Angiotensin-(1-7) with thioether bridge: an angiotensin-converting enzyme-resistant, potent angiotensin-(1-7) analog.

The in vivo efficacy of many therapeutic peptides is hampered by their rapid proteolytic degradation. Cyclization of these therapeutic peptides is an excellent way to render them more resistant against breakdown. Here, we describe the enzymatic introduction of a thioether ring in angiotensin [Ang-(1-7)], a heptapeptide that plays a pivotal role in the renin-angiotensin system and possesses important therapeutic activities. The lactic acid bacterium Lactococcus lactis, equipped with the plasmid-based nisin modification machinery, was used to produce thioether-bridged Ang-(1-7). The resulting cyclized Ang-(1-7) is fully resistant against purified angiotensin-converting enzyme, has significantly increased stability in homogenates of different organs and in plasma derived from pig, and displays a strongly (34-fold) enhanced survival in Sprague-Dawley (SD) rats in vivo. With respect to functional activity, cyclized Ang-(1-7) induces relaxation of precontracted SD rat aorta rings in vitro. The magnitude of this effect is 2-fold larger than that obtained for natural Ang-(1-7). The Ang-(1-7) receptor antagonist D-Pro(7)-Ang-(1-7), which completely inhibits the activity of natural Ang-(1-7), also abolishes the vasodilation by cyclized Ang-(1-7), providing evidence that cyclized Ang-(1-7) also interacts with the Ang-(1-7) receptor. Taken together, applying a highly innovative enzymatic peptide stabilization method, we generated a stable Ang-(1-7) analog with strongly enhanced therapeutic potential.

Renal-selective delivery and angiotensin-converting enzyme inhibition by subcutaneously administered captopril-lysozyme.

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 captopril-lysozyme 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.

Effects of anti-proteinuric therapy with angiotensin-converting-enzyme inhibition on renal protein catabolism in the adriamycin-induced nephrotic rat.

A direct consequence of glomerular protein leakage is an increased exposure of proximal tubular cells to proteins. The aim of the present study was to examine whether chronic proteinuria affects the tubular handling of proteins and whether anti-proteinuric therapy by angiotensin-converting-enzyme (ACE) inhibition restores this tubular function. Renal uptake and catabolic rate of the low-molecular-weight protein (LMWP) myoglobin was determined in anaesthetized control and adriamycin-induced nephrotic rats by external counting after radiolabelling. Proteinuria correlated with the uptake as well as the catabolism of myoglobin. The higher the proteinuria, the lower was the renal uptake of myoglobin (r =0.72, P =0.002). Also, the catabolic rate of myoglobin (r =0.80, P =0.0002) was lower with increasing severity of proteinuria. During treatment with the ACE inhibitor lisinopril, proteinuria was lowered by 79+/-9% (mean+/-S.E.M.). Renal uptake and catabolic rate of the LMWP were not restored by ACE inhibition. The catabolic rate of myoglobin was even decreased further with 48+/-5% compared with pretreatment levels. In summary, adriamycin-induced proteinuria is associated with a lower uptake and a lower catabolic rate of LMWP in the proximal tubule. ACE inhibition lowers proteinuria, but does not restore the affected LMWP uptake and rate of catabolism. The rate of LMWP catabolism is even decreased further. In contrast, the urinary excretion of N -acetyl glucosaminidase, the tubular marker of toxicity, was effectively returned to normal levels during ACE inhibition. Taken together, the data suggest that proteinuria is toxic for the proximal tubular cells and that ACE inhibition protects the remaining functional tubular cells directly against destruction through decreasing hypercatabolism.

Renal targeting of captopril selectively enhances the intrarenal over the systemic effects of ACE inhibition in rats.

1 In previous studies on the renal targeting of the ACE inhibitor captopril, we demonstrated that a 6 fold increased concentration of this drug could be obtained in the kidney after conjugation to the low-molecular-weight protein lysozyme. In this study, we investigated in unrestrained rats whether systemic administration of captopril-lysozyme also results in an enhanced effect on renal parameters, relative to the systemic effects. 2 Renal effects: intravenous infusion of captopril-lysozyme for 6 h resulted in a more pronounced increment of renal blood flow (31+/-2% vs 17+/-4% at 0.5 mg kg(-1) 6h(-1), P<0.01) and an approximately 5 fold enhanced natriuresis (167+/-17% vs 36+/-7% at 1 mg kg(-1) 6 h(-1), P<0.001) in comparison with equimolar amounts of captopril as a free drug. In correspondence with these findings, renal ACE inhibition was potentiated approximately 5 fold (-50+/-4% vs -22+/-3% at 1 mg kg(-1) 6 h(-1), P<0.001). 3 Systemic effects: conjugated captopril did not affect blood pressure in dosages up to 5 mg kg(-1) 6 h(-1). This effect coincided with a less pronounced inhibition of the pressor response to intravenously administered angiotensin I (-12+/-3% vs -66+/-5% at 1 mg kg(-1) 6 h(-1), P<0.001), and a markedly attenuated plasma ACE inhibition (-19+/-2% vs -37+/-3% at 1 mg kg(-1) 6 h(-1), P<0.001) compared to an equivalent dose of free captopril. 4 An experiment of continued intravenous administration of captopril-lysozyme for 7 days in nephrotic syndrome demonstrated that the conjugate is also active in renal disease: the antiproteinuric response was substantially augmented (-67+/-5% vs -15+/-7% at 4 mg kg(-1) 24 h(-1), P<0.001) compared to the free drug, in the absence of blood pressure reduction. 5 These data demonstrate that intravenous administration of a captopril-lysozyme conjugate leads to more selective renal ACE inhibition and enhanced renal effects as well as less systemic effects compared to captopril itself.

