Stability for 6 months and accuracy of a specific enteral caffeine base preparation for a multisite clinical trial.

The 'Intermittent Hypoxia and Caffeine in Infants Born Preterm (ICAF)' study (NCT03321734) uses an extemporaneously compounded enteral caffeine base solution for its study drug. The primary aim of this report is to determine the stability of this specific enteral caffeine base preparation stored for up to 6 months and assess optimal storage temperature. To analyse stability, caffeine solutions were prepared and stored at 4°C and 25°C (room temperature). The caffeine concentrations were analysed over time using high-performance liquid chromatography (HPLC). To confirm the accuracy of compounded caffeine concentrations, study drug samples from three research pharmacies were analysed. Stability results showed that caffeine concentrations are within 5% of the expected concentration when stored for up to 6 months at room temperature. Our results also show that accurate caffeine concentrations were achieved by multiple research pharmacies.

Immature control of breathing and apnea of prematurity: the known and unknown.

This narrative review provides a broad perspective on immature control of breathing, which is universal in infants born premature. The degree of immaturity and severity of clinical symptoms are inversely correlated with gestational age. This immaturity presents as prolonged apneas with associated bradycardia or desaturation, or brief respiratory pauses, periodic breathing, and intermittent hypoxia. These manifestations are encompassed within the clinical diagnosis of apnea of prematurity, but there is no consensus on minimum criteria required for diagnosis. Common treatment strategies include caffeine and noninvasive respiratory support, but other therapies have also been advocated with varying effectiveness. There is considerable variability in when and how to initiate and discontinue treatment. There are significant knowledge gaps regarding effective strategies to quantify the severity of clinical manifestations of immature breathing, which prevent us from better understanding the long-term potential adverse outcomes, including neurodevelopment and sudden unexpected infant death.

Colloidal calcium phosphate in the reconstituted milk micelle may direct wild-type recombinant human beta-casein to fold like the native protein.

Native human beta-casein (CN) at all phosphorylation levels exhibits reproducible behavior and appears to have a unique, stable folding pattern. In contrast, the recombinant non-phosphorylated form of human beta-CN (beta-CN-0P) with the exact amino acid sequence (wild-type), expressed and purified from Escherichia coli, differs greatly in its behavior from the native protein and the complexes formed are unstable to thermal cycling. However, when it was incorporated into reconstituted milk micelles, using bovine kappa-CN at a kappa/beta molar ratio of 1/3 with added Ca2+ ions and inorganic phosphate (P(i)) at levels that would ordinarily precipitate, its association behavior vs. temperature as monitored by turbidity (OD(400 nm)) approximated that of native beta-CN-0P. This suggests that the milk micelle system, and particularly the colloidal calcium phosphate, may act as a 'molecular chaperon' to direct the folding of the molecule into the highly stable conformation found in the purified native human beta-CN molecule.

The formation of casein micelles reconstituted with Ca+2 and added inorganic phosphate is influenced by the non-phosphorylated form of human beta-casein.

The beta-casein (CN) human milk fraction is comprised of a single protein phosphorylated at levels from 0 to 5. Component interactions are dependent on the phosphorylation level. Here, 3 mg/ml of beta-CN-0P, beta-CN-2P, beta-CN-4P, a 2P/4P 1:1 (wt:wt) mixture, or a mixture of all six forms in the ratio in human milk, were mixed with bovine kappa-CN at a kappa/beta molar ratio of 0.33. Measurements were with 0, 5 and 10 mM Ca+2 and 4 and 8 mM added inorganic phosphate (Pi). The turbidity (OD400 nm) and a lack of precipitation as T increased from 4 to 37 degrees C was an index of micelle formation. The results indicate: (1) while micelles will form with Ca+2 alone, added Pi has a significant enhancing effect on micelle formation; (2) the patterns of micelle formation as a function of T are influenced by the beta-CN-0P and beta-CN-1P forms of beta-CN to an unexpected extent.