Our study clearly demonstrates that the stability of oxytocin in aqueous formulations is greatly increased in citrate buffer in combination with divalent metal ions. The improved stability was found to be dependent on the divalent metal ions concentration.
The WHO reported that there is no loss of potency of oxytocin in injection preparation after 12 months refrigerated storage (2–8°C). However, oxytocin lost 14% of its potency after 1 year at 30°C (12
). In another study, the oxytocin concentration in Ringer's lactate solution was reduced by about 10% after 35 days storage at room temperature (near 23°C) (13
). From our observation Ringer's lactate solution was able to stabilize oxytocin in aqueous solution at low temperature (4°C). However at a higher temperature (55°C), the stability of Ringer's lactate solution was poorer than in the presence of citrate or acetate buffers. This result can be attributed to the pH and/or the amount of metal ions in solution. Oxytocin in Ringer's lactate solution has a pH of 6.4 and contains less than 2 mM of divalent metal ions. Hawe et al
) observed that the degradation of oxytocin strongly depends on the pH of the formulation, with the highest stability at pH 4.5. Therefore, a pH of 6.4 might have caused an increased rate of decomposition. However, formulating oxytocin in acetate buffer at pH 4.5 only maintain approximately 30% oxytocin recovered after 1 month storage at 55°C.
The decomposition of oxytocin is mainly caused by deamidation, oxidation, hydrolysis, and dimerization (7
). Deamidation is likely to occur in Gln4
), and Gly9
). Under acidic condition (pH below 3), deamidation of Asn5
occur by direct hydrolysis (7
). Oxidation might occur at Tyr2
) and Cys1,6
), whereas dimerization might occur due to thiol exchange in Cys1,6
Although the specific mechanism has not been elucidate yet, the presence of sufficient amounts of divalent metal ions at pH 4.5 in citrate buffer, however, greatly improved the stability of oxytocin in aqueous solution. In previous studies, the interaction of oxytocin with calcium (20
) and zinc (21
) ions was investigated using NMR and nanoelectrospray mass spectrometry (MS). Those studies which were also supported with molecular modeling, showed that Ca2+
is coordinated by seven carbonyl oxygen atoms (O-Tyr2
, and O-Gly9
) which formed a more compact structure for the oxytocin–Ca2+
complex compared to free oxytocin (20
). Whereas zinc ions formed an octahedral complex with six of the backbone carbonyl oxygen atoms (O-Tyr2
, and O-Gly9
). It was suggested that in the presence of such divalent ions, the hydrophobic groups are situated inside the peptide keeping them away from water molecules. These conformational changes can increase the stability of oxytocin in aqueous medium as they will prevent dimerization and further aggregation by hydrophobic interactions among oxytocin molecules. Metal salts are often used to stabilize peptides or proteins by chelation or ionic interactions (22
). Wang et al
. examined the peptide (P66) stability in the presence of ZnCl2
, and CaCl2
in non aqueous solution, and found that in the presence of 1 mM ZnCl2
, P66 was significantly stabilized. However in the aqueous solution (pure water), these ions did not show any stabilizing effect (22
). In our experiments, the addition of calcium, magnesium, and zinc ions in combination with citrate buffer had a large impact on oxytocin stability in contrast to similar experiments in pure water or acetate buffer. This study suggests that there is a synergistic effect between citrate buffer and the divalent metal ions, possibly due to the protection of the disulfide bridge by complex formation of divalent metal ion and citrate with oxytocin which suppressed intermolecular reaction leading to tri/tetrasulfide formation as well as dimerization (unpublished data).
ITC is a sensitive method for studying the thermodynamics of binding events and quantifying binding reactions. When divalent metal ion are added to oxytocin, the ITC data indicate an interaction between oxytocin and Ca2+
, or Zn2+
ions in the presence of citrate buffer. Both Mg2+
ions demonstrated complex, dual-phase interaction profile, while a single phase was observed for Ca2+
. Each interaction was entropy driven, while both exothermic and endothermic reactions were observed. It may be speculated that the solvation effect, i.e.
, release of structured water molecules plays a key role in binding, while the specific ion–oxytocin interaction further contributes to the complex stability. The latter is also predicted by molecular dynamic simulations (20
). Remarkably, no interaction between oxytocin and either of the tested ions was detected in the acetate buffer or deionized water. These observations underscore the role of a particular environment in the ion–oxytocin interaction and agree well with our findings on the peptide stability. Isothermal titration calorimetric measurements were predictive for the effects observed during the stability study.
In conclusion, this study shows that with a combination of divalent metal salts and citrate buffer, the stability of oxytocin in aqueous solution can be strongly improved. The increased stability of oxytocin aqueous formulations was achieved in the presence of citrate acid buffer and 2 mM or more of the salts CaCl2, MgCl2, or ZnCl2. The oxytocin stability is further increased with increasing concentration of the divalent metals ions up to 50 mM. In combination with citrate buffer, Zn2+ has a superior stabilizing effect as compared with Ca2+ or Mg2+.