Journal of Drug Targeting, April 2006; 14(3): 109115

Degradation of teriparatide by gastro-intestinal proteolytic enzymes

RCH2
MARTIN WERLE1, ANNETTE SAMHABER2, & ANDREAS BERNKOP-SCHNU
Journal of Drug Targeting Downloaded from informahealthcare.com by University of British Columbia on 10/29/14
For personal use only.

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

(Received 19 June 2005; accepted 12 January 2006)

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

parathyroid hormone (Potts et al. 1995) and is

therefore a recombinant human parathyroid hormone
1 34 displaying N and C terminal alpha helices
(Figure 1) which are essential for teriparatide
bioactivity.
The drug is administered daily by s.c. injection.
Because of the obvious advantages of non-invasive
teriparatide administration such as improved patient
compliance and painless administration, the development of such teriparatide delivery systems would be of
great benefit. An interesting approach is the intranasal
administration. Bioavailabilities up to 17.6% were
achieved after intranasal administration of aqueous
teriparatide solutions (Agu et al. 2004). However, the
oral application route is by far the most favoured one.
Although proof for the concept of an oral administration of teriparatide has been previously provided
(Leone-Bay et al. 2001), the full potential of oral
teriparatide delivery is believed to not yet have been
reached. Leone-Bay et al. determined the oral
bioavailability in monkeys to be 2.1% relative to
subcutaneous injection by co-administrating a low

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

110 M. Werle et al.

Journal of Drug Targeting Downloaded from informahealthcare.com by University of British Columbia on 10/29/14
For personal use only.

Figure 1. Structure of teriparatide, the two alpha helices are

highlighted.

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).

Teriparatide (R.t.: 6.6 min) was analysed at 220 nm

using a diode array detector.
Calculations were performed using a calibration
curve with eight calibrators (0.004 0.500 mg/ml),
which correspond to a range of 0.78 100% of the
initial teriparatide concentration used in the experiments. For degradation studies, the area under the
curve (AUC) of the intact teriparatide was determined. Values were calculated using linear regression;
for the calibration curves, the same buffers as for the
experiments were used. All experiments were
performed at least in triplicate.
Enzymatic stability of teriparatide towards isolated secreted
proteases
Enzymatic degradation tests were performed with
trypsin (WORTHINGTON, TPCK treated, E.C.
number 3.4.21.4, 249 p-toluene-sulfonyl-L -arginine
methyl ester (TAME) units/mg solid, from bovine
pancreas), chymotrypsin (WORTHINGTON,
TLCK treated, E.C. number 3.4.21.1, 55.4 benzoylL -tyrosine ethyl ester (BTEE) units/mg solid, from
bovine pancreas), elastase (WORTHINGTON, E.C.
number 3.4.21.36, 4.5 N-succinyl-L -Ala-L -Ala-L -Alap-nitroanilide (Suc Ala3NA) units/mg solid, from
porcine pancreas) and pepsin (SIGMA, E.C. number
3.4.23.1, 4150 hemoglobin units/mg solid, from
porcine gastric mucosa).
Enzyme solutions containing 13.6 TAME U trypsin
and 6.6 BTEE U chymotrypsin dissolved in 120 ml
TRIS buffer (50 mM, pH 6.5) were prepared,
respectively. Elastase solution was prepared by
dissolving elastase in 1% KCl and then adding TRIS
buffer (50 mM, pH 6.5) to obtain an activity of 0.3
(Suc Ala3NA) U elastase per ml. According to the
USPs preparation instructions for artificial gastric
juice, 3.2 mg of pepsin were dissolved in 1.0 ml of
0.08 M HCl. The pH wasaccording to the USP
about 1.5. To each enzyme solution containing
intestinal proteases (120 ml), 120 ml of the teriparatide
solution (1 mg teriparatide in 1 ml 50 mM TRIS
buffer; pH 6.5) was added. Upon being compared to
protease concentrations present in the human small
intestine, all protease concentrations were found to be
in physiological range (Bernkop-Schnurch 1998).
To 120 ml of the pepsin solution, 120 ml of teriparatide
solution (1 mg teriparatide in 1 ml 0.08 M HCl; pH
1.5) was added. The solutions were incubated at 378C
under shaking (at 300 rpm) during the sampling
period. At predetermined time points (0, 5, 15, 30, 60,
120 and 180 min) aliquots (30 ml) were withdrawn
and the reaction was stopped immediately by the
addition of 30 ml of 0.5% trifluor-acetic acid solution
to the intestinal protease solutions and of 30 ml of
0.1 M NaOH to the pepsin solution. The samples
were cooled to 48C and afterwards analysed by HPLC
as described above.

