Polymer conjugation attracts increasing interest in pharmaceutical industry for delivering drugs of simple structure as well as complex compounds such as oligonucleotides, peptides and proteins. However, by far the most active research field is peptide and protein conjugation, mainly because they are therapeutically interesting compounds with very unsatisfactory pharmacokinetics (e.g., low stability, solubility problems, immunogenicity and low residence time in the body).
The objective of this work was to develop a new conjugation technology, which might be able to overcome some of these limitations. This technology uses hydroxyethyl starch, a highly biocompatible semi-synthetic polysaccharide, as a polymeric carrier.
Our experiments demonstrated the feasibility of a covalent coupling between polymer and protein with two different coupling strategies yielding amide (approach A) and amine bonds (approach B), respectively. The protein chosen as a model for the optimisation phase was human serum albumin.
As prerequisite for the desired selective coupling according to approach A, the polysaccharide previously needs to undergo a selective oxidation process. Two different selective oxidation methods were optimised to obtain in high yield one unique carboxylic function per polymer chain. The coupling of the oxidised polymer to HSA according to approach A resulted in yields greater that 90%.
Approach B uses the polymer as such and the coupling exploits a Schiff s base reaction in presence of a selective reducing agent. This strategy gave yields around 40-50%.
Both these reactions were carried out with polymer chains of different size in the range of 10 130 kD.
Besides the chemical feasibility of these new conjugation reactions, it was moreover shown, by using a model enzyme (superoxide dismutase) as target protein, that it is possible to obtain a conjugate which still completely retains the functionality of the original protein after the chemical modification.
Furthermore the conjugates seem to have a much lower reactivity towards antibodies than the original proteins, as shown by a double immunodiffusion testsystem. Probably the polymer chains mask some antigenic epitops by sterically hindering the accessibility of the protein surface.
The coupling technology was furthermore adapted to the conjugation of drugs with low molecular weight, which present administration limits due to their pharmacokinetics. In this case a cleaner coupling strategy was exploited. The selectively oxidised polysaccharide was obtained in the lacton form by simply eliminating a water molecule. The lacton being a reactive form of the carboxylic acid, yielded a stable covalent bond without the need of any other reagent than the polymer and an amino function of the the drug itself.
The conjugation was performed with Amphotericin B, an antifungal drug which is very effective but presents solubility problems that turn out to be the origin of many side effects. The hydroxyethyl starch-Amphotericin B conjugate, besides keeping intact the whole antimycotic potential of the drug, was found to increase the water solubility almost 1000-fold and showed evidences of a better selectivity in distinguishing the pathogen from the host. Moreover, the conjugate showed an unexpected stability in solution. After 90 days at room temperature the antimycotic potential was still the same compared to the original, non-modified drug.
Since this new technology is of considerable commercial interest, the data obtained in this work have lead to two patent applications: WO03074087 Coupling of proteins to a modified polysaccharide;WO03074088 Coupling of low-molecular weight substances to a modified polysaccharide.
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