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Dec 6, 2012 (4 years and 7 months ago)

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Liprotamase
, a Non Porcine Enzyme Therapy, Restores Growth,

and Normalizes Lipid Absorption in Pigs With Exocrine Pancreatic Insufficiency (EPI)

RESULTS

Grujic D,
1

Piedra
J
,
2,4

Szymanczyk

S,
2,4

Szwiec

K,
2

Bala

T,
2

Uschakova

G,
2,3

Osdachenko

I,
2,3

Kovalenko T,
2

Pierzynowski S
2,3,4

1
Alnara Pharmaceuticals, Cambridge, MA, USA;
2
SGPlus, Malmo, Sweden;
3
Dept Biology, Lund, Sweden;
4
Dept Medical Biology, IMW, Lublin, Poland

The American College of Gastroenterology (ACG) Annual Scientific Meeting and Postgraduate Course; San Antonio, TX, USA; Octob
er
15

20, 2010

Sponsored
by
Alnara

Pharmaceutical Inc.,
a wholly
-
owned
subsidiary of Eli
Lilly and
Company

INTRODUCTION


Liprotamase

is a novel biotechnology
-
derived, non
-
porcine enzyme
replacement therapy that contains three (3) highly purified and stable
enzymes: crystalline cross
-
linked recombinant lipase (32,500 USP
units/capsule), crystalline protease (25,000 USP units/capsule), and
amorphous amylase (3,750 USP units/capsule). The particular enzymes
selected for inclusion in
liprotamase

were based on their stability in the

pH of the gastrointestinal tract without enteric coating and their broad
substrate specificity without the need for co
-
factors.
ref

1,2

Because of its
purity and stability, recently we were able to develop a novel liquid
formulation, rapid disintegrating tablets (
LipRDT
), meant for treatment of
the pediatric population and others who have difficulty swallowing
capsules.


Liprotamase

RDT was tested in a pig surgical model of exocrine
pancreatic insufficiency (EPI).
3,4
Total EPI in pigs dramatically decreases
the levels of digestive enzymes and bicarbonate in the small intestine,
causing bile salt precipitation and inhibition of micelle formation resulting
in poor lipid absorption,
steatorrhea

and arrested growth, similar to
symptoms seen in patients with pancreatic insufficiency (PI).

OBJECTIVE


To assess the efficacy of a new, RDT formulation as a PERT, we
conducted a cross over design study in young EPI pigs. The effectiveness
for fat digestion was demonstrated by change in coefficient of fat
absorption (%CFA). As a novel way of efficacy expression, we monitored
fat and protein absorption, expressed as postprandial changes in
lipaemic

index (LI), non
-
esterified

fatty acid (NEFA), triglyceride (TG) and somatic
growth.

METHODS


Efficacy of two daily doses of
liprotamase
, high (HRDT: 290,000 U Lipase,
24,000 U Amylase, 194,400 U Protease) and low (LRDT: 146,000 U Lipase,
12,000 U Amylase, 102,400 U Protease) were tested in eight (8) EPI pigs
that were fed a high fat diet (HFD, ~40g fat/meal). Prior to dosing, tablets
were dissolved into a small amount of water, (4 tablets/meal) and offered in
the middle of the morning and afternoon meals. Study included 1 control
and 2 treatment periods, each one week. During the fifth week all pigs
were placed on LRDT.

Study Design:


For the fecal balance study, stool was collected on days 5, 6, and 7 of the
respective control and treatment weeks from EPI and healthy pigs and the
%CFA was calculated using a standard gravimetric method.
5


Fat absorption was assessed by postprandial changes in LI, TG and NEFA
in blood samples collected before the morning meal and then at 30 min
and 1h intervals on the last day of the control and treatment weeks in both
EPI and healthy piglets.


Changes in growth (body mass increase kg/week) and overall health were
recorded after seven (7) days of treatment.


Fat digestion, expressed as %CFA for 72h collections is shown in Fig.1.
Treatment CFA was 84% and 79% with HRDT and LRDT (ext. LRDT)
respectively, vs. 93% in healthy pigs.

Figure

1. Mean CFA in EPI Pigs During Control,


LipRDT

Weeks, Wash Out, and Comparison


with Healthy Pigs (n=7
-
10, *p<0.05)


Based on comparison analysis with healthy pigs,
liprotamase

treatment
resulted in effective digestion of fat as shown by normalization of postprandial
lipid profiles demonstrated by LI (Fig. 2), TG (Fig. 3) and NEFA (Fig. 4).

Figure

2.
Lipaemic

Index From Blood Taken Before


and After the Meal on the Last Day


of Control and
LipRDT

Weeks (n=5
-
6 pigs)

Figure 3. TG Concentration in Blood Taken Before


and After the Meal on the Last Day

of Control and
LipRDT

Weeks (n=5
-
6 pigs)

Figure

4. NEFA Concentration in Blood Taken Before and
After the Meal on the Last Day of Control and
LipRDT

Weeks (n=5
-
6)


As

demonstrated,

postprandial

changes

in

LI,

NEFA

and

TG

very

much

resemble

lipid

absorption

profiles

of

healthy

pigs,

suggesting

effective

digestion

and

absorption

in

the

proximal

part

of

the

small

intestine
.

Figure

5.
Ceco
-
illeal

Loop Showing “Natural” Lipid
Absorption in
LipRDT
-
treated Pig


Comparative analyses between healthy pigs (of the same age and breed) fed
high fat feed vs. EPI
liprotamase

RDT treated pigs (Table 1).

