Inhibition of Octreotide Acylation Inside PLGA Microspheres by Derivatization of the Amines of the Peptide with a Self-Immolative Protecting Group

Article abstract of DOI:10.1021/acs.bioconjchem.5b00598

Acylation of biopharmaceuticals such as peptides has been identified as a major obstacle for the successful development of PLGA controlled release formulations. The purpose of this study was to develop a method to inhibit peptide acylation in poly(d,l-lac

Full text of DOI:10.1021/acs.bioconjchem.5b00598

Article
pubs.acs.org/bc
Inhibition of Octreotide Acylation Inside PLGA Microspheres by
Derivatization of the Amines of the Peptide with a Self-Immolative
Protecting Group
Mehrnoosh Shirangi,† Marzieh Najafi, Dirk T. S. Rijkers, Robbert Jan Kok, Wim E. Hennink,
,§
†
‡
†
†
,†
and Cornelus F. van Nostrum*
†
‡
Department of Pharmaceutics and Medicinal Chemistry & Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, 3512 JE Utrecht, The Netherlands
§
Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Science, Tehran 1417614411, Iran
S Supporting Information
*
ABSTRACT: Acylation of biopharmaceuticals such as peptides has been identified as a major
obstacle for the successful development of PLGA controlled release formulations. The purpose of
this study was to develop a method to inhibit peptide acylation in poly(D,L-lactide-co-glycolide)
(
(
PLGA) formulations by reversibly and temporarily blocking the amine groups of a model peptide
octreotide) with a self-immolative protecting group (SIP), O-4-nitrophenyl-O′-4-acetoxybenzyl
carbonate. The octreotide with two self-immolative protecting groups (OctdiSIP) on the N-
terminus and lysine side chain was synthesized by reaction of the peptide with O-4-nitrophenyl-O′-
4
-acetoxybenzyl carbonate, purified by preparative RP-HPLC and characterized by mass
spectrometry. Degradation studies of OctdiSIP in aqueous solutions of different pH values
showed that protected octreotide was stable at low pH (pH 5) whereas the protecting group was
eliminated at physiological pH, especially in the presence of an esterase, to generate native
octreotide. OctdiSIP encapsulated in PLGA microspheres, prepared using a double emulsion
solvent evaporation method, showed substantial inhibition of acylation as compared to the
unprotected octreotide: 52.5% of unprotected octreotide was acylated after 50 days incubation of
microspheres in PBS pH 7.4 at 37 °C, whereas OctdiSIP showed only 5.0% acylation in the same time frame. In conclusion, the
incorporation of self-immolative protection groups provides a viable approach for inhibition of acylation of peptides in PLGA
delivery systems.
INTRODUCTION
since PEG could in principle reduce the accumulation of acid
degradation products in the degrading matrices and peptide
adsorption onto the PLGA surface. However, this approach did
not show a favorable effect concerning peptide acylation inside
■
(
Biodegradable and biocompatible poly(D,L-lactide-co-glycolide)
PLGA) has been widely used for the design of sustained
release formulations for therapeutic peptides and proteins.
1
−4
11
degrading PLGA microspheres. Houchin et al. studied the
effects of pH-modifying excipients, such as a proton sponge
compound (1,8-bis(dimethylamino)naphthalene), magnesium
hydroxide, ammonium acetate, and magnesium acetate, on the
chemical stability and acylation of a model peptide (VYPNGA)
in PLGA films. They showed that addition of these excipients
to PLGA formulations does not prevent acylation reactions,
and even an increased acylation was observed likely due to the
However, the stability of peptides and proteins within PLGA
matrices has been identified as a major challenge for the
5
−7
successful development of PLGA controlled release systems.
PLGA can interact with nucleophiles, such as the N-terminus
and primary amine groups of lysine residues of peptides/
proteins, resulting in an acylation reaction between the
peptide/protein and the polymer. This reaction involves the
nucleophilic attack of an amine of the peptide on a lactate or
glycolate ester of the polymer resulting in aminolysis, followed
by hydrolysis of the conjugated polymer chain. Finally, this
results in covalent connection of a glycolyl or lactyl group
12
increased nucleophilicity of the peptide amine. Sophocleous
et al. suggested that peptide sorption to PLGA is the first step
to peptide acylation. They prevented peptide sorption to PLGA
by adding a divalent cationic salt to a peptide/PLGA mixture,
8
,9
through an amide bond on the peptide. Peptide acylation
may potentially result in loss of activity, a change of receptor
13,14
which indeed resulted in attenuation of acylation.
In
10
another study from this group, the prevention of acylation of
octreotide in the presence of divalent cationic salt inside PLGA
affinity, or even immunogenicity and should therefore be
avoided.
