M Haas

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-Pro7-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.

Angiotensin-(1-7) with thioether-bridge: an ACE-resistant, potent Ang-(1-7) analogue

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-Pro7-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.

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.

POTENTIALS AND LIMITATIONS OF THE LOW-MOLECULAR-WEIGHT PROTEIN LYSOZYME AS A CARRIER FOR RENAL DRUG TARGETING

Selective targeting of drugs to the kidney may enable an increasedrenal effectiveness combined with a reduction of extrarenal toxicity. Intrarenaldelivery to the proximal tubular cell can be achieved using lowmolecular-weightproteins, such as lysozyme. Administration of high dosages of lysozyme, requiredto study the effects of such conjugates in vivo,however, is restricted since a partial escape of the renal reabsorption andthe occurrence of unwanted effects on systemic blood pressure and renal functionmay occur. The purpose of this study was to investigate the optimal parenteraladministration schedule and the maximum dose of lysozyme, providing the mostoptimal tubular reabsorption and at the same time a minimal effect on bloodpressure and renal hemodynamics, comparing continuous infusion of lysozymewith 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 injectionsof in total 250 mg.kg−1.6 h−1(51.8±3.7% of the dose) compared to the same dose administered by continuousinfusion (11.7±2.4%, P<0.001).Continuous infusion of lysozyme up to 1000 mg.kg−1in 6 hours had no effect on systemic blood pressure, whereas a bolus injectionof lysozyme (167 mg.kg−1) resulted in reversibleblood pressure lowering of 52.2±2.2% (P<0.001).A dose-dependent decline of the glomerular filtration rate was observed atdosages of lysozyme higher than 100 mg.kg−1.6h−1, with a maximal reduction of 53.0±3.7%after infusion of 1000 mg.kg−1.6 h−1.Effective renal plasma flow was less affected and only lowered statisticallysignificant at dosages of 500 (–12.6±3.3%, P<0.05)to 1000 mg.kg−1.6 h−1(–17.2±3.9%, P<0.01). Weconclude that bolus injections of lysozyme should not be used for renal targetingpurposes since it results in considerable tubular loss of lysozyme in theurine as well as cardiovascular side effects. In contrast, continuous infusionof lysozyme using dosages sufficient for renal drug targeting (maximally 15mg.kg−1.h−1)only has minimal effects on blood pressure and renal hemodynamics, with aminimal urinary lysozyme loss as well.

Dynamic and kinetic differences of the vascular and myocardial effects of calcium antagonists in the rat heart.

We studied the effects of nifedipine, nimodi-pine, verapamil, D600 (gallopamil), D888 (desmethoxy-verapamil), D890 (quaternized verapamil), bepridil, and diltiazem on the coronary flow and the left ventricular pressure in the retrogradely perfused paced rat heart; in addition, we investigated the time course of onset and recovery of these effects. We found a clear difference in potency order for the vascular and cardiac effects as well as widely different kinetics of coronary flow increase and negative inotropic activity. Furthermore, positive inotropism at low doses of some calcium antagonists seemed to be related to the vascular effects of these compounds. We conclude that the rat heart contains a hydrophylic and readily accessible, vascular “dihydropyridine” site and a more hydrophobic, possibly intramembraneous or intracellular, myocardial “verapamil” site with a lower accessibility for verapamil derivatives and bepridil.

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 -acetyl-glucosaminidase 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 (IC 50 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.

Drug delivery to the kidneys and the bladder with the low molecular weight protein lysozyme

