Therapeutic Activity of Peg Albumin in Transgenic Mouse Model of Sickle Cell Disease Is a Function of the Pattern of Pegylation

Background Conjugation of Albumin (Alb) with six copies of PEG 5K chains using extension arm chemistry (EAF) generates a novel semisynthetic supra perfusion resuscitation fluid (EAF P5K6 Alb) that performs better than the conventional colloidal plasma expanders in extreme hemodilution and hemorrhagic shock animal models. The superior activity of EAF P5K6 Alb is attributed to its property of increasing functional capillary density. This product has been shown to increase the efficacy of blood transfusion when given in combination with blood. The influence of pattern of surface decoration on SCD therapeutic activity of PEG Alb with one copy of PEG 30K (P30K1Alb) and six copies of PEG5K (EAF P5K6 Alb) has been investigated in the chronic mouse model of SCD, BERK low ϒ. Two important questions posed here are: 1) Does the pattern of PEGylation influence the increase in hydrodynamic volume and packing density of PEG shell in the PEG30KAlb adducts and 2) Does the P30K1Alb and EAF P5K6 Alb have the similar SCD therapeutic activity. Methods P30K1 Alb was prepared by PEGylation at Cys34 of Alb using maleimidopropionamido PEG30K followed by ion exchange chromatography purification. EAF P5K6 Alb was purified by tangential flow filtration method. Intravital microscopy analysis for the assessment of therapeutic effects of both the PEG Alb adducts was examined in the cremaster muscle of transgenic SCD mice. Berk low ϒ mice were infused with 10 % top load of a 4 gm % PEG Alb solutions (either EAF P5K6 Alb or P30K1 Alb) by IP and the attenuation of vaso-occlusion, as well as improvement in blood flow has been compared. Results P30K1 Alb elutes as a very resolved material in ion exchange column and its 1 copy of PEG30K was confirmed by NMR studies. The hydrodynamic volume of P30K1 Alb is higher than EAF P5K6 Alb as reflected by size exclusion chromatography, even though the overall total mass to the Alb is same. This reflects that P30K1 is more flexible than EAF P5K6 Alb. The molecular radius and COP of EAFP5K6Alb is higher whereas viscosity is less than that of P30K1Alb (Table 1). Accordingly, plasma expander like properties of PEG Alb when surface decorated with a given total mass of PEG is a function of pattern of PEGylation. EAF P5K6 Alb exhibited a significant therapeutic activity after 3 hours (hrs) of treatment, as seen by the attenuation of the vaso-occlusion (Fig 1). This effect remained the same after 24 hrs. There was no noticeable change in the blood flow or RBC velocity in spite of significant reduction in leukocyte adhesion. Discussions and Conclusions The correlation of pattern of PEGylation of PEG Alb with the supra perfusion activity of EAF P5K6 Alb has been investigated by Tsai et. al. (unpublished results) by designing EAF P3K10. It was concluded that EAF P5K6 is the better pattern of PEGylation to induce the supra perfusion. Results suggest that the PEGylation induced increase in the hydrodynamic volume under flow is higher with a single copy of PEG30K than 6 copies of PEG 5K. Under flow conditions, PEG 30K and P30K1Alb exhibit comparable hydrodynamic volume, conjugating Alb to PEG 30K has not much influence on the overall hydrodynamic volume of PEGAlb. On the other hand, under static conditions, the packing density of its PEG shell is lower than that of EAF P5K6 Alb. There is significant reduction in adhesion of leucocytes in 3 hrs of treatment with EAF P5K6 Alb as seen in the intravital microscopy studies. However very little effect is seen in terms of changes in the blood flow. After 24 hrs of treatment, there is increase in emigration of leukocytes. Surprisingly, P30K1 has little or no SCD therapeutic activity. The amount of PEG Alb used here is only small (10 %) as compared to about 25 % in hemorrhagic shock models; accordingly, the contribution of viscosity is small, if any. We suggest that the SCD therapeutic activity of EAF P5K6 Alb is primarily related to the "parachute effect" of PEG Alb with multiple copies of PEG chains. We have also shown that EAF hexaPEGylation of Tempol (antioxidant) conjugated Alb, synergistically increased the antioxidant therapeutic activity Alb Tempol in attenuating the experimentally induced vaso-occlusive crisis in a transgenic mouse model of SCD, NY1DD. The P30K1 Alb lacks such a parachute-like effect. These results raise the important question whether the "mushroom" structure of EAF P3K10 Alb may show more SCD therapeutic activity than EAF P5K6 Alb and EAF P3K10 Alb conjugated with antioxidant. Disclosures No relevant conflicts of interest to declare.

Synthesis of Poly(2-methyl-2-oxazoline) Star Polymers with a β-Cyclodextrin Core

Synthesis of star polymers with a β-cyclodextrin (CD) core was undertaken using the arm-first, then the core-first strategy. Cationic ring opening polymerisation (CROP) of 2-methyl-2-oxazoline (MeOx) was first initiated by allyl bromide, and then quenched with heptakis(6-deoxy-6-amino)β-CD in order to get a 7-arm star polymer. Then heptakis(6-deoxy-6-iodo-2,3-di-O-acetyl)β-CD was synthesised in order to get an initiator for the CROP of MeOx. Initiation and propagation kinetic measurements were undertaken and the ratio kp/ki was found to be too high to provide a controlled polymerisation. Using iodine as co-initiator allowed a decrease of the kp/ki ratio that gave better control of the polymerisation. DOSY NMR and viscosity characterisations were undertaken, and both techniques lead to the demonstration of a lower hydrodynamic volume of the star polymers versus the linear counterparts, for compounds of the same molecular weight.