Christina Avanti

A New Strategy To Stabilize Oxytocin in Aqueous Solutions: II. Suppression of Cysteine-Mediated Intermolecular Reactions by a Combination of Divalent Metal Ions and Citrate

A series of studies have been conducted to develop a heat-stable liquid oxytocin formulation. Oxytocin degradation products have been identified including citrate adducts formed in a formulation with citrate buffer. In a more recent study we have found that divalent metal salts in combination with citrate buffer strongly stabilize oxytocin in aqueous solutions (Avanti, C.; et al. AAPS J.2011, 13, 284-290). The aim of the present investigation was to identify various degradation products of oxytocin in citrate-buffered solution after thermal stress at a temperature of 70 °C for 5 days and the changes in degradation pattern in the presence of divalent metal ions. Degradation products of oxytocin in the citrate buffer formulation with and without divalent metal ions were analyzed using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). In the presence of divalent metal ions, almost all degradation products, in particular citrate adduct, tri- and tetrasulfides, and dimers, were greatly reduced in intensity. No significant difference in the stabilizing effect was found among the divalent metal ions Ca(2+), Mg(2+), and Zn(2+). The suppressed degradation products all involve the cysteine residues. We therefore postulate that cysteine-mediated intermolecular reactions are suppressed by complex formation of the divalent metal ion and citrate with oxytocin, thereby inhibiting the formation of citrate adducts and reactions of the cysteine thiol group in oxytocin.

Stability of lysozyme in aqueous extremolyte solutions during heat shock and accelerated thermal conditions

The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70°C), betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55°C), firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account.

The formation of oxytocin dimers is suppressed by the zinc–aspartate–oxytocin complex

The aim of this study was to investigate the effect of divalent metal ions (Ca, Mg(2+) , and Zn(2+) ) on the stability of oxytocin in aspartate buffer (pH 4.5) and to determine their interaction with the peptide in aqueous solution. Reversed-phase high-performance liquid chromatography and high-performance size-exclusion chromatography measurements indicated that after 4 weeks of storage at 55°C, all tested divalent metal ions improved the stability of oxytocin in aspartate-buffered solutions (pH 4.5). However, the stabilizing effects of Zn(2+) were by far superior compared with Ca(2+) and Mg(2+) . Liquid chromatography-tandem mass spectrometry showed that the combination of aspartate and Zn(2+) in particular suppressed the formation of peptide dimers. As shown by isothermal titration calorimetry, Zn(2+) interacted with oxytocin in the presence of aspartate buffer, whereas Ca(2+) or Mg(2+) did not. In conclusion, the stability of oxytocin in the aspartate-buffered solution is strongly improved in the presence of Zn(2+) , and the stabilization effect is correlated with the ability of the divalent metal ions in aspartate buffer to interact with oxytocin. The reported results are discussed in relation to the possible mode of interactions among the peptide, Zn(2+) , and buffer components leading to the observed stabilization effects.

Aspartate buffer and divalent metal ions affect oxytocin in aqueous solution and protect it from degradation

Oxytocin is a peptide drug used to induce labor and prevent bleeding after childbirth. Due to its instability, transport and storage of oxytocin formulations under tropical conditions is problematic. In a previous study, we have found that the stability of oxytocin in aspartate buffered formulation is improved by the addition of divalent metal ions (unpublished results). The stabilizing effect of Zn(2+) was by far superior compared to that of Mg(2+). In addition, it was found that stabilization correlated well with the ability of the divalent metal ions to interact with oxytocin in aspartate buffer. Furthermore, LC-MS (MS) measurements indicated that the combination of aspartate buffer and Zn(2+) in particular suppressed intermolecular degradation reactions near the Cys(1,6) disulfide bridge. These results lead to the hypothesis that in aspartate buffer, Zn(2+) changes the conformation of oxytocin in such a way that the Cys(1,6) disulfide bridge is shielded from its environment thereby suppressing intermolecular reactions involving this region of the molecule. To verify this hypothesis, we investigate here the conformation of oxytocin in aspartate buffer in the presence of Mg(2+) or Zn(2+), using 2D NOESY, TOCSY, (1)H-(13)C HSQC and (1)H-(15)N HSQC NMR spectroscopy. Almost all (1)H, (13)C and (15)N resonances of oxytocin could be assigned using HSQC spectroscopy, without the need for (13)C or (15)N enrichment. (1)H-(13)C and (1)H-(15)N HSQC spectra showed that aspartate buffer alone induces minor changes in oxytocin in D2O, with the largest chemical shift changes observed for Cys(1). Zn(2+) causes more extensive changes in oxytocin in aqueous solution than Mg(2+). Our findings suggest that the carboxylate group of aspartate neutralizes the positive charge of the N-terminus of Cys(1), allowing the interactions with Zn(2+) to become more favorable. These interactions may explain the protection of the disulfide bridge against intermolecular reactions that lead to dimerization.

A Retrospective Surveillance of the Antibiotics Prophylactic Use of Surgical Procedures in Private Hospitals in Indonesia

Background: According to international guidelines, prophylactic antibiotics in elective surgery should be given as a single dose 30 to 60 minutes before the operation is conducted. Postoperative administration of antibiotics should be discontinued 24 hours after surgery to minimize bacterial resistance and to keep control over hospitalization costs. There is a lack of data on the actual antibiotic use around surgical procedures in Indonesia. Objective: This retrospective surveillance study aimed to obtain defined daily doses (DDD) and DDDs per 100 bed days (DDD-100BD) for prophylactically used antibiotics in two private hospitals in Surabaya, East Java. These hospitals are considered to be representative for the current situation in Indonesia. Method: Data from a total of 693 patients over a nearly 1-year period (2016) were collected and evaluated. Results: The overall DDD per patient was 1.5 for hospital A and 1.7 for hospital B. The overall DDD-100BD was 30 for hospital B. Of the 24 antibiotics given prophylactically, ceftriaxone was the most commonly used in both hospitals. Conclusion: There was a clear discrepancy between daily practice in both hospitals and the recommendations in the guidelines. This study shows that better adherence to antibiotic stewardship is needed in Indonesia. Substantial improvements need to be made toward guided precision therapy regarding quantity (dose and frequency), route of administration (prolonged intravenous), and choice of the type of antibiotic.