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.
We studied the therapeutic efficacy of an intravenously injected antifibrotic agent encapsulated in liposomes on inhibiting collagen accumulation in hypertensive blood vessels. cis-4-Hydroxy-L-proline (cHyp) in liposomes was injected into rats exposed to 10% 02, and drug effect was evaluated by measuring right ventricular pressure and hydroxyproline content of the pulmonary artery. Right
Inhibitors of collagen such as cis-4-hydroxy-L-proline (cHyp) may ameliorate bleomycin (bleo)-induced pulmonary fibrosis. An alternating polymer of poly(ethylene glycol) (PEG)-lysine (PEG-Lys) with cHyp attached as a pendant side chain was prepared for intratracheal delivery with bioinactive trans-Hyp (tHyp) polymer as control. To test whether the cHyp polymer has prolonged lung retention and sustained antifibrotic activity, we first instilled 3H- and 14C-labeled cHyp polymer in normal rats. Lung retention was 86 +/- 9% at 6 h and 29 +/- 3% at 7 d (n = 5). Next, rats were instilled intratracheally with either saline (sal) or 1.2 U bleo, and the following treatment groups were studied: Bleo/sal; Bleo/cHyp polymer; Bleo/tHyp polymer; and Bleo/PEG-Lys + cHyp. The dose of the test agents was 150 mg/kg polymer containing 8.5 mg/kg cHyp or tHyp instilled intratracheally at 7 and 14 d after bleo. At 21 d, hydroxyproline content (mg/lung) was: Control, 1.8 +/- 0.1; Bleo/sal 4.0 +/- 0.1*; Bleo/cHyp polymer, 2.8 +/- 0.3*+; Bleo/tHyp polymer, 4.4 +/- 0.2*; and Bleo/PEG-Lys + cHyp, 4.0 +/- 0.1* (*p < 0.05 versus Control; +p < 0.05 versus Bleo/sal; n = 5/group). The cHyp polymer also reduced lung total protein content, but the decrease was not significant. The dose required to produce 50% inhibition of lung collagen was approximately 700-fold less than monomeric cHyp. Thus, the cHyp polymer is a potent, long-acting antifibrotic agent which may be useful in treating lung fibrosis.
The proline analogue cis-4-hydroxy-L-proline (cHyp) inhibits collagen accumulation but diffuses out of tissues. To prolong the antifibrotic effect, we used a copolymer of cHyp attached to a backbone of poly(ethylene glycol) (PEG) and lysine. The copolymer was encapsulated in liposomes conjugated with PEG or in liposomes coated with the polysaccharide amylopectin to improve uptake by lungs after intravenous infusion. Amylopectin-liposomes had approximately 3-fold greater uptake in cultured endothelial cells compared with PEG-liposomes and greater lung retention 1 wk after infusion (5.2 +/- 0.8% versus 2.7 +/- 0.2%, p < 0.05). Sustained antifibrotic activity, assayed by growth inhibition of smooth muscle cells and fibroblasts over 4 d, was greater for amylopectin-liposomes/copolymer than PEG-liposomes/copolymer. Inhibition of collagen accumulation in pulmonary arteries of hypoxic (10% O2) rats was used to assess antifibrotic activity. Amylopectin-liposomes/copolymer attenuated increased right ventricular pressure by approximately 50% and completely prevented excess vascular collagen 1 wk after a single intravenous injection. The copolymer in liposomes was > 1,000-fold more effective by weight than unencapsulated monomeric cHyp. Thus, the copolymer, a potent, long-acting antifibrotic agent, totally prevented collagen accumulation for 1 wk in pulmonary arteries undergoing vascular remodeling when delivered in amylopectin-liposomes.
We studied whether the therapeutic efficacy of the antifibrotic agent cis-4-hydroxy-L-proline (cHyp) in preventing bleomycin-induced pulmonary fibrosis in rats is enhanced by intratracheal delivery in liposomes. Dual-radiolabeled liposomes were used to study the distribution and stability of liposomes after intratracheal instillation. Lung retention was > 20% 1 wk after intratracheal instillation of 9 mumol phospholipid, and liposomes were intact as indicated by the ratio of the lipid and aqueous-phase markers remaining unchanged. For the fibrosis study, groups of rats were instilled with 1.2 U bleomycin (Bleo) and treated 1 and 2 wk later by single intratracheal instillation of test compounds. The control group received 0.3 ml saline (Bleo/sal). The treated groups received 9 mumol phospholipid in 0.3 ml of the following liposome preparations: empty liposomes (Bleo/lip), liposomes and 100 mg/kg of free unencapsulated cHyp (Bleo/lip/cHyp), and 100 mg/kg of liposome-encapsulated cHyp (Bleo/lip-cHyp). At 3 wk, fibrosis (mg hydroxyproline/g weight lung) by groups was as follows: control, 2.6 +/- 0.1 (SEM); Bleo/sal, 3.2 +/- 0.1, Bleo/lip, 3.2 +/- 0.1, and Bleo/lip/cHyp, 3.1 +/- 0.1, p < 0.05 compared with control; Bleo/lip-cHyp, 2.6 +/- 0.1, p < 0.05 compared with Bleo/sal, n = 3 to 6. Histologic grading of fibrosis did not show decreased fibrosis in the Bleo/lip-cHyp group, probably because of the focal nature of the fibrotic lesions. We conclude that cHyp encapsulated in liposomes prevents bleomycin-induced fibrosis by biochemical measurements. Delivery of antifibrotic agents to the lung in carrier vehicles promotes retention and may enhance their efficacy in treating bleomycin-induced pulmonary fibrosis.
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