Several methods to minimize peptide acylation within PLGA
microparticles have been proposed. Lucke et al. investigated
whether a blockcopolymer of poly(ethylene glycol) (PEG) and
poly(D,L-lactic acid) (PLA) would reduce peptide acylation,
Received: November 4, 2015
Revised: December 30, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.bioconjchem.5b00598
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Article
microspheres prepared by a double emulsion solvent
evaporation method was investigated. Addition of divalent
cationic salts to both the inner and outer water phase of the
double emulsion indeed decreased octreotide acylation: after 28
days, 76.3% of octreotide was in its native form inside the
microspheres without any salt, as compared to 90.9% that was
Dextran sulfate A-octreotide and dextran sulfate B-octreotide
complexes that were encapsulated in PLGA microparticles
showed <7% acylation during in vitro release in a gel matrix
based on a ABCBA pentablock polymer of polylactic acid (A),
poly(ε-caprolactone) (B), and PEG (C). However, they did not
show the extent of acylation of unprotected octreotide as a
15
33
in its native form using MnCl as excipient. In a more recent
reference under the same conditions in the gel matrix.
2
1
6
study they evaluated the effect of addition of divalent cation
salts as well as carboxymethyl chitosan (CMCS) on inhibition
of acylation of octreotide, salmon calcitonin (sCT), and human
parathyroid hormone (hPTH). Divalent cation salts decreased
the extent of acylation of octreotide and hPTH but not of sCT.
Addition of CMCS alone was ineffective, whereas a
combination of inorganic cations with CMCS improved the
stability of octreotide and sCT but it had no effect on hPTH
Despite all efforts, none of the approaches described above
could fully and safely prevent acylation of peptides in PLGA
matrices. Therefore, a new approach for modifying the peptide
by an enzyme-cleavable/self-immolative protecting group is
investigated in this paper. Self-immolative prodrugs have been
widely used to generate in active drug form an inactive
34
precursor/prodrug in the body. Self-immolative elimination is
the spontaneous and irreversible fragmentation of a compound
into its building blocks through a cascade of electronic
elimination processes. This phenomenon is most commonly
observed for electron-rich aromatic species. The electron
donating substituent is required to lower the energy barrier
1
6
stability. From this study it may be concluded that the effects
of excipients on inhibition of acylation varied depending on the
properties of the individual peptides. Therefore, selective
addition of such stabilizers to PLGA formulations needs to
be considered on an individual peptide on a case-by-case
35
of dearomatization.
1
6
basis. Feng et al. evaluated the inhibitory effect of dications on
This study aims for the synthesis of a new self-immolative
protecting group and its attachment to the amine functionalities
of the therapeutic peptide octreotide. The structure of the
investigated group is shown in Scheme 1 and is based on 4-
the acylation of an acidic peptide (exenatide) outside or inside
2
+
PLGA microspheres. They showed that Ca did not prevent
2+
acylation and Mn resulted in some inhibition of acylation,
whereas Zn2 possessed the greatest inhibitory effect.
+
17
Minimizing peptide acylation (with some degree of success)
has been reported by modifications of polymer composition
with alternating the polymer with more hydrophilic
Scheme 1. Self-Immolative Conversion of OctdiSIP into
Native Octreotide
1
8−20
domains.
Chemical modification (e.g., PEGylation) of peptide
therapeutics is an attractive option to improve their physical
stability and resistance to proteases, to reduce immunogenicity,
2
1−24
and to increase half-life.
also prevent acylation; however, it may cause changes in the
In some cases, PEGylation may
25,26
affinity of the peptide/protein for the target receptor.
Na et
al. presented a PEGylation strategy to prevent acylation of
octreotide. However, PEGylation of the lysine residue of
octreotide resulted in significant loss of its biological
2
7,28
activity.
It should be remarked that in this study PEGylated
octreotide was incubated with PLGA polymer dispersed in
phosphate buffer pH 7.4 instead of encapsulating the
PEGylated derivatives in the microparticles.
Reversibly or temporarily blocking the amine groups could
be an interesting approach to prevent acylation. Ahn et al.
investigated reversible blocking of the amine groups of
octreotide with maleic anhydride (MA) to prevent its acylation
in PLGA particles. The hydrolysis of maleoyl amides is acid
2
9
catalyzed. The hydrolytic degradation of PLGA results in the
formation of acid degradation products that accumulate in the
3
0,31
polymer matrix, which results in pH decrease,
and the MA
hydroxybenzyl alcohol which has been previously used for the
35−37
conjugate, because of its acid-sensitivity, was supposed to be
converted into intact peptide that is subsequently released into
the surrounding medium. Indeed, a substantial inhibition of the
formation of acylated octreotide was observed. However, the
maleoylated octreotide was released faster than it was converted
into intact octreotide inside the PLGA film. Since deblocking
after release is rather slow at physiological conditions, the
possible toxicity of maleoylated octreotide should be evaluated.
Vaishya et al. reported minimizing octreotide acylation by
masking the reactive nucleophilic amine of octreotide with a
reversible hydrophobic ion-pairing complex, which is stable at
acidic pH inside degrading the PLGA particles and can
dissociate at physiological pH to yield native octreotide.
development of self-immolative polymers.
