Jonas Fransson

Solvent Effects on the Solubility and Physical Stability of Human Insulin-Like Growth Factor I

AbstractPurpose. The solubility and physical stability of human Insulin-like Growth Factor I (hIGF-I) were studied in aqueous solutions with different excipients. Methods. The solubility of hIGF-I was determined by UV-absorption and quantification of light blocking particles. The physical stability of hIGF-I was studied with differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Results. Human IGF-I precipitated at low temperature in the presence of 140 mM benzyl alcohol and 145 mM sodium chloride. CD data showed that the tertiary structure of hIGF-I during these conditions was perturbed compared to that in 5 mM phosphate buffer. In the presence of benzyl alcohol 290 mM mannitol stabilized hIGF-I. Sodium chloride or mannitol by themselves had no effect on either the solubility or the tertiary structure. Benzyl alcohol was attracted to hIGF-I, whereas sodium chloride was preferentially excluded. The attraction of benzyl alcohol was reinforced by sodium chloride leading to salting-out of hIGF-I. The CD-data indicated interactions of benzyl alcohol with phenylalanine in hIGF-I. Thermal denaturation of hIGF-I occurred in all solutions with sodium chloride, whereas mannitol or benzyl alcohol had no effect on the thermal stability. The thermal stability of hlGF-I was thus decreased in 145 mM sodium chloride although it was excluded from hIGF-I. Conclusions. The self-association and thermal aggregation of hIGF-I is driven by hydrophobic interactions. Benzyl alcohol is attracted to hIGF-I and induces changes in the tertiary structure causing hydrophobic attraction of the protein at low temperatures.

Injection-site reactions upon Kineret (anakinra) administration: experiences and explanations

Anakinra (Kineret), a recombinant form of human interleukin-1 (IL-1) receptor antagonist, is approved for the treatment of rheumatoid arthritis (RA) in combination with methotrexate. Kineret is self-administered by daily subcutaneous injections in patients with active RA. The mechanism of action of anakinra is to competitively inhibit the local inflammatory effects of IL-1. Kineret is generally safe and well tolerated and the only major treatment-related side effects that appear are skin reactions at the injection site. Due to the relatively short half-life of anakinra, daily injection of the drug is required. This, in combination with the comparably high rates of injection-site reactions (ISRs) associated with the drug, can become a problem for the patient. The present review summarises published data concerning ISRs associated with Kineret and provides some explanations as to their cause. The objective is also to present some clinical experiences of how the ISRs can be managed.

Oxidation of human insulin-like growth factor I in formulation studies, II. Effects of oxygen, visible light, and phosphate on methionine oxidation in aqueous solution and evaluation of possible mechanisms

AbstractPurpose. The oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I) in aqueous solution was studied with respect to oxygen, visible light and sodium phosphate. Methods. Aqueous solutions of hIGF-I were prepared with different amounts of phosphate and dissolved oxygen. The solutions were stored either in darkness or exposed to artificial visible light. The oxidized hIGF-I was quantified by RP-HPLC. A two level full factorial experimental design, with two levels of each of the three factors studied, was used. Results. Oxidation was found to be positively correlated with light, oxygen content and, interestingly, phosphate. The increasing effect of phosphate on the oxidation appears not to originate from metal contaminants. The influence of both oxygen and phosphate increased with time. The pH dependence of oxidation indicated the formation of a phosphorylated sulfonium ion as an oxidation intermediate. A significant interaction effect between phosphate and visible light suggested participation of radicals. Conclusions. Factorial experiments provide a valuable tool when studying complex mechanisms with interacting factors. The oxidation of methionine in hIGF-I is significantly affected by light but also by the presence of phosphate buffer.