Low Molecular Weight Protein Lysozyme Research Articles

Imatinib-ULS-lysozyme: A proximal tubular cell-targeted conjugate of imatinib for the treatment of renal diseases

The anticancer drug imatinib is an inhibitor of the platelet-derived growth factor receptor (PDGFR) kinases, which are involved in the pathogenesis of fibrotic diseases. In the current study we investigated the delivery of imatinib to the proximal tubular cells of the kidneys and evaluated the potential antifibrotic effects of imatinib in tubulointerstitial fibrosis.Coupling of imatinib to the low molecular weight protein lysozyme via the platinum (II)-based linker ULS yielded a 0.8:1 drug-carrier conjugate that rapidly accumulated in the proximal tubular cells upon intravenous and intraperitoneal administration. The bioavailability of intraperitoneally administered imatinib-ULS-lysozyme was 100%. Renal imatinib levels persisted for up to 3days after a single injection of imatinib-ULS-lysozyme. Compared with an equal dose imatinib mesylate, imatinib-ULS-lysozyme resulted in a 30- and 15-fold higher renal exposure of imatinib, for intravenous and intraperitoneal administration respectively. Imatinib-ULS-lysozyme could not be detected in the heart, which is the organ at risk for side-effects of prolonged treatment with imatinib.The efficacy of imatinib-ULS-lysozyme in the treatment of tubulointerstitial fibrosis was evaluated in the unilateral ureteral obstruction (UUO) model in mice. Three days UUO resulted in all signs of early fibrosis, i.e. an increased deposition of matrix and production of profibrotic factors. Although a moderately increased activity of PDGFR-β was observed, the profibrotic phenotype could not be inhibited with imatinib mesylate or with imatinib-ULS-lysozyme. Further evaluation of imatinib mesylate and imatinib-ULS-lysozyme is therefore warranted in an animal model of renal disease in which the activation of PDGFR-β is more pronounced.

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.

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.

Renal Drug Delivery With Low-Molecular-Weight Proteins: The Effect of Charge Modifications on the Body Distribution of Drug-Lysozyme Conjugates

Low-molecular-weight proteins (LMWPs) can be used as drug carriers for renal drug delivery. The covalent attachment of drug molecules is commonly achieved via reaction to the primary amino groups of the protein. However, these positively charged groups are also important for the renal reabsorption of proteins. In this study, we investigated the effect of the modification of primary amino groups on the pharmacokinetics of drug-LMWP conjugates, using the organic anion fluorescein isothiocyanate (FITC) as a model drug and the LMWP lysozyme (LZM) as a drug carrier. We prepared a series of conjugates with different amounts of primary amino groups: a more negatively charged conjugate was prepared by derivatizing the primary amino groups of LZM with succinic anhydride, while a more positively charged conjugate was prepared by derivatizing the carboxylic acid groups with ethylendiamine. The final conjugates had an average amount of primary amino groups of 0.2 (FITC-Suc-LZM), 6.5 (FITC-LZM), or 8.5 (FITC-cat-LZM). We studied the renal handling of the conjugates in freely moving Wistar rats. After i.v. injection of 33 mg/kg conjugate, the FITCLZM and the FITC-Suc-LZM conjugates were incompletely reabsorbed in the kidney, resulting in the excretion of the intact conjugate into the urine (29 4% versus 45 4% of the injected dose, p < 0.05). In contrast, the FITC-cat-LZM conjugate was not excreted into the urine (0.3 0.1% of the injected dose, p < 0.001 distributed selectively to the kidney, and significant amounts of this versus FITC-LZM). An organ distribution study with 125I radiolabeled cat-LZM showed that the cationized LMWP accumulated in the kidney (12 0 . 3% of the dose). However, cat-LZM no longer LMWP were found in the liver and spleen (53 1% and 5 2%, respectively). We conclude that charge modifications influence the biodistribution of drug-LZM conjugate. First, reducing the amount of primary amino groups results in decreased tubular reabsorption and increased excretion of the conjugate into the urine. Finally, increasing the amount of primary amino groups results not only in improved renal reabsorption, but also in an increased extrarenal distribution of the conjugate and therefore in a partial loss of renal selectivity.

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 amino-groups per molecule. We studied the pharmucokinetics 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-LAMP 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.

Drug-targeting to the kidney: Renal delivery and degradation of a naproxen-lysozyme conjugate in vivo

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.