The design of
this protecting group is such that it is stable at acidic pH (inside
degrading PLGA microspheres) and decomposition is triggered
by either enzymatic hydrolysis, e.g., esterases, or the higher pH
outside the microspheres. The first trigger step, which is the
hydrolysis of the acetate group, is rate-limiting in the generation
32
35
of the parent octreotide, as the remaining part of the self-
immolative group is rapidly eliminated and split into nontoxic
compounds like carbon dioxide, acetic acid, and 4-hydroxy-
38
benzyl alcohol (Scheme 1). Octreotide was used as a model
peptide to protect its most susceptible sites of acylation (the N-
18
terminus and lysine residue ) using this self-immolative
protecting group.
B
DOI: 10.1021/acs.bioconjchem.5b00598
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Bioconjugate Chemistry
Article
RESULTS AND DISCUSSION
Synthesis and Characterization of Self-Immolative
Protected Octreotide. As shown in Scheme 2, the first step
was carried out by NMR analysis (Figure S1 and S2, Supporting
Information). Subsequently, the self-immolative protecting
group (SIP, O-4-nitrophenyl-O′-4-acetoxybenzyl carbonate,
compound 5) was successfully synthesized by the reaction of
the 4-acetoxybenzyl alcohol with 4-nitrophenyl chloroformate
■
Scheme 2. Synthesis of the Self-Immolative Protecting
Group and Its Conjugation to Octreotide
(
step 2). SIP was obtained in a yield of 60% and characterized
by NMR (Figures S3 and S4). A single, sharp melting point was
observed by DSC at 114.7 °C (ΔH = 87.1 J/g) (Figure S5).
The carbonate intermediate in compound 5 is highly reactive
toward amines because it provides a good leaving group (4-
nitrophenol) to form a carbamate bond between an amine
group of octreotide and carbonate of SIP. Thus, compound 5
was conjugated to octreotide by substitution of nitrophenyl
carbonate by the two primary amines of the peptide, i.e., the N-
terminus and the ε-amine of the lysine residue. A mixture of
products was obtained, including diprotected peptide and some
byproducts. The desired diprotected octreotide (OctdiSIP) was
isolated by preparative HPLC in a yield of 21% and with a
purity of >95% according to the areas under the curve of its
UPLC chromatogram (Figure S6). The product was identified
by mass spectrometry and showed the correct molecular weight
of 1403 Da.
In Vitro Hydrolytic and Enzymatic Degradation of
OctdiSIP. To study the rate of the conversion of the OctdiSIP
into native octreotide, the kinetics of the hydrolytic degradation
of OctdiSIP was determined in (2:1 v/v) buffer/acetonitrile
mixture at different pH values at 37 °C. It should be mentioned
that acetonitrile was added as cosolvent since OctdiSIP has a
very low solubility in water. Figure 1 shows the UPLC
chromatograms of the incubation mixtures at pH 7.4 at 0, 48,
and 120 h, respectively. The main degradation products that
gradually appeared in time were identified by UPLC-MS as
being the two expected monoprotected peptides, with the
remaining SIP on either the N-terminus or the ε-amine of the
lysine residue (OctmonoSIP, m/z 1212), and native octreotide
(m/z 1020). However, minor peaks were observed that were
identified by MS as octreotide with one acetyl group
in the synthesis of the protecting group was O-acetylation of
the phenol group of 4-hydroxybenzyl alcohol to yield 4-
acetoxybenzyl alcohol. The yield was 52% and the identification
Figure 1. UPLC of 75 μg/mL OctdiSIP incubated in buffer/acetonitrile (2:1) (v/v) at pH 7.4 and 37 °C: (A) t = 0; (B) t = 48 h; and (C) t = 120 h.
Fluorescent detection was used with excitation at λ = 280 nm and emission at λ = 330 nm. The identification of the numbered peaks is shown in
Table 1.
C
DOI: 10.1021/acs.bioconjchem.5b00598
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Bioconjugate Chemistry
acetylated octreotide, m/z 1062) and octreotide with one SIP
and one acetyl group (two isomers of acetylated OctmonoSIP,
m/z 1256: acetyl on N-terminus with SIP on lysine or SIP on
N-terminus with acetyl on lysine) (Table 1). The formation of
Article
(
The effect of pH on the degradation kinetics and hydrolysis
rate constants of OctdiSIP was analyzed by monitoring the
degradation of OctdiSIP in buffer/acetonitrile at pH 5.0, 7.4,
and 9.0 by UPLC (Figure 2); the UPLC chromatograms of the
incubation mixtures at pH 5 and 9 at 0, 48, and 120 h are
shown in Figure S7 and S8 in the Supporting Information.