Degradation of teriparatide

Journal of Drug Targeting Downloaded from informahealthcare.com by University of British Columbia on 10/29/14
For personal use only.

Enzymatic stability of teriparatide towards rat intestinal

mucosa
Degradation studies with rat intestinal mucosa were
performed similarly, as described previously (Bernkop-Schnurch et al. 1997). In order to prevent
permeation, a parafilm layer was mounted onto the
acceptor chamber of an Ussing chamber system. Parts
of the first 15 cm of freshly excised small intestine of
laboratory rats were mounted onto the Ussing
chambers, the basolateral side of the mucosa facing
the parafilm layer. The donor and acceptor compartments of the chambers were filled with 0.75 ml of
freshly prepared incubation medium containing
250 mM NaCl, 2.6 mM MgSO 4, 10 mM KCl,
40 mM glucose and 50 mM NaHCO3. This solution
was buffered with 40 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and pH was
adjusted to 6.5. Experiments were performed at 378C
and were started 15 min after the mounting of the
tissue. Solutions of the donor chambers were replaced
by 0.75 ml of 0.375 mg/ml teriparatide solution in
incubation medium. Samples were withdrawn every
30 min for 3 h and then analysed via HPLC as
described above.
Enzymatic stability of teriparatide towards isolated
aminopeptidase N
Aminopeptidase N degradation studies were
performed similar to a method described previously
(Valenta et al. 2002). Teriparatide was dissolved in
50 mM TRIS buffer pH 7.0 to obtain a concentration
of 1 mg/ml. To 200 ml of this solution, 200 ml of
aminopeptidase N (SIGMA, E.C. number 3.4.11.2,
25 L-leucine-p-nitroanilide units/mg, from porcine
kidney) solution in buffer was added to obtain a final
aminopeptidase N concentration of 120 mU in the
incubation medium (400 ml). Experiments were
carried out at pH 7.0, 378C and under shaking
(at 300 rpm). Samples were withdrawn after 0, 60,
120, 180, 240, 300 and 360 min of incubation.
Enzymatic degradation was stopped by the addition
of 0.5% TFA solution and samples were analysed by
HPLC as described above.
In addition, samples were analysed via the 2,4,6trinitrobenzenesulfonic acid (TNBS) test. TNBS
reagent reacts with primary amino groups to a
compound detectable at 405 nm. Therefore, the
TNBS test was used in order to quantify the amount
of free primary amino groups in solution. Exopeptidases such as aminopeptidase N cleave the N-terminal
amino acid of a peptide and therefore an increase of
primary amino groups indicates teriparatide cleavage.
To 200 ml of a 1 mg/ml teriparatide solution, 200 ml of
aminopeptidase N solution (as described above) was
added. Experiments were carried out at pH 7.0, 378C
and under shaking (at 300 rpm). Samples were
withdrawn after 0, 60, 120, 180, 240, 300 and