Targeting of doxorubicin to the urinary bladder of the rat shows increased cytotoxicity in the bladder urine combined with an absence of renal toxicity.

Targeting of anti-tumor drugs to the urinary bladder for the treatment of bladder carcinoma may be useful, since these agents generally have a low degree of urinary excretion and are highly toxic elsewhere in the body. The anti-tumor drug doxorubicin was coupled to the low-molecular weight protein lysozyme via the acid-sensitive cis-aconityl linker. All free amino groups of the lysozyme were used for drug attachment to achieve intact excretion of the doxorubicin-aconityl-lysozyme conjugate into the bladder. In the bladder, the cytotoxic drug should be regenerated through acidification of the urine. First, the doxorubicin-aconityl-lysozyme conjugate was tested in rats for its target specificity and general toxicity. Wistar rats were injected intravenously with 2 mg/kg free doxorubicin or 10 mg/kg lysozyme-conjugated doxorubicin. Total urinary excretion of doxorubicin was about 10 times higher if the drug was coupled to lysozyme (39 +/- 3% versus 4.4 +/- 0.4%). Free doxorubicin had no detectable toxic effects on heart, liver and lung but caused severe renal damage (proteinuria, N-acetylglucosaminidase excretion and glomerulosclerosis). None of the rats injected with doxorubicin-lysozyme conjugate showed such renal toxicity. Second, we tested whether doxorubicin could be released from the conjugate in the bladder through acidification of the urine and if the released doxorubicin could still exert a cytotoxic effect. Doxorubicin-aconityl-lysozyme (2 mg/kg conjugated doxorubicin, i.v.) was administered in rats with acidified urine (pH 6.1 +/- 0.1) and in rats with a high urinary pH (8.2 +/- 0.4). Ten times more doxorubicin was released from the conjugate in the group with acidified urine (15 +/- 7% versus 1.7 +/- 0.1%). In agreement with this, cytotoxicity was also higher in the low pH group (IC50 of 255 +/- 47 nM versus 684 +/- 84 nM doxorubicin). In conclusion, a specific delivery of doxorubicin to the urinary bladder combined with a reduced toxicity of doxorubicin in the kidneys can be achieved by coupling this anti-tumor drug to the low-molecular weight protein lysozyme via an acid-labile linker. A release of cytotoxic doxorubicin in the urinary bladder can be achieved by acidification of the urine. This technology, after further optimization, may provide an interesting tool for the treatment of bladder carcinoma.

Specific drug delivery to the kidney.

The mesangial cells of the glomerulus, the proximal tubular cells and the interstitial fibroblasts are the first choice targets for renal drug delivery since they play a pivotal role in many disease processes in the kidney. In the present review, only targeting to the proximal tubular cell is addressed because only this has been studied extensively. Two approaches of drug delivery to the proximal tubular cell have been studied up to now, the prodrug/softdrug and low-molecular-weight protein (LMPWP) approach. Most research on tubular specific drug delivery has focused on the development of amino-acid prodrugs that, after delivery, require activation by more or less kidney-selective enzymes. Large differences in renal selectivity are found. For some prodrugs, a rapid removal of the released drug from the kidney explained the low renal selectivity whereas for others, cleavage in non-target tissue and insufficient transport across the cell to the enzyme site seemed mainly responsible. The LMWP approach is based on drug attachment to a protein (<30 kD) that is freely filtered through the glomerulus and after accumulation is selectively catabolized in the lysosomes of the proximal tubular cell. Using LMWPs as drug carriers, a higher renal selectivity can be attained and a broader range of drugs can be attached while the rate of drug release can also be manipulated. The studies with captopril-lysozyme and naproxen-lysozyme clearly showed that targeting resulted in a higher renal selectivity and that drugs delivered into and regenerated in the proximal tubular cell exert renal selective pharmacological activity. Further testing will provide more definite data on the added value of this delivery technology.