OctdiSIP is quite stable in pH 5 (Figure 2A) while the
conversion into OctmonoSIP, native octreotide, and the other
byproducts occurs at elevated pH (7.4 and 9.0; Figure 2B and
C, respectively). These figures also show that the amount of the
monoprotected intermediate products first increases and then
decreases over time because these intermediates are formed
from hydrolysis of one of the protecting groups at either the N-
terminus or lysine and are subsequently converted into native
octreotide or one of the acylated products. Eventually, the final
degradation products are native octreotide and acetylated
octreotide, which are formed in almost equal amounts (Figure
Table 1. Identification of the Observed Peaks in Figure 1 by
UPLC-MS
number of
peaks
observed [M + H]+
m/z
Δm (Da)
assigned structure
octreotide (native)
1
1020
1062
0
2, 3
+42
acetylated
octreotide
4
6
, 5
, 7
1212
1254
+192
OctmonoSIP
+234 (192 +
acetylated
OctmonoSIP
4
2)
8
1404
+384 (192 +
92)
OctdiSIP
1
2
B and C). Table 2 shows the rate constants of the degradation
acetylated products can be explained by aminolysis of an acetate
group of a remaining SIP unit by a free amine that is formed
after deprotection (either by an inter or an intra molecular
reaction). Scheme 3 depicts the full reaction scheme of
degradation of OctdiSIP in neutral (or basic) media. The two
isomeric peptides with one acetyl and one SIP group
of OctdiSIP (c = 75 μg/mL in buffer/acetonitrile 2:1) at
different pH at 37 °C. This table shows that the rate constant at
pH 5.0 is much lower than at neutral and basic pH. The half-life
of OctdiSIP in pH 5.0 at 37 °C is estimated to be 1050 h in
comparison with 63 h at pH 7.4. Ester hydrolysis is catalyzed by
H or OH ions, and for that reason the trigger to release the
SIP group by hydrolysis of the acetate unit is pH dependent,
with the lowest hydrolysis rate at around pH 5.0. This is in line
with the general principle of ester hydrolysis with the minimum
+
−
(
acetylated OctmonoSIP) are obtained by reaction between
an amine of OctmonoSIP and a SIP-acetate group from another
peptide molecule (Scheme 3: k , k ). After deprotection of the
5
6
39,40
remaining SIP unit (k , k ) these intermediate isomers will
7
8
rate constant at ∼pH 4.5.
finally result in monoacetylated octreotide. On the other hand,
intramolecular reaction of the amine of OctmonoSIP with the
remaining SIP on the same peptide will directly result in
monoacetylated octreotide (k11 and k12 in Scheme 3; see
Scheme 4 for the proposed reaction mechanism). Also, reaction
of native octreotide with the acetate group of protected
octreotide (k , k ), will finally result in the formation of
It must be stressed that the values derived from Figure 2 are
the rate constants for hydrolysis in buffer/acetonitrile (2:1)
mixtures. The influence of the dielectric constant (ϵ) of the
medium on the k of ester hydrolysis was studied by de Jong
obs
41
et al. They have found higher kobs values at higher dielectric
constant of the medium ranging from ϵ = 47.7 to 68.3. The
dielectric constant of buffer/acetonitrile (2:1) that we used in
9
10
acetylated octreotide as the main byproduct.
our studies was 64.8 according to the formula ϵ = ϵacetonitrile
×
Scheme 3. Scheme of Proposed Degradation Pathway for OctdiSIP in Neutral or Basic Media
D
DOI: 10.1021/acs.bioconjchem.5b00598
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Article
Scheme 4. Proposed Mechanism of Intramolecular Aminolysis
Table 2. Reaction Rate Constants of Degradation of
OctdiSIP (c = 75 μg/mL) at 37 °C at Different pH Values
buffer/acetonitrile (2:1)
(
measured)
buffer (calculated)
pH
k (h−1)
t1/2 (h)
k (h−1
)
t1/2 (h)
3
3
3
−3
−3
5.0
7.4
9.0
0.66 × 10−
11.0 × 10
93 × 10−
1050
63
4.16 × 10
69.3 × 10
160
10
−
7.4
590 × 10−3
1.2
than in buffer/acetonitrile 2:1. The reaction rate constants and
half-life times in both media are summarized in Table 2. We
thus estimated the half-life time of OctdiSIP to be around 10 h
at physiological pH in water.
Importantly, the pH value inside degrading PLGA micro-
42−44
spheres was reported to be approximately 4.
For successful
application of OctdiSIP to minimize acylation of peptide inside
degrading PLGA microspheres, the stability of the protecting
group at lower pH (Figure 5A) is a clear advantage. In the ideal
case, the protecting group must be eliminated completely after
release to yield native octreotide. The half-life time of OctdiSIP
in pH 7.4 is around 63 h in buffer/acetonitrile 2:1 and
estimated to be 10 h in buffer, although the formation of the
acetylated byproducts was unexpected and is not desired.