111

360 min of incubation. The reaction was stopped by

adding 40 ml 1 M HCl to 50 ml of each sample. Then
300 ml of 0.1% TNBS in 5% NaHCO3 and 60 ml 1 M
NaOH was added to each sample and the mixture was
transferred onto a microtitration plate. The reaction
was allowed to proceed for an incubation time of 2 h at
room temperature. The absorption was measured at
405 nm and the concentration of primary amino
groups was calculated using a standard curve obtained
from increasing concentrations of cysteine.
Thin layer chromatography
Solutions of teriparatide, aminopeptidase N and
teriparatide/aminopeptidase N in 50 mM TRIS buffer
pH 7.6 were incubated for 3 h at 378C and 500 rpm.
Thin layer chromatography (TLC) was performed as
described in the European Pharmacopeia using silica
gel 60 F254 and a mixture of H2O, acetic acid and
butanol (20:20:60) as mobile phase. After developing,
ninhydrin reagent (1.0 g of ninhydrin dissolved in
ethanol/acetic acid 5/1) was sprayed on the dry
plate to dye single amino acids. Reference amino
acids were serine, valine, glutamic acid, isoleucine and
glutamine.
Statistical data analysis
Statistical data analysis was performed using the
Students t-test, with p , 0.05 as the minimal
significance unless indicated otherwise.
Results and discussion
In order to achieve sufficient oral bioavailability of
peptides and proteins, their extensive degradation
during GI passage must be significantly suppressed or
circumvented. Regarding the multitude of possible
strategies to overcome enzymatic degradation, the
design and optimization of an oral delivery system,
however, has to be based on an exact knowledge of the
enzymatic degradation process of the peptide drug
within the GI tract (Schilling and Mitra 1991;
Ikesue et al. 1993; Lu et al. 1999). Therefore, the
degradation of teriparatide has been evaluated in this
study in detail under conditions mimicking those of
the GI tract. Due to individual variations in enzyme
output and differences in published data concerning
enzymatic secretion in humans, the design of in vitro
assays that reflect actual enzyme concentrations along
the GI tract in vivo is quite difficult. The concentrations of pancreatic secreted enzymes used within
this study relate to the mean secretion in human
pancreas as previously reviewed (Bernkop-Schnurch
1998). A pH of 6.5 for experiments with intestinal
proteases was chosen in accordance with the mean pH
present in the human duodenum. Pepsin degradation
studies were performed according to the USP (1990).

Journal of Drug Targeting Downloaded from informahealthcare.com by University of British Columbia on 10/29/14
For personal use only.

112 M. Werle et al.

Regarding the specificity of the utilized proteolytic
enzymes (Figure 2), a prediction of expected cleavage
of teriparatide was made. However, the in vitro results
obtained in this study correlated only to a limited
extent with the preferred, theoretical cleavage sites.
Although teriparatide contains several elastase cleavage sites, elastase caused only minor damage to
teriparatide. In contrast, although no typical pepsin
cleavage sites occur within the molecule, pepsin
degraded teriparatide extensively. After 5 min of
incubation in acidic pepsin solution, intact teriparatide could no longer be detected. Considering that no
acidic hydrolysis of teriparatide occurred by preparing
the calibration curve at the same pH as used for the
experiment, the degradation must have been caused
exclusively by pepsin. Preferred pepsin cleavage sites
are the amide bonds between two aromatic amino
acids. Nevertheless, besides these preferential cleavage
sites, pepsin exhibits a broad substrate spectrum
(Sachdev and Fruton 1970). Suspected pepsin
cleavage sites within the teriparatide molecule are
the aromatic amino acids phenylalanine and tryptophan. However, pepsin-caused degradation can be
easily avoided by coating the dosage form with a
gastric fluid resistant layer. It has been demonstrated
in numerous studies that drugs can be protected
properly from gastric degradation by an enteric
coating of the dosage form with agents such as
polymethacrylates (Gupta et al. 2001) or cellulose
acetate phthalate (Malm and Hiatt 1951). Referring to
the evaluated intestinal proteases, trypsin and chymotrypsin degraded teriparatide the most. Within 5 min
of incubation in solutions containing either of those
proteases, teriparatide was degraded completely.
Teriparatide bears five typical trypsin cleavage
sitespreferred amino acids for trypsin cleavage are
R and K (Bode 1979)whereas the molecule displays
no preferential chymotrypsin cleavage sites. A rest
activity of trypsin in the chymotrypsin solution was
excluded by using 1-chloro-3-tosylamido-7-amino-2heptanone (TLCK) treated chymotrypsin. However,
apart of a preferred cleavage at F and Y moieties,
chymotrypsin is also known to cleave peptides bearing
L, M, N, Q and W (Baumann et al. 1970; Berezin and

