Beruflich Dokumente
Kultur Dokumente
ThioMatrix GmbH, Research Center Innsbruck, Mitterweg 24, 6020 Innsbruck, Austria, and 2Department of Pharmaceutical
Technology, Leopold-Franzens-University, Innrain 52, Josef Moller Haus, 6020 Innsbruck, Austria
Abstract
Teriparatide, a recombinant parathyroid hormone (1 34) is the first approved agent for the treatment of osteoporosis that
stimulates new bone formation. Currently, the drug is administered daily by s.c. injection. Because of the obvious advantages
of oral teriparatide administration, the development of such a delivery system would be of great benefit. Besides other barriers,
the enzymatic barrier caused by gastro-intestinal (GI) proteolytic enzymes is believed to be responsible for negligible
teriparatide oral bioavailability. It was therefore the aim of the study to evaluate the stability of teriparatide towards a variety of
GI proteases under physiological conditions. Results indicate that teriparatide is entirely degraded by trypsin, chymotrypsin
and pepsin within 5 min. In contrast, even after 3 h of incubation with elastase about 85% of undegraded teriparatide could
still be detected. Within an incubation period of 3 h in the presence of rat small intestinal mucosa, approximately half of the
teriparatide was degraded. Experiments with isolated aminopeptidase N demonstrated that this membrane bound peptidase is
primarily involved in the degradation process. Results gained from and recorded in this study provide a precise
characterisation of the enzymatic barrier for oral teriparatide administration and represents a prerequisite for the development
of oral teriparatide delivery systems.
Keywords: Teriparatide, parathyroid hormone (134), PTH, osteoporosis, oral peptide delivery
Introduction
Osteoporosis is a disease that decreases bone density
and consequently leads to bone fractures. The goal of
an effective osteoporosis treatment is an improvement
in bone density and strength. Estrogen hormone
replacement therapy (HRT), biphosphonates, calcitonin and selective estrogen receptor modulators
(SERMs) are drugs that mainly improve bone density
by inhibiting bone turnover or osteoclastic resorption
activity (Sato et al. 1999). In comparison to these
drugs, teriparatidemarketed under the name FORTEOTMis the first approved agent for the treatment
of osteoporosis that stimulates new bone formation
mediated by its anabolic properties. It stimulates the
bone formation activity of osteoblasts, replacing lost
bone in both osteopenic, ovariectomized rats and
osteoporotic humans (Dobnig and Turner 1997).
Teriparatide was approved by the FDA for the
treatment of osteoporosis in 2002. It has an identical
sequence to the 34 N-terminal amino acids (pharmacological active region) of the 84 amino acid human
Correspondence: A. Bernkop-Schnurch, Department of Pharmaceutical Technology, Leopold-Franzens-University, Innrain 52, Josef Moller
Haus, 6020 Innsbruck, Austria. Tel: 43 512 507 5383. Fax: 43 512 507 2933. E-mail: andreas.bernkop@uibk.ac.at
ISSN 1061-186X print/ISSN 1029-2330 online q 2006 Taylor & Francis
DOI: 10.1080/10611860600647934
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molecular weight delivery agent. The oral administration of teriparatide without co-administration of
any auxiliary agent did not lead to any elevation in
circulating blood parathyroid hormone levels. The
inability of teriparatide from reaching systemic
circulation can be explained by various barriers that
are encountered by the oral route. These barriers
include the diffusion barrier of the mucus layer
covering gastro-intestinal (GI) epithelia (BernkopSchnurch and Fragner 1996), as well as the absorption
barrier (Bernkop-Schnurch 1998). These have to be
overcome in order to achieve sufficient oral bioavailability. The most significant barrier for teriparatide,
however, seems to be the enzymatic barrier (Woodley
1994) caused by luminally secreted and membrane
bound proteolytic enzymes. Several therapeutic
proteins such as insulin or epidermal growth factor
(Playford et al. 1995) are degraded after oral
administration by pepsin in the stomach. The small
intestinal milieu is bearing a variety of proteases
including trypsin, chymotrypsin, elastase and brush
border membrane bound enzymes (BernkopSchnurch 1998). It was therefore the aim of the
current study to evaluate the stability of teriparatide
towards GI proteases in order to provide substantial
information for the development of an efficient oral
delivery system. The stability of teriparatide towards
several isolated secreted proteasessuch as trypsin,
chymotrypsin, elastase and pepsin as well as
membrane bound peptidases of rat small intestinal
mucosa and isolated aminopeptidase Nwas therefore evaluated in physiological concentrations and
physiological pH.
Materials and methods
HPLC analysis
HPLC analyses were performed with Nucleosil 5 C18
columns (250 4.6 mm). A flow rate of 1 ml/min was
maintained, using solvents A (0.1% TFA in destilled
H2O) and B (0.1% TFA in acteonitril). The following
gradient was used: 0 10.5 min (80 35% A), 10.5
12 min (35 80% A) and 12 17 min (80% A).
Degradation of teriparatide
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Degradation of teriparatide
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Figure 6. TLC of (A) aminopeptidase N in 50 mM TRIS buffer pH 7.5 after 3 h of incubation at 378C, (B) teriparatide in 50 mM TRIS
buffer pH 7.5 after 3 h of incubation at 378C and (C) teriparatide and aminopeptidase N in 50 mM TRIS buffer pH 7.5 after 3 h of incubation
at 378C; reference amino acids: D serine, E glutamic acid, F valine, G isoleucine, H glutamine.
Table I. Summary of teriparatide degradation by various luminally secreted and membrane bound proteolytic enzymes; experimental
conditions are explained within the text; each point represents the ^SD of at least three experiments.
Proteolytic enzyme
Aminopeptidase N
Trypsin
Chymotrypsin
Elastase
Pepsin
Membrane bound peptidases
Protease/teriparatide
molar ratio (mol/mol)
1:3000
1:12
1:6
1:100
1:1.3
n.a.
30 min
60 min
120 min
180 min
n.a.
0
0
94 ^ 4
0
n.a.
n.a.
0
0
95 ^ 2
0
94 ^ 3
89 ^ 4
0
0
92 ^ 2
0
84 ^ 11
78 ^ 23
0
0
86 ^ 3
0
59 ^ 8
50 ^ 5
0
0
84 ^ 5
0
51 ^ 19
Degradation of teriparatide
Acknowledgements
The Austrian Nano-Initiative co-financed this work
as part of the Nano-Health project (no. 0200), the
sub-project Nano-Pep-0254 (NANO-N-0254) being
financed by the Austrian Forschungsforderungs-Fond
fur die gewerbliche Wirtschaft (FFF).
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