(Glyco)-protein drug carriers with an intrinsic therapeutic activity: The concept of dual targeting

Dual targeting can in principle be achieved by using intrinsically active carriers that not only deliver the conjugated drug but also otherwise influence the pathological process. Potential carriers of this kind are monoclonal antibodies, certain interferons and interleukins, as well as certain enzymes, peptide hormones and antivirally active (glyco)-proteins. The low molecular weight protein lysozyme, that is easily filtered and reabsorbed in kidney tubular cells, was used as a carrier for renal delivery of various drugs including anti-bacterial agents. The carrier itself can add to the anti-infective effect through its lysing effect on bacterial cell walls. Albumins derivatized with various sugars were used for delivery of the anti-inflammatory drug naproxen to hepatocytes, endothelial cells and Kupffer cells. Naproxen-albumin exhibited a potent hepatoprotective effect if delivered to sinusoidal cell types. Negatively charged and enzymatically active protein carriers can contribute to therapeutic effects, among others, through receptor antagonism and detoxification of endotoxins. Some (glyco)-proteins, designed as a drug carrier for anti-HIV agents, exhibited an intrinsic antiviral activity even without coupling of antiviral drugs. This activity was caused by an increased negative charge of the particular (glyco)-proteins. The in vitro IC 50 values of some of these polyanionic proteins were in the nanomolar concentration range. The mechanism of action was found to be inhibition of a post binding virus/cell fusion event. The particular negatively charged albumins (NCAs) showed, in the therapeutic dose range, favorable pharmacokinetics with regard to lymphatic distribution and residence time in the blood stream. They have little acute toxicity and, in contrast to other polyanionic compounds, such as dextran sulfate are readily biodegradable. The NCAs lack an anticoagulant activity and exhibit low immunogenicity. Importantly, a high activity against primary clinical HIV isolates was observed. We detected a high affinity binding to the V3 loop of the envelope glycoprotein gpl20, explaining the potent effect on virus/cell fusion and HIV-I replication as well as the significant activity on syncytium-inducing HIV variants. The particular antiviral proteins can in principle be applied as intrinsically active drug carriers for the targeting of nucleoside analogues such as AZT, protease inhibitors and glycosidase inhibitors. A major advantage would be the concomitant blocking of sequential steps in the viral replication process with the aims to obtain synergism and prevention of drug resistance.

Low molecular weight proteins as carriers for renal drug targeting: naproxen coupled to lysozyme via the spacer L-lactic acid.

Low molecular weight proteins (LMWPs) are potential carriers for targeting drugs to the kidney. To test whether ester bonds are suitable for the reversible drug conjugation, the antiinflammatory drug naproxen (Nap) was conjugated to the LMWP lysozyme (LYSO) via an ester bond using an L-lactic acid spacer (Nap-lact-LYSO, 1:1:1). The distribution and degradation of the conjugate in rats were compared to those of an equimolar mixture of free drug and LMWP and of a directly coupled conjugate without spacer (Nap-LYSO). The plasma clearance of Nap-lact-LYSO closely resembled that of Nap-LYSO and LYSO itself. Its major accumulation site appeared to be the kidney as demonstrated by extracorporal gamma-camera counting of the LMWP. Renally released naproxen was excreted in the urine as 6-desmethyl-naproxen-sulfate (6-DMN-S). Apparently the kidneys represent the main sites of demethylation and sulfation after administration of the LMWP-coupled drug. In addition, the renal excretion of naproxen (including its metabolites) was significantly delayed and sustained as compared to that after injection of uncoupled naproxen. Using the L-lactic acid spacer LMWP conjugation, the renal selectivity of Nap was increased 5.6 +/- 0.41-fold. Additional in vitro studies with Nap-lact-LYSO revealed that renal generation of the parent drug coincided with formation of low molecular weight catabolites, mainly as naproxen-L-lactic acid-lysine (Nap-lact-Lys). This indicated that in vitro the rate of cleavage of the ester bond is significantly slower than digestion of the carrier backbone itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal specific delivery of sulfamethoxazole in the rat by coupling to the low molecular weight protein lysozyme via an acid-sensitive linker

Sulfamethaxazole (SM) was converted to a renal specific drug targeting preparation by coupling the drug to egg-white lysozyme via an acid-sensitive cis-aconityl linker (1:1). Due to this chemical manipulation SM was rapidly distributed to the kidney. Both in vitro and in vivo data indicate that SM was uncoupled from the carrier by chemical hydrolysis in the lysosomes of proximal tubular cells, resulting in parent active drug at the target site. This concept is applicable to other drug-polypeptide conjugates which rapidly distribute to the kidney and might enable selective manipulation of renal (patho)physiology.

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process

1. 1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the nitration method. 3. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant K d of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.

Renal ultrastructural and biochemical injuries induced by aminoglycosides.