Figure 2. Preferred theoretical cleavage sites of various luminally

secreted proteases and aminopeptidase N.

Figure 3. Degradation of teriparatide by elastase (-W-) and by

membrane bound proteases of rat small intestinal mucosa (-X-) at
378C and pH 6.5; elastase incubation medium contained 240 ml
50 mM TRIS buffer pH 6.5, 120 mg teriparatide and 0.036 Suc
Ala3NA U elastase; membrane bound protease incubation medium
contained 0.375 mg/ml teriparatide, 250 mM NaCl, 2.6 mM
MgSO4, 10 mM KCl, 40 mM glucose, 50 mM NaHCO3 and
40 mM HEPES buffer; each point indicates the mean ^ SD of at
least three experiments.

Martinek 1970). Teriparatide displays five L, two M,

three N and one W, which are believed to be the target
of cleavage. Besides chymotrypsin and trypsin,
another important serine protease secreted by the
pancreas is elastase. As shown in Figure 3, teriparatide
is degraded by elastase to a certain degree. Within 3 h
of incubation in elastase solution, only about 15% of
teriparatide was degraded, although teriparatide displays ten typical elastase cleavage sites. Aside from the
varying specificities of the utilized proteases, a further
explanation for the relatively minor degradation
caused by elastase in comparison to that caused by
trypsin and chymotrypsin may be found in the
comparatively lower amount of elastase secreted by
the pancreas and consequently small elastase concentrations used within this study. The approximate
molar ratio of chymotrypsin to trypsin to elastase used
in this study was 16:8:1.
Degradation of teriparatide caused by membrane
bound peptidases was also evaluated in this study. The
time-dependent degradation of teriparatide by membrane-bound peptidases of rat small intestinal mucosa
is shown in Figure 3. Within 3 h of incubation, about
50% of teriparatide was degraded. To gain further
knowledge about degradation caused by membranebound peptidases, degradation studies were also
performed with isolated aminopeptidase N, which is
the most abundant membrane-bound peptidase.

Journal of Drug Targeting Downloaded from informahealthcare.com by University of British Columbia on 10/29/14
For personal use only.

Degradation of teriparatide

Figure 4. Degradation of teriparatide by aminopeptidase N at

378C and pH 7.0; incubation medium contained 400 ml 50 mM
TRIS buffer pH 6.5, 200 mg teriparatide and 120 L-p-nitroanilide
mU aminopeptidase N; each point indicates the mean ^ SD of at
least three experiments.