The low molecular weight protein (LMWP) lysozyme is a suitable drug carrier for renal drug targeting. When the tubular reabsorption of a LMWP can be prevented, the protein will be excreted in the urine. In this way, lysozyme (LZM) conjugates might also be used as carriers for targeting to the urinary tract. Since positive domains on the protein surface are important for the interaction with the tubular uptake-receptor, we studied the urinary excretion of a drug-LZM conjugate with and without positive charge on the LMWP. We synthesized two conjugates with the fluorescent compound fluorescein. A positively charged conjugate was obtained by reacting fluorescein isothiocyanate (FITC) with LZM at a 1:1 molar to molar ratio; this conjugate contained six free primary aminogroups. The conjugate without positively charged groups was obtained by reacting the remaining free primary aminogroups of the FITC-LZM with succinic anhydride (Suc). The Suc-FITC-LZM contained only 0.2 free primary aminogroups per molecule. We studied the pharmacokinetics of the conjugates in freely moving Wistar rats. The FITC-LZM conjugate was excreted intactly into the urine for 29 +/- 4% of the injected dose. The Suc-FITC-LZM was excreted into the urine intactly for 45 +/- 4%. These data indicate that the excretion of a drug-LMWP conjugate into the urine can be increased by decreasing the positive charge on the carrier surface. Such a carrier may be an attractive candidate for drug targeting to the bladder.

Urine collection in the freely moving rat: Reliability for measurement of short-term renal effects

Studies on short-term renal responses to (pharmacological) intervention require accurate and multiple collection of urine samples. Several invasive techniques have been described for frequent urine collection of the conscious rat, each having their own limitations. No data are available about the feasibility of the spontaneously voiding, freely moving rat for this purpose. In the present study, bladder voidings of six rats were time-registered and collected separately for several days. The data show a considerable 24-h variation coefficient of both the voided volume and the bladder collection time with a poor correlation between the two parameters. Forced diuresis induced by continuous i.v. infusion (2 ml/h) increased the frequency of urine voiding and thus the time-resolution of the urine-production pattern. However, this method failed to reduce the variation coefficient of the voided volume, the collection time, and the correlation between the two parameters. The fact that variations in creatinine excretion paralleled the variation in urinary flow suggests that both phenomena are likely be due to incomplete bladder emptying. Correction for this incomplete bladder collection, using the creatinine excretion, indeed reduced the variation coefficient of sodium excretion successfully from 61 +/- 17% to 29 +/- 5% during normal diuresis and from 56 +/- 19% to 22 +/- 6% during forced diuresis. In conclusion, the spontaneously voiding, freely moving rat can be used for short-term renal response studies if the collected urine samples are corrected for incomplete bladder emptying using urinary creatinine concentrations. This procedure allows the detection of changes in a urinary parameter if this exceeds a 40% deviation of the normal value.

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

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. 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). 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. 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. 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.

STEREOISOMERS OF CALCIUM-ANTAGONISTS DISTINGUISH A MYOCARDIAL AND VASCULAR MODE OF PROTECTION AGAINST CARDIAC ISCHEMIC-INJURY

Concentration-dependent effects of the enantiomers of the calcium antagonists, gallopamil, diltiazem, and bepridil have been studied in the Langendorffperfused rat heart, subjected to 30 min of global ischemia. It is shown that the time course, as well as the height of the energy deprivation-induced left ventricular diastolic contracture that develops during ischemia, can be selectively inhibited by negative inotropic concentrations of the calcium antagonist enantiomers. The time needed for recovery from the diastolic contracture during the reperfusion phase can be shortened significantly by lower, vasodilating concentrations of the drugs. In normoxically perfused hearts, stereoselectivity factors (sf) of the enantiomers of the compounds amounted to 63, 10, and 2 for the negative inotropic and 12.6, 79, and 4 for the vasodilating activities of gallopamil, cis-diltiazem, and bepridil, respectively. The sf values of negative inotropism proved to be remarkably similar to sf values of 50 and 7.9 for gallopamil and cis-diltiazem in the protection of the ischemic contracture during ischemia, whereas the sf values of coronary now increase closely paralleled the values of 7.9, 63, and 2.5 for gallopamil, cis-diltiazem, and bepridil, respectively, in protection during the reperfusion phase. The results strongly suggest that at reperfusion the vasoselective enantiomers of calcium antagonists provide protection related to improved tissue perfusion, and thereby possibly restoring the distorted ionic and energetic homeostasis, whereas the other enantiomers are more involved in a direct energy-saving activity, resulting in protection during the ischemic period.