However, esterases present in the body can affect the
conversion by catalyzing the hydrolysis of the acetate group
45
of the SIP units, and thereby trigger the deprotection.
Therefore, an enzymatic degradation study was done, in which
we reduced the amount of cosolvent to 3% acetonitrile to
minimize enzyme inactivation (as compared to the hydrolytic
degradation study where 33% acetonitrile was used). Under
these conditions, the enzymatic activity was determined to be 5
units/mg, in comparison with 12.5 units/mg in PBS. 4.2 μg of
the esterase (or 0.02 units) was added to 15 μg (0.01 μmol) of
OctdiSIP in 1 mL PBS buffer pH 7.4 containing 3% acetonitrile,
an amount of enzyme that in principle is suitable to hydrolyze
the two protecting groups in octreotide in 1 min (one unit of
enzyme is able to hydrolyze one μmol ester bonds per minute,
so 0.02 unit which is also a physiologically relevant
Figure 2. Degradation profile of OctdiSIP (75 μg/mL buffer/
acetonitrile (2:1)) at (A) pH 5, (B) pH 7.4, and (C) pH 9.
46
facetonitrile + ϵwater × fwater, with f being the volume fractions and
ϵacetonitrile = 37.5 and ϵwater = 78.5. Assuming the same
dependence of ϵ on the degradation rate constant as found
concentration is able to hydrolyze 0.02 μmol ester bonds
per minute). Figure 3 shows the relative peak areas in the
UPLC chromatogram of OctdiSIP before and after addition of
the esterase. The conversion was indeed complete after 1 min
in pH 7.4 at 37 °C after addition of the enzyme and, most
4
1
by de Jong et al. for oligo(lactic acid) hydrolysis, we anticipate
that the rate constant of OctdiSIP is 6.3-fold higher in buffer
E
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Article
Figure 3. Relative peptide content (A) before adding esterase and (B) 1 min after adding esterase to a solution of OctdiSIP in PBS pH 7.4 containing
% acetonitrile.
3
Figure 4. UPLC chromatograms of extracted peptides from PLGA microspheres after 50 days incubation in PBS buffer pH 7.4 at 37 °C: (A)
Octreotide and (B) OctdiSIP loaded microspheres.
importantly, without formation of any acetylated octreotide, as
opposed to the hydrolytic degradation study presented above.
This is an important observation, which means that because of
the carboxyl esterase in the body the conversion is expected
to be completely to native octreotide.
Preparation and Characterization of Microspheres.
PLGA microspheres loaded with octreotide or OctdiSIP were
prepared using a double emulsion/solvent evaporation
technique. The average particle sizes of octreotide and OctdiSIP
loaded microspheres were 23.5 ± 0.6 μm and 46.9 ± 11.2 μm,
respectively. The OctdiSIP microspheres had a bigger particle
size probably due to the addition of 50% DMF to the OctdiSIP
solution to increase the solubility of the peptide. The loading
efficiencies were 86.5 ± 2.4% and 61.3 ± 6.7% and loading
capacities were 3.9 ± 0.1% and 3.0 ± 0.3% for octreotide and
OctdiSIP, respectively (n = 3).
spheres, the peptide and its degradation products were
extracted from the microspheres at different time points of
incubation in PBS buffer pH 7.4 at 37 °C. The extracted
samples were analyzed by UPLC and MS and compared with
peptides that were extracted from microspheres containing
unprotected octreotide. Figure 4A,B shows the chromatograms
of extracted peptides that were still present in the PLGA
microspheres after 50 days, for microspheres loaded with native
and protected peptide, respectively. Figure 4A shows a main
peak eluting after approximately 2.6 min which corresponds to
native octreotide (m/z 1020), while extra peaks with longer
retention times emerged that were attributed by MS analysis to
acylated octreotide (glycolyl and lactyl adducts or both, mainly
glycol duct at m/z 1078). Figure 4B shows the main peak
eluting after approximately 5 min that corresponds to
remaining OctdiSIP (m/z 1404), while some conversion to
OctmonoSIP (around 4 min, m/z 1212) and native octreotide
(2.6 min, m/z 1020) had occurred. As opposed to unprotected
46
Stability of OctdiSIP in the PLGA Microspheres. To
determine the stability of OctdiSIP inside the PLGA micro-
F
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Article
octreotide, only some small peaks related to acylation (i.e.,
glycolyl and lactyl adducts) with retention times of 3 min
protected peptide against acylation once loaded in PLGA
microspheres was investigated. The kinetics of OctdiSIP
deprotection was investigated in buffer/acetonitrile mixtures
of different pH and in the presence of an esterase. The
protecting group was stable at the acidic pH inside degrading
microspheres and decomposed to yield octreotide at physio-
logical pH. The protecting group was able to significantly
inhibit the formation of lactyl and glycolyl adducts of octreotide
in PLGA microspheres, as compared to unprotected octreotide
in PLGA. The formation of unexpected acetylated byproducts
was reduced by the action of an esterase, which triggered the
elimination of the protecting groups. The self-immolative
protecting group presented here was successfully designed and
can be a versatile and generally applicable approach to inhibit
the interaction and subsequent acylation of amines of peptides
and proteins in polyester-based controlled release systems. In
vivo release, conversion, and pharmacokinetics of OctdiSIP
from PLGA microspheres should be studied further.