Results of these studies, as shown in Figure 4, indicate

that aminopeptidase N is at least partly responsible for
teriparatide degradation. About 20% of intact
teriparatide was detected within a six-hour incubation
period with aminopeptidase N.
In comparison to luminally secreted proteases
which are mainly endopeptidases, the most abundant
membrane-bound peptidases, such as aminopeptidase
N, are exopeptidases. It is well known that the
terminal located helices of teriparatide are essential for
bioactivity which is mediated by an activation of
cAMP/protein kinase-A (PKA) as well as protein
kinase-C (PKC). C-truncated derivatives such as
PTH 1 31 are able to stimulate intracellular cAMP
accumulation. However, it has been demonstrated
that PTH 1 31 is less potent to increase serum
calcium levels in mice in comparison to PTH 1 34
(Mohan et al. 2000). Fujimori et al. demonstrated that
activation of cAMP/PKA system requires the N-terminal amino acids 1 and 2 whereas the phospholipaseC/PKC system is coupled to a longer domain of the
hormones N-terminus (Fujimori et al. 1992). Also
Tsomaia et al. (2004) showed that the N-terminal
residues (1 4) of the signalling domain plays a
significant role in PTH action. Furthermore it has
been demonstrated, that the truncated fragment PTH
2 34 was only 67% as potent as PTH 1 34 and
deletion of the first two amino acids at the N terminus
abolished the hormones ability to stimulate cAMP
production in UMR-106-01 cells (Civitelli et al.
1994). Moreover, it was shown that also the PTH
analogues 3 34, 7 34 and 13 34 do not stimulate
cAMP production (Yu and Chandrasekhar 1997).
It was therefore also of particular interest to verify
whether degradation caused by aminopeptidase N

113

Figure 5. Increase of primary amino groups in presence of isolated

aminopeptidase N at 378C and pH 7.0; incubation medium
contained 400 ml 50 mM TRIS buffer pH 6.5, 200 mg teriparatide
and 120 L-p-nitroanilide mU aminopeptidase N; each point
indicates the mean ^ SD of at least three experiments.

stops after cleavage of the first N-terminal amino acid,

or whether further amino acids are cleaved from
teriparatide. Hence, samples of aminopeptidase
N-degradation studies were also analysed via the
TNBS test. This reagent reacts with primary amino
groups. Taking into account that each teriparatide
molecule contains three K residues which react with
TNBS, an increase in primary amino groups of
approximately factor 1.6 indicates that aminpeptidase
N-cleavage does not stop after cleavage of the
N-terminal amino acid of teriparatide. Results of this
study are shown in Figure 5. Moreover, these findings
were supported by TLC. After incubation of
teriparatide in presence of aminopeptidase N, at
least three different free amino acids were detected,
whereas no free amino acids were detected after
incubation of teriparatide or aminopeptidase N,
respectively. Results of this study are shown in
Figure 6. The first five amino acids of the N-terminus
were used as recerences (D serine, E glutamic
acid, F valine, G isoleucine, H glutamine).
Comparison of the retention times of the free amino
acids caused by aminopeptidase N (C) and the
retention times of the reference amino acids (D H)
indicated that at least the first four amino acids are
cleaved from the teriparatide N-terminus. Although it
has been demonstrated by several studies that slight
chemical modifications of the N-terminal amino acid
can stabilize a peptide drug towards aminopeptidasecaused degradation (Wallace 1992), the effect of such
modifications upon pharmacological activity has to be
investigated thoroughly.
A summary of the stability of teriparatide towards
GI proteases gained in the current study is provided

Journal of Drug Targeting Downloaded from informahealthcare.com by University of British Columbia on 10/29/14
For personal use only.

114 M. Werle et al.

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.

in Table I. Although teriparatide is degraded by

pepsin, gastric degradation can be overcome by
enteric coating. Teriparatide is degraded extensively
by trypsin and chymotrypsin and to a certain extent
by elastase. In order to reach sufficient high oral
bioavailability, intestinal degradation must be minimized or even excluded. Attempts to reduce
enzymatic degradation include the use of analogues,
prodrugs, and formulations such as nanoparticles,
microparticles and liposomes that shield therapeutic
peptides and proteins from luminal enzymatic attack.
The design of delivery systems targeting the colon,
where proteolytic activity is relatively low (Ikesue
et al. 1993), is also an important approach to
circumvent excessive enzymatic degradation. Moreover, the co-administration of enzyme inhibitors has
become of increasing interest. Due to such excipients, various in vivo studies demonstrated a
significantly improved bioavailability of drugs after
oral dosing (Fujii et al. 1985; Morishita et al. 1992;
Yamamoto et al. 1994). In order to avoid systemic
toxic side effects of such enzyme inhibitors, their

