A renal-specific controlled release of an active drug may enable a reduction of the required dose and may provide a reduction of extra-renal toxicity. To achieve renal specific targeting of the NSAID naproxen, the low-molecular-weight protein (LMWP) lysozyme was employed as carrier since it is mainly taken up and catabolized in the proximal tubules of the kidney. A conjugate was synthesized with an average coupling degree of 2 mol naproxen per 1 mol lysozyme in which the drug was directly coupled to the protein via a peptide bond. First, we investigated whether naproxen conjugation affects the renal disposition of lysozyme. As native lysozyme, the conjugate was predominantly and rapidly (within 20 min) taken up by the kidney. The subsequent decrease in renal content reflecting the renal degradation of the conjugated lysozyme molecules appeared also to be similar to that of native lysozyme with a half life of four hours. Second, the effect of lysozyme conjugation on the body distribution of naproxen was studied. An important observation with regard to the aimed reduction in extra-renal side effects was that no detectable amounts of free naproxen were present in the plasma after administration of conjugate. Conjugation of naproxen to lysozyme resulted in a pronounced (70-fold) increase of naproxen accumulation in the kidney. In agreement with the protein disposition study, the conjugate was rapidly taken up by the kidney and subsequently degraded. In conclusion, renal selective targeting of the NSAID naproxen can be obtained by conjugation with the LMWP lysozyme. This concept of drug delivery to the kidney has the potential to improve drug efficacy and safety.
Selective targeting of drugs to the kidney may enable an increased renal effectiveness combined with a reduction of extrarenal toxicity. Intrarenal delivery to the proximal tubular cell can be achieved using low-molecular-weight proteins, such as lysozyme. Administration of high dosages of lysozyme, required to study the effects of such conjugates in vivo, however, is restricted since a partial escape of the renal reabsorption and the occurrence of unwanted effects on systemic blood pressure and renal function may occur. The purpose of this study was to investigate the optimal parenteral administration schedule and the maximum dose of lysozyme, providing the most optimal tubular reabsorption and at the same time a minimal effect on blood pressure and renal hemodynamics, comparing continuous infusion of lysozyme with single dose injections. Urinary lysozyme excretion increased dose-dependently, both during continuous infusion and intravenous bolus injections. However, this loss of intact lysozyme into the urine was much higher after 3 injections of in total 250 mg x kg(-1) x 6 h(-1) (51.8+/-3.7% of the dose) compared to the same dose administered by continuous infusion (11.7+/-2.4%, P < 0.001). Continuous infusion of lysozyme up to 1000 mg x kg(-1) in 6 hours had no effect on systemic blood pressure, whereas a bolus injection of lysozyme (167 mg x kg(-1)) resulted in reversible blood pressure lowering of 52.2+/-2.2% (P<0.001). A dose-dependent decline of the glomerular filtration rate was observed at dosages of lysozyme higher than 100 mg x kg(-1) x 6 h(-1), with a maximal reduction of 53.0+/-3.7% after infusion of 1000 mg x kg(-1) x 6 h(-1). Effective renal plasma flow was less affected and only lowered statistically significant at dosages of 500 (-12.6+/-3.3%, P<0.05) to 1000 mg x kg(-1) x 6 h(-1) (-17.2+/-3.9%, P<0.01). We conclude that bolus injections of lysozyme should not be used for renal targeting purposes since it results in considerable tubular loss of lysozyme in the urine as well as cardiovascular side effects. In contrast, continuous infusion of lysozyme using dosages sufficient for renal drug targeting (maximally 15 mg x kg(-1) x h(-1)) only has minimal effects on blood pressure and renal hemodynamics, with a minimal urinary lysozyme loss as well.
ABSTRACT:In previous studies, we have demonstrated that the low molecular weight protein lysozyme can be used as a renal-selective drug carrier for delivery of the angiotensin-converting enzyme (ACE) inhibitor captopril. Typically, such macromolecular drug-targeting preparations are administered intravenously. In the present study, we investigated the fate of captopril-lysozyme following subcutaneous administration, a convenient route for long-term treatment. The absorption from the subcutaneous injection site and renal uptake of lysozyme were determined by gamma scintigraphy in rats. Bioavailability, renal accumulation, and stability of the captopril-lysozyme conjugate were evaluated by high performance liquid chromatography analysis and by ACE activity measurements.Lysozyme was absorbed gradually and completely from the subcutaneous injection site within 24 h and accumulated specifically in kidneys. After subcutaneous injection of the captopril-lysozyme conjugate, higher renal captopril levels and lower captoprillysozyme levels in urine indicated the improved renal accumulation in comparison with intravenous administration of the conjugate, as well as its stability at the injection site. After both treatments, captopril-lysozyme conjugate effectuated renal ACE inhibition, whereas plasma ACE was not inhibited. In conclusion, our results demonstrate that we can use the subcutaneous route to administer drug delivery preparations like the captopril-lysozyme conjugate.
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