(
majority glycolyl octreotide, m/z 1078) and 4.3 min (glycolyl
OctmonoSIP, m/z 1270) were observed. Importantly, no
acetylated compounds (like those that emerged in the
hydrolysis study presented above) could be detected.
Figure 5 shows the relative peak areas of the peptides that
were extracted from degrading PLGA microspheres loaded with
MATERIALS AND METHODS
■
Chemicals. All materials were purchased from Sigma-
Aldrich (Zwijndrecht, The Netherlands) unless stated other-
wise and used without further purification. Octreotide acetate
(
H N-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, molecular
2
weight of 1019.2 Da) was obtained from Biorbyt (USA). 4-
Hydroxybenzylalcohol was obtained from Aldrich (Switzer-
land), 4-nitrophenyl chloroformate was purchased from Sigma
(
China), and triethylamine from Sigma-Aldrich (Belgium).
Poly(vinyl alcohol) (PVA; MW 30 000−70 000; 88% hydro-
lyzed) and esterase from porcine liver were obtained from
Sigma-Aldrich (USA). Disodium hydrogen phosphate dihy-
drate (Na HPO ·2H O) and sodium dihydrogen phosphate
2
4
2
monohydrate (NaH PO ·H O) were obtained from Merck.
2
4
2
Sodium azide (NaN , Bio Ultra, ≥99.5%) was purchased from
3
Sigma (Germany). HPLC and MS grade acetonitrile (ACN),
peptide grade dichloromethane (DCM), dimethylformamide
(
DMF), tetrahydrofuran (THF), and ethyl acetate were
Figure 5. Change of relative peptide content in time, of PLGA
microspheres loaded with (A) octreotide and (B) OctdiSIP, during
incubation in PBS 7.4 at 37 °C. The contents are expressed as relative
peak areas from UPLC analysis.
purchased from Biosolve (The Netherlands). PLGA (acid
terminated 5004A with D,L-lactide/glycolide molar ratio of
5
0:50, IV = 0.4 dL/g) was obtained from Purac.
Synthesis of Self-Immolative Protecting Group.
Scheme 2 shows the pathway of the synthesis of the protecting
OctdiSIP, at different time points. The peptides that remained
in the PLGA particles mostly consisted of the fully protected
form (76.4% after 50 days) and some monoprotected
degradation products (13.5% after 50 days). Native and
acylated octreotide were only found back in minor amounts
group. The chemical structures of intermediate and final
1
13
compounds were confirmed by H NMR (400 MHz) and
C
NMR (100 MHz) analysis measured on an Agilent 400-MR
NMR spectrometer (Agilent Technologies, Santa Clara, USA).
4-Acetoxybenzyl alcohol (3). 4-Hydroxybenzyl alcohol 2
(0.62 g, 5 mmol) and triethylamine (0.4 mL, 3 mmol, 0.6
equiv) were dissolved in THF (13 mL) which was dried over 3
Å molecular sieve, and purged with dry nitrogen for 15 min.
The solution was cooled on ice and acetic acid anhydride 1
(0.57 mL, 6 mmol, 1.2 equiv) followed by triethylamine (0.4
mL, 3 mmol, 0.6 equiv) were added dropwise during 3 min.
The mixture was stirred at 0 °C under nitrogen atmosphere
until the spot of 4-hydroxybenzyl alcohol could not be detected
on TLC (∼30 min). The solvent was evaporated and the
residue was redissolved in ethyl acetate (50 mL). The organic
layer was washed with brine three times and dried over MgSO4,
filtered, and evaporated to dryness. The residue was purified by
silica gel chromatography, using ethyl acetate/n-hexane (9:1 v/
(at day 50:4.9% native octreotide, 1.5% acyl (mostly glycolyl)
adducts of octreotide, and 3.5% are acyl (mostly glycolyl)
adducts of OctmonoSIP). Importantly, the total amount of
acylated products was significantly lower in the microspheres
loaded with the protected peptide as compared to the
unprotected peptide (5.0% and 52.5%, respectively). Therefore,
it can be anticipated accordingly that the released peptide from
the OctdiSIP loaded microspheres will display a lower degree of
acylation as well. The released protected octreotide will rapidly
be converted in vivo by esterase to form native octreotide
exclusively according to the previous experiments.