Table 1. EPI Pigs Treated With
LipRDT

vs. Healthy Pigs

EPI P
IGS

N=6
-
8

H
EALTHY

P
IGS

N
=
5
-
10

L
IPROTAMASE

RDT + HFD

HFD

HRDT
(
DAILY

DOSE
)

LRDT
(
D
AILY

DOSE
)

D
AILY

D
OSE
:

L
IPASE
-
CLEC

PROTEASE

A
MYLASE


290,000 U

194,400 U

24,000 U


146,000 U

102,400 U

12,000 U

Treatment CFA

Mean
±

SD

Change

(
Basal

Treat
)


84.0
±

5%

60% CFA

(24%

84%)


79.0
±

8.0%

55% CFA

(24%

79%)


92.9
±

1.2%

Growth (Weight Increase)

6%

8%

14%

Stool Weight


From Basal

37%

30%

-

Lipaemic

Index (Mean
±

SD)

Maximum (
mmol
/L)

AUC


2.86
±

2.04

17.3
±

6.4


2.58
±

2.02

12.9
±

7.1


2.12
±

1.0

12.8.0
±

3.4

Triglyceride (TG) (Mean
±

SD)

Maximum (
mmol
/L)

AUC


0.71
±

0.46

4.1
±

2.0


0.75
±

0.43

4.5
±

1.6


0.69
±

0.2

3.8
±

0.7

NEFA (Mean
±

SD)

Maximum (
mmol
/L)

AUC


0.25
±

0.11

1.6
±

0.4


0.24
±

0.10

1.6
±

0.6


0.26
±

0.06

1.4
0
±

0
.1


1 week of treatment with
liprotamase

RDT resulted in significant growth
and phenotypic changes shown in Fig. 6 and Fig. 7.

Figure 6. Mean Body Mass Increase in EPI Pigs after
1 Week of Treatment (*p<0.05)

Figure 7. Sick EPI Pig vs. “Happy“ Healthy and Alert
Liprotamase

RDT Pig













CONCLUSIONS

This

in

vivo

activity

study

of

a

novel

formulation

of

liprotamase

in

a

EPI

pig

model

that

mimics

PI

disease

in

human

subjects

resulted

in
:

1.
Normalization

of

fat

absorption

based

on

postprandial

LI,

and

blood

concentrations

of

NEFA

and

TG

with

both

tested

doses

2
.

Reversal

of

growth

retardation

from

control

week,

with

a

mean

body

mass

increase

of

6
%

(
0
.
9
kg/week)

and

8
%
(
1
kg/week)

with

HRDT

and

LRDT

respectively


3
.

Significantly

improved

fat

digestion

to
:



HRDT
:

84
%

CFA
;

from

basal

of

24
%

to

treatment

of

84
%
,

(
Δ
60
%
)


LRDT
:

79
%

CFA
;

from

basal

of

24
%

to

treatment

of

79
%
,

(
Δ
55
%
)

4
.

Evidence

that

postprandial

LI,

TG

and

NEFA,

are

viable

endpoints

that

better

reflect

nutritional

status

and

health,

and

should

be

explored

in

human

trials

instead

of

%
CFA

which

has

limitations

in

the

clinical

setting

5
.

Successful

in

vivo

activity

of

a

novel

RDT

formulation

of

liprotamase

designed

for

the

treatment

of

EPI

in

infants,

children,

patients

with

a

G
-
tube,

and

others

who

have

difficulty

swallowing

capsules

References:

1.
Borowitz

D,
et al.
Study of a novel pancreatic enzyme replacement therapy in pancreatic insufficient
subjects with cystic fibrosis.
J
Pediatr
.
2006; 49:658
-
662.

2.
Borowitz

D,
et al.
An international open
-
label study of the long
-
term safety of
liprotamase

for
treatment of pancreatic insufficiency in cystic fibrosis, Poster at NACF meeting 2009.

3.
Donaldson, J
et al.

The effectiveness of enzymatic replacement therapy measured by turbidimetry and
the lipaemic index in exocrine pancreatic insufficient young growing pigs, fed a high

fat diet. (2009)
Advances in Medical Science, 54 (1),p 7
-
13.

4.
Rengman

S.
et al.

An elemental diet fed,
enteral

or parental, does not support growth in young pigs
with exocrine pancreatic insufficiency. (2009), Clinical Nutrition; 1
-
6.

5.
van de
Kamer
, J. Total Fatty Acids in Stool, Standard Methods of Clinical Chemistry, vol. 2, (1958).

0.0
0.1
0.2
0.3
0.4
0'
30'
1h
2h
3h
4h
6h
8h
NEFA (mmol/L)

CONT
d7 HRDT
d7 LRDT
Healthy pigs
14
15
16
17
18
19
20
21
CONT
HRDT
LRDT
Wash out
Weekly Body Weight Gain (kg)

*

*

6%

8%

Healthy Pigs= 14%

0.2
0.4
0.6
0.8
1.0
1.2
basal
30'
1h
2h
3h
4h
6h
8h
TG (mmol/L)

CONT
d7 HRDT
d7 LRDT
Healthy pigs
0.0
1.0
2.0
3.0
4.0
5.0
basal
30'
1h
2h
3h
4h
6h
8h
Lipaemic

Index

CONT EPI
7d HRDT
7d LRDT
Healthy pigs
Adaptation
7 days
Control
7 days
n=8
HRDT
7 days
n=4
End
LRDT
7 days
n=4
Wash out
7 days
n=8
HRDT
7 days
n=4
LRDT
7 days
n=4
LRDT
7 days
n=8
Adaptation

7 days

0
10
20
30
40
50
60
70
80
90
100
CONT
HRDT
LRDT
Wash Out
Ext.LRDT
Healthy
Pigs
CFA(%)


*

*

*