growth, and develop radio-
graphically and histologically apparent rickets together with
epileptic seizures and apnea, and die between postnatal days
10 and 12 [11,12,14].
Th e first disease efficacy study utilize d SC ENB-0 040
injections of newborn Akp2 / mice daily for 15 days at
1 mg/kg per dose [23]. m CT anal ysis of animal s treat ed at
this dosage showed no attenuation of skeletal disease in the
calvarium , and the proxim al tibia l growth p late (phys is)
showed excessive widening of the hypertr ophic zone, con-
sistent with early rick ets in both ENB-0 040- and vehicle-
injected Akp2 / animal s. Increas ing the daily dose to
2 mg/kg improved the general appea rance, body weight
and tail length of treated Akp2 / mice, whi ch also showed
a normal growth rate. Next, Akp2 / mice underwe nt 15
days of daily SC injectio ns at 8.2 mg/kg ENB -0040. Treated
animals had grea ter body weight than vehicle-treated mice
and had plasma PP i concen trations in the n ormal range . In
this short-t erm experimen t, ENB-0040 treat ment minimized
hypomine ralization in the feet and reduced the numb er of
mice exhibiting severely d ysmorphic rib cages. Similarly,
the hind limbs appea red heal thy in all treated animal s [23].
Long-ter m (52 day) survival and com plete prevention of
skeletal defects and epileps y were obta ined with daily SC
injections of 8.2 mg/kg ENB-0040, an outcome accompa -
nied by normal p lasma PL conce ntrations and unremar kable
calcium conce ntrations .
ENB-0040 treatment also pr even t e d h y p o m i n e r a l i z a -
tion of al veolar bone and dent in [23], and a cellular cemen-
tum, which is typical ly missi ng or reduced in Akp2 /
mice, f ormed normally [24]. M icrocomput ed tomogr aphy
r evea l e d a c o n s i s t e n t r e d u c t i o n o f m i n e r a l i z a t i o n i n b o t h
alve olar bone and root dentin (and root analog dentin in the
incisor) in t h e c ontrol (ve hicle alone inj ected) Akp2 /
mice (Figur e 28.2, left panels)—a feature not present in the
EN B-0040-treated Akp2 / mice, which showe d c omplete
mineralizat ion of a lveol ar b one a nd dent in. Imm uno-
hi stochemical staining f or osteopontin, revea l e d a b s e n c e
of acellular c em entum a long th e r oot s urface in untreated
Ak p 2 / mice, w hi le ENB-0040-treated Akp2 /
mice had an unrem ar kable acellular cementum l ayer com-
pa rable w ith that seen in WT mice (Figure 28.2, right
pa n e l s) [ 24 ] .

3 .2 mg/kg/ day for feet, 2.8 mg/kg/day for rib cage, and

2 .9 mg/kg/ day for lower limbs. These ED 80 doses are
consistent with our previous experience showing enhanced
skeletal integrity fol lowi ng short term, 15-day treat ment of
Akp2 / animals usin g 2 mg/kg/ day of ENB-0 040 [23].
Studies using ENB-0040 produced under cGMP condi-
tions confirm and extend our previous data regarding preven-
tion of HPP in Akp2 / mice by administration of the bone-
targeted form of TNAP from birth [23] and helped to set the
stage for clinical trials now underway for HPP patients. 2
The ED 80 along with enzyme concentrations measured in
animal efficacy studies and ENB-0040 PK data were used to
estimate the minimum effective concentrations for Akp2 /
mice. Initial dosing of ENB-0040 for clinical trials was then
based on: (1) dose-response data [28] in the Akp2 / mouse
model of infantile HPP, (2) efficacy observed in treated
Akp2 / mice that achieved serum ALP activity in the range
of 650-1000 U/L; (3) the no-observed-adverse-effect level
(NOAEL) in more sensitive species (rats and monkeys)
established in 1-month IV toxicology studies and 1-month
IV/SC bridging and tolerability studies in rats; (4) a safety
factor of 10 applied to the NOAEL; and (5) results from a
multicenter, open-label, dose-escalating study of the safety,
tolerability, and pharmacology of ENB-0040 in six adults
withHPPwho received one dose of 3mg/kg IVand then either
1 or 2mg/kg ENB-0040 SC once weekly for 3 weeks.
An investigational new drug (IND) application to use
ENB-0040 to treat HPP was filed in June of 2008. To date,
two clinical studies have been completed, three are ongoing,
and two additional studies are being initiated. 2 Preliminary
clinical data have been presented only as abstracts at

2 http:// www.clinicaltrials.gov