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Purpose: To investigate the stability of KSL, an antimicrobial decapeptide, and its analogues, in human saliva and simulated gastric fluid for delivery in the oral cavity., Materials and Methods: The degradation products of KSL in human saliva and simulated gastric fluid were separated by reversed-phase HPLC and their structures were identified by electrospray ionization-mass spectrometry. Analogues of KSL were synthesized by solid-phase synthesis procedure. Their enzymatic stabilities and antimicrobial activities were studied., Results: KSL was degraded by the peptide bond cleavages at Lys(6)-Val(7) in the human saliva and Phe(5)-Lys(6) in simulated gastric fluids. Three analogues of KSL were synthesized; the Lys(6) residue was either methylated (KSL-M), or replaced with Trp (KSL-W), or the d-form of Lys (KSL-D). The KSL analogues were much more stable than the native KSL, with the rank order of stability being KSL-D > KSL-W > KSL-M > KSL in human saliva. However, in simulated gastric fluid, while KSL-D was still stable, KSL-W was significantly degraded. In addition, KSL-D significantly lost the antimicrobial activity, whereas KSL-W completely preserved the activity against several oral bacteria. In a chewing gum formulation, KSL-W showed a more sustained release profile as compared with the native KSL., Conclusion: This study suggests that KSL-W could be used as an antiplaque agent in a chewing gum formulation.

Purpose: : To determine the optimal polyethylene glycol (PEG)-conjugate of octreotide by evaluating the effects of PEGylation chemistry on the biological activity and pharmacokinetic properties., Methods: : Octreotide was chemically modified by reaction with succinimidyl propionate monomethoxy PEG (SPA-mPEG, molecular weight 2000) or succinimidyl butyraldehyde-mPEG (ALD-mPEG, molecular weight 2000 and 5000). The structural conformation of PEG-octreotides was evaluated by circular dichroism (CD), the biological activity was assessed by measuring the decrease of serum insulin-like growth factor-I levels in rats, and a pharmacokinetic study was performed after subcutaneous administration in rats. The stability against acylation was investigated with poly(D,L -lactide-co-glycolide) (PLGA)., Results: : ALD-mPEG was site-specific in PEGylating octreotide at the N-terminus. The mono-PEG-octreotides prepared with ALD-mPEG (mono-ALDPEG-octreotide), which alkyl bond preserves the amine's positive charge, showed complete preservation of biological activity, whereas the PEG-octreotides prepared with SPA-mPEG showed lower activity. In the CD analysis, the spectra of the mono-ALDPEG-octreotides were nearly superimposable with that of native octreotide. The mono-ALDPEG-5K-octreotide showed significantly improved pharmacokinetic properties compared with mono-ALDPEG-2K-octreotide as well as native octreotide. Both mono-ALDPEG-2K- and mono-ALDPEG-5K-octreotides were stable against acylation by degrading PLGA., Conclusions: : The mono-PEGylation of octreotide at N-terminus with ALD-mPEG produced a conjugate that is biologically and structurally active and stable against acylation by PLGA, and therefore it may serve as a candidate for somatostatin microsphere formulations.

Purpose: To investigate the mechanism by which polyethylene glycol (PEG) conjugation (PEGylation) prevents the acylation of octreotide by poly(d,l-lactide-co-glycolide) (PLGA)., Methods: Octreotide was chemically modified by reaction with succinimidyl propionate-monomethoxy PEG. Each PEGylated octreotide species with different PEG number and modified position was separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with endoproteinase Lys-C digestion. Acylation of octreotide and PEGylated octreotides was observed with hydrophobic and hydrophilic PLGA., Results: Two mono- and one di-PEGylated octreotides were separated by RP-HPLC. MALDI-TOF MS of the PEGylated products after Lys-C digestion at different pH revealed that the two mono-PEGylated octreotides were modified at the N-terminus and Lys(5) residue, respectively. The interaction of octreotide with PLGA involved an initial adsorption followed by acylation and the subsequent release of octreotide and acylated octreotide. The initial adsorption of octreotide was dependent on the acidity of PLGA. PEGylation of octreotide significantly inhibited the initial adsorption and acylation by PLGA. In particular, the acylation could be completely prevented by mono-PEGylation at the N-terminus of octreotide., Conclusions: This study shows that the N-terminus of octreotide is the preferred PEGylation site to prevent acylation in degrading PLGA microspheres. The mono-N-terminally PEGylated octreotide may possibly serve as a new source for somatostatin microsphere formulation.

Purpose: To separate and characterize the different positional isomers of mono-PEGylated salmon calcitonins (mono-PEG-sCTs) and to evaluate the effects of the PEGylation site on the stability of different mono-PEG-sCTs in rat kidney homogenate., Methods: Mono-PEG-sCTs were prepared using succinimidyl carbonate monomethoxy polyethylene glycol (5,000 Da) and separated by gel-filtration HPLC followed by reversed-phase HPLC. To characterize PEGylated sCTs, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and reversed-phase HPLC of the trypsin digested samples were performed. Mono-PEG-sCTs and sCT in rat kidney homogenates were measured by column-switching reversed-phase HPLC with on-line detection of the radioiodinated samples using a flow-through radioisotope detector., Results: Three different mono-PEGylated sCTs were separated by reversed-phase gradient HPLC. From the MALDI-TOF MS analysis, the average molecular weight of mono-PEG-sCTs was confirmed as around 8650 Da. The presence of PEG moiety in the mono-PEG-sCTs was also manifested by the fact that the distance between two adjacent mass spectum lines was 44 Da which corresponds to PEG monomer unit. Tryptic digestion analysis demonstrated that these mono-PEG-sCTs are 3 positional isomers of N-terminus, Lys18- and Lys11-residue modified mono-PEGylated sCTs. The degradation half-life of these 3 positional isomers in rat kidney homogenates significantly increased in order of the N-terminus (125.5 min), Lys11- (157.3 min), and Lys18 residue modified mono-PEGylated sCT (281.5 min) over the native sCT (4.8 min)., Conclusion: Three positional isomers of mono-PEGylated sCTs were purified and characterized. Of these, the resistance to proteolytic degradation was highest for the Lys18-residue modified mono-PEG-sCT. These studies demonstrate that the in vivo stability of PEGylated sCTs is highly dependent on the site of PEG molecule attachment.