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Identification and Assessment of Octreotide Acylation in Polyester Microspheres by LC-MS/MS.

Shirangi M, Hennink WE, Somsen GW, van Nostrum CF - Pharm. Res. (2015)

Bottom Line: Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres.Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

ABSTRACT

Purpose: Polyesters with hydrophilic domains, i.e., poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and a multiblock copolymer of poly(ε-caprolactone)-PEG-poly(ε-caprolactone) and poly(L-lactide) ((PC-PEG-PC)-(PL)) are expected to cause less acylation of encapsulated peptides than fully hydrophobic matrices. Our purpose is to assess the extent and sites of acylation of octreotide loaded in microspheres using tandem mass spectrometry analysis.

Methods: Octreotide loaded microspheres were prepared by a double emulsion solvent evaporation technique. Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres. To scrutinize the structural information and localize the actual modification site(s) of octreotide, liquid chromatography ion-trap mass spectrometry (LC-ITMS) was performed on the acylated adducts.

Results: Hydrophilic microspheres showed less acylated adducts in comparison with PLGA microspheres. LC-MS/MS showed that besides the N-terminus and primary amine of lysine, the primary hydroxyl of the end group of octreotide was also subjected to acylation. Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.

Conclusions: Hydrophilic polyesters are promising systems for controlled release of peptide because substantially less acylation occurs in microspheres based on these polymers. LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

No MeSH data available.


In vitro release of octreotide from (a) PLGA, (b) PLGHMGA, (c) PLHMGA and (d) (PC-PEG-PC)-(PL) microspheres in PBS pH 7.4: native octreotide (squares), acylated octreotide (triangles) and total octreotide (sum of native and acylated adducts, circles).
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Fig5: In vitro release of octreotide from (a) PLGA, (b) PLGHMGA, (c) PLHMGA and (d) (PC-PEG-PC)-(PL) microspheres in PBS pH 7.4: native octreotide (squares), acylated octreotide (triangles) and total octreotide (sum of native and acylated adducts, circles).

Mentions: Figure 5 shows the cumulative release of octreotide from the different microspheres in PBS pH 7.4 at 37°C. These graphs show that acylation of octreotide is significantly higher when released from PLGA microspheres than of the others. Peptide release from PLGA microspheres started after 15–20 days without burst release and continued for the next 70 days until a plateau at 80% of the loaded amount was reached. UPLC analysis showed that only 31% of the released peptide was in its native form, while 69% was acylated (assuming the same UV-absorbance response for native and acetylated product). PLGHMGA microspheres showed after a burst release of 10% consisting of only native peptide, a phase of low release of about 25 days (~10% of the loading was released). Faster release of octreotide (both native and acylated) started at day 25 reaching 90% of the loading at day 60. UPLC analysis showed that 72% of the released octreotide was in its native form while only 28% was acylated. For PLHMGA microspheres, in line with the result of PLGHMGA, initially only native octreotide was released, and both native and acylated peptide started to be released after day 25. Finally, around 78% of the released peptide was native octreotide, whereas 22% was acylated adducts. For (PC-PEG-PC)-(PL) microspheres, the release was faster than from the other formulations and it seems that the release is governed by diffusion rather than polymer degradation (26). This is understandable because PEG increases the hydrophilicity of the polymer matrix which in turn results in water absorption allowing diffusion of peptide through the (channels or pores of) hydrated particles. The (PC-PEG-PC)-(PL) microspheres particles released the peptide in a continuous manner for 30 days and >90% of the loading was released at that time point. UPLC analysis showed that 75% of the released octreotide was in its native form while 25% was acylated. Figure 4d also shows that both native and acylated peptides were released from the start of the experiment. Although the presence of polymer degradation products can catalyze peptide acylation, the role of water should be considered as well. Liang et al. showed that a bell shape relation exists between the water content and the extent of acylation of exenatide (a polypeptide drug with a molecular weight of 4200 Da, which is clinically used as an adjunct for glycemic control in type 2 diabetes) when drug-loaded PLGA microspheres were incubated at different relative humidity. They showed that acylation kinetics depends on the water content of the microspheres. At low water contents, water acts as plasticizer resulting in more acylation of the unfolded peptide (unfolding occurs due to the hydrophobic polymer matrix). According to the authors, at higher water contents, bulk water present in the matrix will cause conformation recovery of the peptide resulting in a state in which it is less susceptible for acylation reaction (31). The presence of PEG in (PC-PEG-PC)-(PL) microspheres will result in rapid hydration of the particles during the initial stage of the incubation in buffer and facilitate acylation of octreotide. This explains that already at early time points of release acylated octreotide adducts were detected (Fig. 5d). However at later time points the ratio between native and acylated octreotide did not change suggesting that acylation occurs only during the initial stages likely because in later stages the water content of the micrsopheres became so high that acylation is prevented. Further the high water content facilitates release of the polymer degradation products.Fig. 5


Identification and Assessment of Octreotide Acylation in Polyester Microspheres by LC-MS/MS.

