Conjugates of cis-4-Hydroxy-L-proline and Poly(PEG-Lys), a Water Soluble Poly(ether urethane): Synthesis and Evaluation of Antifibrotic Effects in vitro and in vivo

Abstract

Synthetic approaches for the preparation of macromolecular conjugates of the antifibrotic agent cis-4-hydroxy-L-proline (cHyp) were explored, and the efficacy of the conjugates in inhibiting collagen accumulation was investigated in vitro and in vivo. In one approach, poly(PEG-Lys), an alternating copolymer of poly(ethylene glycol) (PEG) and lysine, was used as the carrier. To prepare pendent chain systems, cHyp was attached to poly(PEG-Lys) through an amide linkage [poly(PEG-Lys-cHyp amide)] or through an ester linkage [poly(PEG-Lys-cHyp ester)]. In an alternative approach, cHyp was incorporated into the backbone of a linear copolymer consisting of PEG, succinic acid, and cHyp units [poly(PEG-succinate-cHyp)]. Bioactivity in vitro was assessed by the ability of the cHyp conjugates to inhibit growth of cultured smooth muscle cells (SMC) and rat lung fibroblasts (RLF). Cell numbers were compared to control experiments in the presence of biologically inactive trans-4-hydroxy-L-proline (tHyp). After a 5 day period, the presence of 8 micrograms/mL of cHyp delivered by poly(PEG-Lys-cHyp amide) resulted in a 47% reduction in the number of SMC (p < 0.05), the presence of 36 micrograms/mL of cHyp delivered by poly(PEG-Lys-cHyp ester) resulted in a 38% reduction in the number of SMC (p < 0.05), while the presence of 118 micrograms/mL of cHyp delivered by poly(PEG-succinate-cHyp) resulted in a 31% reduction in the number of cells (p < 0.05). An identical trend was observed for the inhibition of RLF growth. In general, poly(PEG-Lys-cHyp amide) was most active, followed by poly(PEG-Lys-cHyp ester) and the backbone system, poly(PEG-succinate-cHyp). Specifically, poly(PEG-Lys-cHyp amide) was over 100-fold more active in inhibiting cell growth than free cHyp. Bioactivity in vivo was evaluated by measuring collagen accumulation in subcutaneously implanted poly(vinyl alcohol) sponges in rats. Among the tested conjugates, poly(PEG-Lys-cHyp amide) was most active, reducing collagen accumulation in the sponge by 33% after 14 days relative to controls (p < 0.05). This result indicates that the covalent attachment of cHyp to poly(PEG-Lys) carries may be a useful strategy for the local inhibition of collagen accumulation in tissues.

Conjugates Bearing Multiple Formyl-Methionyl Peptides Display Enhanced Binding to but Not Activation of Phagocytic Cells

“…PEG-based copolymers have been found to be biocompatible and are currently being developed for multiple in vivo applications (Poiani et al, 1994). These include the use of biotin targeting moieties to promote sodium-dependent multivitamin transporter (SMVT) mediated transport of molecules across the intestinal epithelium (Ramanathan et al, 2001).…”

The Role of Collagen Synthesis in Ventricular and Vascular Adaptation to Hypoxic Pulmonary Hypertension

“…Previous work has demonstrated that collagen accumulation is a key contributor to proximal pulmonary artery stiffening in HPH [5][6][7] and may play a role in distal arterial remodeling as well [8]. Agents that impair the synthesis of new collagen have been shown to limit the severity of HPH and RV hypertrophy [9][10][11][12], but their effects on RV contractility, RV filling, and the efficiency of hemodynamic coupling between the RV and the pulmonary vasculature remain unknown. Overall, the role of collagen in the progression of PAH remains largely unknown.…”

Water-soluble polyacetals derived from diphenols

“…Typically PEG 1 (e.g. 3 400 g mol 21 ) rapidly undergoes an exothermic copolymerisation with triethylene glycol divinyl ether 2 in the presence of catalytic p-toluene sulfonic acid (pTSA) to give polyacetal 3 as a white solid (Scheme 1). 4 Weight average molecular weights up to 120 000 g mol 21 (M w /M n = 1.6-1.8; aqueous GPC with PEG standards) are typically isolated.…”