immobilization to unabsorbable polymeric carriers

such as polyacrylates might be a promising strategy.
Proof for the concept of such systems has already
been provided by various in vivo studies (Marschutz
et al. 2000; Guggi et al. 2003).
Conclusion
Within the current study, substantial information
about the stability of teriparatide towards various
luminally secreted and membrane-bound proteases of
the GI tract has been provided. Trypsin, chymotrypsin
as well as pepsin have been identified to degrade
teriparatide extensively. Elastase also degrades teriparatide to a certain, but comparatively less degree.
Membrane-bound peptidases have equally been
shown to cause degradation to teriparatide. Furthermore, aminopeptidase N was found to be involved in
this degradation process. Information gained within
this study represents an important prerequisite for the
development of potential oral teriparatide delivery
systems.

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.

Percent of remaining intact teriparatide

5 min

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).

Journal of Drug Targeting Downloaded from informahealthcare.com by University of British Columbia on 10/29/14
For personal use only.

References
Agu RU, Valiveti S, Earles DC, Klausner M, Hayden PJ,
Wermeling DP, Stinchcomb AL. 2004. Intranasal delivery of
recombinant human parathyroid hormone [hPTH (1 34)],
teriparatide in rats. Endocr Res 30(3):455467.
Baumann WK, Bizzozero SA, Dutler H. 1970. Specificity of alphachymotrypsin dipeptide substrates. FEBS Lett 8(5):257260.
Berezin IV, Martinek K. 1970. Specificity of alpha-chymotrypsin.
FEBS Lett 8(5):261262.
Bernkop-Schnurch A. 1998. The use of inhibitory agents to
overcome the enzymatic barrier to perorally administered
therapeutic peptides and proteins. J Control Rel 52(12):116.
Bernkop-Schnurch A, Fragner R. 1996. Investigations into the
diffusion behaviour of polypeptides in native intestinal mucus
with regard to their peroral administration. Pharm Sci 2(1):
361 363.
Bernkop-Schnurch A, Paikl C, Valenta C. 1997. Novel bioadhesive
chitosan-EDTA conjugate protects leucine enkephalin from
degradation by aminopeptidase N. Pharm Res 14:917922.
Bode W. 1979. Activation, activity and inhibition of bovine trypsin.
Naturwissenschaften 66(5):251258.
Civitelli R, Bacskai BJ, Mahaut-Smith MP, Adams SR, Avioli LV,
Tsien RY. 1994. Single-cell analysis of cyclic AMP response to
parathyroid hormone in osteoblastic cells. J Bone Miner Res
9(9):14071417.
Dobnig H, Turner RT. 1997. The effects of programmed
administration of human parathyroid hormone fragment
(1 34) on bone histomorphometry and serum chemistry in
rats. Endocrinology 138(11):46074612.
Fujii S, Yokohama T, Ikegaya K, Salo F, Yakoo N. 1985. Promoting
effect of the new chymotrypsin inhibitor FK-448 on the
intestinal absorption of insulin in rats and dogs. J Pharm
Pharmacol 37:545549.
Fujimori A, Cheng SL, Avioli LV, Civitelli R. 1992. Structurefunction relationship of parathyroid hormone: Activation of
phospholipase-C, protein kinase-A and -C in osteosarcoma cells.
Endocrinology 130(1):2936.
Guggi D, Kast CE, Bernkop-Schnurch A. 2003. In vivo evaluation
of an oral salmon calcitonin-delivery system based on a thiolated
chitosan carrier matrix. Pharm Res 20(12):19891994.
Gupta VK, Beckert TE, Price JC. 2001. A novel pH- and timebased multi-unit potential colonic drug delivery system.
I. Development. Int J Pharm 213(12):83 91.
Ikesue K, Kopeckova P, Kopecek J. 1993. Degradation of proteins
by guinea pig intestinal enzymes. Int J Pharm 95:171179.
Leone-Bay A, Sato M, Paton D, Hunt AH, Sarubbi D, Carozza M,
Chou J, McDonough J, Baughman RA. 2001. Oral delivery of