CONCLUSION
■
Octreotide was successfully protected by a self-immolative
group at the two amine functionalities that can undergo
acylation side reactions in PLGA, and resistance of the
v) as eluent. Alcohol 3 was a faint yellow oil and was obtained
1
in 52% yield. H NMR (CD CN): δ = 2.28 (s, 3H, OC(O)-
3
CH ), 4.61 (s, 2H, CH O), 7.09 (d, 2H, -C H ), 7.41 (d, 2H,
3
2
6
4
G
DOI: 10.1021/acs.bioconjchem.5b00598
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
-
(
2
C H ) (Figure S1, Supporting Information); 13C NMR
To evaluate the instability of the protecting group in the
presence of an esterase, OctdiSIP was incubated at pH 7.4 with
porcine liver esterase. In detail, 4.2 μL from a stock solution of
1 mg/mL of porcine esterase in PBS 7.4 was added to 1 mL of
a solution of 15 μg/mL of OctdiSIP in PBS pH 7.4 that also
contained 3% acetonitrile to solubilize the peptide. After 1 min
of incubation at 37 °C the solution was analyzed by UPLC
measurement. Prior to investigation of the esterase-catalyzed
conversion of OctdiSIP, the enzymatic activity of porcine liver
esterase in PBS buffer and in the presence of 3% acetonitrile
was determined by measuring the enzyme catalyzed hydrolysis
of p-nitrophenyl acetate (pNPA) to p-nitrophenol (pNP),
which has an absorption maximum about 405 nm, using a
Shimadzu UV-2450 spectrophotometer (Shimadzu Corpora-
tion, Kyoto, Japan). Enzyme assays were carried out in 100 mM
PBS pH = 7.4 at 37 °C. A pNP dilution series was used for
calibration. One unit of the enzyme is defined as the amount of
6
4
CD CN): δ = 169.77, 149.94, 139.69, 127.81, 121.64, 63.24,
3
0.33 (Figure S2).
O-4-Nitrophenyl-O′-4-acetoxybenzyl carbonate (5). 4-
Nitrophenyl chloroformate 4 (1.1 g, 5.5 mmol, 1.1 equiv)
dissolved in 3 mL dry acetonitril was added dropwise during 20
min to a stirred solution of 4-acetoxybenzyl alcohol 3 (0.83 g, 5
mmol) and triethylamine (0.8 mL, 6 mmol, 1.2 equiv) in
anhydrous acetonitrile (10 mL). The obtained reaction mixture
was stirred at room temperature under nitrogen atmosphere for
1
h, during which triethylamine hydrochloride precipitated
from the reaction mixture. Subsequently, ethyl acetate (50 mL)
was added and the obtained solution was washed with cold
water (3 × 15 mL). The organic layer was dried over anhydrous
MgSO , filtered, and evaporated to yield the crude product as a
white solid, which was recrystallized from ethyl acetate/
petroleum ether in 60% yield. Melting point was determined
at 114.7 °C using differential scanning calorimetry (DSC)
Q2000 apparatus (TA Instruments, New Castle, DE, USA),
4
47
enzyme that releases 1 μmol p-nitrophenol per min in pH 7.4 at
48
3
7 °C.
1
(
Figure S5). H NMR (CD CN): δ = 2.27 (s, 3H, OC(O)-
3
Preparation and Characterization of PLGA Micro-
CH ), 5.30 (s, 2H, -CH OC(O)O), 7.16 (d, 2H, -C H OAc),
3
2
6
4
spheres Loaded with OctdiSIP or Octreotide. PLGA
microspheres loaded with octreotide or OctdiSIP were prepared
by a double emulsion (W/O/W) solvent evaporation
7
.46 (d, 2H, -C H OAc), 7.51 (d, 2H, -C H NO ), 8.30 (d, 2H,
6 4 6 4 2
1
3
-
C H NO ) (Figure S3); C NMR (CD CN): δ = 169.49,
6 4 2 3
1
7
55.66, 152.46, 151.25, 145.65, 132.54, 129.84, 125.30, 122.26,
0.00, 20.25 (Figure S4).
Conjugation of the Protecting Group (Compound 5)
18
technique. Briefly, 10 mg of an octreotide solution in 50 μL
Milli-Q water was emulsified in 500 μL of dichloromethane
(
DCM) solution of PLGA (220 mg, 25% w/w) by using an
to Octreotide. A solution of compound 5 (16 mg, 0.05 mmol
in 0.5 mL DMF) was added dropwise to a mixture of octreotide
(
IKA homogenizer (IKA Labortechnik Staufen, Germany) for 30
s at the highest speed (30 000 rpm) to get the primary
emulsion. In the case of OctdiSIP, which has poor solubility in
water due to the hydrophobic protecting groups, 10 mg of
OctdiSIP was dissolved in 50 μL of 50:50 (v/v) water/DMF
prior to emulsification in the organic solution. Next, 500 μL of
a PVA solution (1% w/w in 30 mM phosphate buffer, pH 7.4)
was added slowly, and the mixture was vortexed for 30 s at
20 mg, 0.02 mmol) and triethylamine (0.015 mL, 0.11 mmol)
in DMF (0.5 mL) during 10 min. The obtained reaction
mixture was stirred at room temperature for 24 h. During this
period of stirring, the reaction mixture turned yellow due to
formation of 4-nitrophenol. Preparative HPLC (Akta purifier)
was used for purification of the desired product. Separation was
carried out on a C-18 column (19 × 150 mm, 5 μm, Sunfire). A
linear gradient elution was run from 30% to 60% (v/v)
acetonitrile in water, each containing 0.1% (v/v) TFA, for 27
min. The flow rate was 10 mL/min, and UV detection at λ 280
nm was used for monitoring the separation. The collected
fractions were identified by direct infusion into an ion-trap mass
spectrometer (Agilent 1100 Series LC/MSD SL) equipped
with an electrospray ionization source (Agilent Technologies)
using a syringe pump of Cole-Parmer (Vernon Hill, IL, USA).