Shirangi M, Hennink WE, Somsen GW, van Nostrum CF - Pharm. Res. (2015)

In vitro release of octreotide from (a) PLGA, (b) PLGHMGA, (c) PLHMGA and (d) (PC-PEG-PC)-(PL) microspheres in PBS pH 7.4: native octreotide (squares), acylated octreotide (triangles) and total octreotide (sum of native and acylated adducts, circles).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4526596&req=5

Fig5: In vitro release of octreotide from (a) PLGA, (b) PLGHMGA, (c) PLHMGA and (d) (PC-PEG-PC)-(PL) microspheres in PBS pH 7.4: native octreotide (squares), acylated octreotide (triangles) and total octreotide (sum of native and acylated adducts, circles).
Mentions: Figure 5 shows the cumulative release of octreotide from the different microspheres in PBS pH 7.4 at 37°C. These graphs show that acylation of octreotide is significantly higher when released from PLGA microspheres than of the others. Peptide release from PLGA microspheres started after 15–20 days without burst release and continued for the next 70 days until a plateau at 80% of the loaded amount was reached. UPLC analysis showed that only 31% of the released peptide was in its native form, while 69% was acylated (assuming the same UV-absorbance response for native and acetylated product). PLGHMGA microspheres showed after a burst release of 10% consisting of only native peptide, a phase of low release of about 25 days (~10% of the loading was released). Faster release of octreotide (both native and acylated) started at day 25 reaching 90% of the loading at day 60. UPLC analysis showed that 72% of the released octreotide was in its native form while only 28% was acylated. For PLHMGA microspheres, in line with the result of PLGHMGA, initially only native octreotide was released, and both native and acylated peptide started to be released after day 25. Finally, around 78% of the released peptide was native octreotide, whereas 22% was acylated adducts. For (PC-PEG-PC)-(PL) microspheres, the release was faster than from the other formulations and it seems that the release is governed by diffusion rather than polymer degradation (26). This is understandable because PEG increases the hydrophilicity of the polymer matrix which in turn results in water absorption allowing diffusion of peptide through the (channels or pores of) hydrated particles. The (PC-PEG-PC)-(PL) microspheres particles released the peptide in a continuous manner for 30 days and >90% of the loading was released at that time point. UPLC analysis showed that 75% of the released octreotide was in its native form while 25% was acylated. Figure 4d also shows that both native and acylated peptides were released from the start of the experiment. Although the presence of polymer degradation products can catalyze peptide acylation, the role of water should be considered as well. Liang et al. showed that a bell shape relation exists between the water content and the extent of acylation of exenatide (a polypeptide drug with a molecular weight of 4200 Da, which is clinically used as an adjunct for glycemic control in type 2 diabetes) when drug-loaded PLGA microspheres were incubated at different relative humidity. They showed that acylation kinetics depends on the water content of the microspheres. At low water contents, water acts as plasticizer resulting in more acylation of the unfolded peptide (unfolding occurs due to the hydrophobic polymer matrix). According to the authors, at higher water contents, bulk water present in the matrix will cause conformation recovery of the peptide resulting in a state in which it is less susceptible for acylation reaction (31). The presence of PEG in (PC-PEG-PC)-(PL) microspheres will result in rapid hydration of the particles during the initial stage of the incubation in buffer and facilitate acylation of octreotide. This explains that already at early time points of release acylated octreotide adducts were detected (Fig. 5d). However at later time points the ratio between native and acylated octreotide did not change suggesting that acylation occurs only during the initial stages likely because in later stages the water content of the micrsopheres became so high that acylation is prevented. Further the high water content facilitates release of the polymer degradation products.Fig. 5

Bottom Line: Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres.Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

ABSTRACT

Purpose: Polyesters with hydrophilic domains, i.e., poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and a multiblock copolymer of poly(ε-caprolactone)-PEG-poly(ε-caprolactone) and poly(L-lactide) ((PC-PEG-PC)-(PL)) are expected to cause less acylation of encapsulated peptides than fully hydrophobic matrices. Our purpose is to assess the extent and sites of acylation of octreotide loaded in microspheres using tandem mass spectrometry analysis.

Methods: Octreotide loaded microspheres were prepared by a double emulsion solvent evaporation technique. Release profiles of octreotide from hydrophilic microspheres were compared with that of PLGA microspheres. To scrutinize the structural information and localize the actual modification site(s) of octreotide, liquid chromatography ion-trap mass spectrometry (LC-ITMS) was performed on the acylated adducts.

Results: Hydrophilic microspheres showed less acylated adducts in comparison with PLGA microspheres. LC-MS/MS showed that besides the N-terminus and primary amine of lysine, the primary hydroxyl of the end group of octreotide was also subjected to acylation. Nucleophilic attack of the peptide can also occur to the carbamate bond presented in (PC-PEG-PC)-(PL) since 1,4-butanediisocyanate was used as the chain extender.

Conclusions: Hydrophilic polyesters are promising systems for controlled release of peptide because substantially less acylation occurs in microspheres based on these polymers. LC-ITMS provided detailed structural information of octreotide modifications via mass analysis of ion fragments.

No MeSH data available.