115

biologically active parathyroid hormone. Pharm Res

18(7):964970.
Lu RH, Kopeckova P, Kopecek J. 1999. Degradation and
aggregation of human calcitonin in vitro. Pharm Res
16(3):359367.
Malm CJJE, Hiatt GD. 1951. Cellulose acetate phthalate as an
enteric coating material. J Am Pharm Assoc 40(10):520525.
Marschutz MK, Caliceti P, Bernkop-Schnurch A. 2000. Design and
in vivo evaluation of an oral delivery system for insulin. Pharm
Res 17(12):1468 1474.
Mohan S, Kutilek S, Zhang C, Shen HG, Kodama Y, Srivastava AK,
Wergedal JE, Beamer WG, Baylink DJ. 2000. Comparison of
bone formation responses to parathyroid hormone(134), (1
31), and (2 34) in mice. Bone 27(4):471478.
Morishita I, Morishita M, Takayama K, Machida Y, Nagai T. 1992.
Hypoglycemic effect of novel oral microspheres of insulin with
protease inhibitor in normal and diabetic rats. Int J Pharm
78:9 16.
Playford RJ, Marchbank T, Calnan DP, Calam J, Royston P,
Batten JJ, Hansen HF. 1995. Epidermal growth factor is
digested to smaller, less active forms in acidic gastric juice.
Gastroenterol 108(1):92101.
Potts JT, Bringhurst FR, Gardella T, Nussbaum S, Segre G,
Kronenberg H. 1995. Parathyroid hormone: Physiology,
chemistry, biosynthesis, secretion, metabolism, and mode of
action. 3rd ed. Philadelphia, P.A. W.B. Saunders.
Sachdev GP, Fruton JS. 1970. Secondary enzyme-substrate
interactions and the specificity of pepsin. Biochemistry
9(23):44654470.
Sato M, Grese TA, Dodge JA, Bryant HU, Turner CH. 1999.
Emerging therapies for the prevention or treatment of
postmenopausal osteoporosis. J Med Chem 42(1):124.
Schilling RJ, Mitra AK. 1991. Degradation of insulin by trypsin and
alpha-chymotrypsin. Pharm Res 8(6):721 727.
Tsomaia N, Pellegrini M, Hyde K, Gardella TJ, Mierke DF. 2004.
Toward parathyroid hormone minimization: Conformational
studies of cyclic PTH(114) analogues. Biochemistry 43(3):
690 699.
USP, C 1990. United States Pharmacopeia XXII-NFXVII. US
Pharmacopeial Conv (40a):1788.
Valenta C, Marschutz M, Egyed C, Bernkop-Schnurch A. 2002.
Evaluation of the inhibition effect of thiolated poly(acrylates) on
vaginal membrane bound aminopeptidase N and release of the
model drug LHRH. J Pharm Pharmacol 54(5):603610.
Wallace RJ. 1992. Acetylation of peptides inhibits their degradation
by rumen micro-organisms. Br J Nutr 68(2):365 372.
Woodley JF. 1994. Enzymatic barriers for GI peptide and protein
delivery. Crit Rev Ther Drug Carrier 11(23):6195.
Yamamoto A, Taniguchi T, Rikyun K, Tsuji T, Fujita T,
Murakami M, Muranishi S. 1994. Effects of various protease
inhibitors on the intestinal absorption and degradation of insulin
in rats. Pharm Res 11:14961500.
Yu XP, Chandrasekhar S. 1997. Parathyroid hormone (PTH 134)
regulation of rat osteocalcin gene transcription. Endocrinology
138(8):30853092.