The fraction of interest, i.e., octreotide with two self-immolative
protecting (SIP) groups (OctdiSIP), was collected, freeze-dried,
and molecular weight measured by mass spectrometry (Figure
S6B). The yield was 21%. The purity of the obtained OctdiSIP
was calculated by UPLC analysis from the areas under the curve
of the UPLC chromatogram (Figure S6A).
Hydrolytic and Enzymatic Deprotection of OctdiSIP.
The hydrolytic deprotection of OctdiSIP was studied in
aqueous solutions of pH 5.0, 7.4, and 9.0 using acetate buffer
for pH 5.0 and phosphate buffer for pH 7.4 and 9.0. The buffer
concentration was 100 mM and the ionic strength was adjusted
to 0.3 with sodium chloride. A solution of OctdiSIP (75 μg/
mL) was prepared in buffer/acetonitrile 2:1 (v/v), divided in
different Eppendorf tubes, and incubated at 37 °C with mild
shaking. At selected time intervals, samples were taken and
stored in a freezer at −20 °C in order to prevent further
hydrolysis prior to analysis with UPLC. The degradation rate
constant kobs was determined from the slope of the plot of the
natural logarithm of the residual OctdiSIP concentration versus
time.
3
0 000 rpm. The emulsion was transferred into an external
aqueous solution (5 mL) containing PVA 0.5% (w/w) in 30
mM phosphate buffer pH 7.4 while stirring. Continuous stirring
at room temperature for 2 h resulted in the extraction and
evaporation of DCM. Finally, hardened microspheres were
collected by centrifugation (Laboratory centrifuge, 4 K 15
Germany) at 3000 g for 3 min, subsequently washed 3 times
with 100 mL RO water and freeze-dried at −50 °C and at 0.5
mbar in a Chris Alpha 1−2 freeze-dryer (Osterode am Harz,
Germany) for 16 h. The dried microspheres were stored at −25
°
C.
The size of the microspheres and the size distribution were
analyzed by a laser blocking technology (Accusizer 780, Optical
particle sizer, Santa Barbara, California, USA) after dispersing
the freeze-dried microspheres in water.
The octreotide or OctdiSIP loading was determined by
dissolving about 10 mg of microspheres (accurately weighted)
in 2 mL of acetonitrile with gentle shaking. Subsequently, 2 mL
of solution of 0.2% w/v glacial acetic acid, 0.2% w/v sodium
acetate, and 0.7% w/v sodium chloride in water was added to
precipitate the polymer. Next, the mixture was incubated at
room temperature for 20 min, and the precipitated polymer was
spun down by centrifugation at 5000 g for 2 min. The
octreotide or OctdiSIP content in the supernatant was
measured by ultraperformance liquid chromatography
(UPLC, vide infra). Loading efficiency (LE) of the peptide in
the microspheres is reported as the encapsulated peptide
divided by the total amount of peptide used for encapsulation.
H
DOI: 10.1021/acs.bioconjchem.5b00598
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Loading capacity (LC) is defined as the encapsulated amount
of peptide divided by dry weight of the microspheres.
with an acetyl adduct (Δm + 42 Da); UPLCultra performance
liquid chromatography
Peptide Stability Inside PLGA Microspheres. For
stability study of the peptides inside the microspheres, freeze-
dried microspheres (4−10 mg) were suspended in 1.5 mL PBS
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.5b00598.
■
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Corresponding Author
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ABBREVIATIONS
■
polyester microspheres by LC-MS/MS. Pharm. Res. 32, 3044−3054.
OctdiSIPoctreotide with two self-immolative protecting groups;
OctmonoSIPoctreotide with one self-immolative protecting
group; PLGApoly(D,L-lactide-co-glycolide); Acylglycolyl or
lactyl; acylated octreotideoctreotide with a glycolyl or lactyl
adduct (Δm + 58 or +72 Da); acetylated octreotideoctreotide
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E. (2012) Controlled release of octreotide and assessment of peptide
acylation from poly(D,L-lactide-co-hydroxymethyl glycolide) com-
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