Aminoglycoside-induced proteinuria may result from general renal damage or may reflect alterations in specific steps in the renal handling of proteins. To differentiate between the two possibilities, experiments were designed to quantify the effects of nephrotoxic doses of gentamicin, tobramycin and netilmicin in the intact rat, isolated perfused rat kidney (IPK) and kidney slices using the cationic low molecular weight protein lysozyme as a model protein. Each aminoglycoside was administered IP to male Wistar rats (15 or 30 mg/kg/day) for 5 or 7 days. Scanning and transmission electron microscopy indicated that gentamicin and tobramycin induced a decrease in the number and diameter of endothelial fenestrae and degranulation of the myoepithelioid cells of the juxtaglomerular apparatus. Concurrently, gentamicin and tobramycin decrease the glomerular sieving coefficient of lysozyme from 0.8 to 0.6 and 0.5, respectively. Netilmicin did not affect the percentage reabsorption of lysozyme whereas gentamicin and tobramycin induced a 50% decrease in lysozyme reabsorption by the IPK. Gentamicin and tobramycin decreased equally lysozyme degradation by the IPK; this decrease was time- and dose-dependent when evaluated in slices from renal cortex. Perfusion of rat kidneys with gentamicin induced a dose-dependent decrease in reabsorption and catabolism of lysozyme. In conclusion, these studies demonstrate that polycationic aminoglycosides alter ultrastructure and glomerular permeability, tubular reabsorption and intracellular digestion of proteins.

Renal Ultrastructural and Biochemical Injuries Induced by Aminoglycosides

Aminoglycoside-induced proteinuria may result from general renal damage or may reflect alterations in specific steps in the renal handling of proteins. To differentiate between the two possibilities, experiments were designed to quantify the effects of nephrotoxic doses of gentamicin, tobramycin and netilmicin in the intact rat, isolated perfused rat kidney (IPK) and kidney slices using the cationic low molecular weight protein lysozyme as a model protein. Each aminoglycoside was administered IP to male Wistar rats (15 or 30 mg/kg/day) for 5 or 7 days. Scanning and transmission electron microscopy indicated that gentamicin and tobramycin induced a decrease in the number and diameter of endothelial fenestrae and degranulation of the myoepithelioid cells of the juxtaglomerular apparatus. Concurrently, gentamicin and tobramycin decrease the glomerular sieving coefficient of lysozyme from 0.8 to 0.6 and 0.5, respectively. Netilmicin did not affect the percentage reabsorption of lysozyme whereas gentamicin and tobramycin induced a 50% decrease in lysozyme reabsorption by the IPK. Gentamicin and tobramycin decreased equally lysozyme degradation by the IPK; this decrease was time- and dose-dependent when evaluated in slices from renal cortex. Perfusion of rat kidneys with gentamicin induced a dose-dependent decrease in reabsorption and catabolism of lysozyme. In conclusion, these studies demonstrate that polycationic aminoglycosides alter ultrastructure and glomerular permeability, tubular reabsorption and intracellular digestion of proteins.

Inhibition of renal accumulation of lysozyme (basic low molecular weight protein) by basic proteins and other basic substances.

Together the two rat kidneys accumulated a total of 31.7 +/- 1.6% of the intravenously injected amount of 7 nmoles egg-white-lysozyme (measured as iodine 125 lysozyme) within 10 min. The low molecular weight protein lysozyme and other basic substances were injected simultaneously in order to evaluate whether these basic substances can inhibit the renal lysozyme accumulation. The inhibitory effect of various basic compounds was dose-dependent with a maximal reduction of lysozyme accumulation to 11.7 +/- 0.08%. The basic substances could be divided into three groups depending upon the micromolar amount injected at which a 50% inhibition was achieved (0.3-1.2 micromoles: cytochrome C, ribonuclease; 10.9 micromoles; spermine; 501-688 micromoles: L-arginine, L-lysine). The neutral myoglobin had no effect on renal lysozyme accumulation. The inhibitory potency appeared to increase with increasing molecular weight and pI value of the substance tested. Microperfusion experiments of proximal convoluted tubules of rat kidney revealed that luminal reabsorption of the basic lysozyme can be inhibited by the basic protein cytochrome C in a dose-dependent fashion. In these experiments the perfusion solution contained 57 micromol .l-1 lysozyme, an intratubular lysozyme concentration at which the tubular lysozyme reabsorption was found to be about 80% saturated. A 50% inhibition of the tubular endocytic lysozyme reabsorption was achieved a cytochrome C concentration of 102 micromol.l-1.

Lysozymuria in Patients with Proximal Tubular Dysfunction : The Relationship between Lysozyme and Sodium Reabsorption in the Proximal Tubule

Activity of the low molecular weight protein lysozyme was measured by the lyso-plate method in the serum and urine of 25 normal adults and 51 patients suffering from various renal diseases, i.e. 22 primary glomerular, 19 primary interstitial, and 10 primary diseases. No correlation was found between serum and urinary lysozyme activities, although lysozymuria was present in all of the azotemic patients with serum lysozymeactivities greater than 90μg/ml, a threefold increase above normal serum levels. CLy fract was significantly higher in diseases, such as adult Fanconi syndrome, acute tubular necrosis, and transplanted kidney, than in glomerular diseases. In eight patients with primary tubular dysfunction, there were significantly correlated increases in CLy fract and CNa fract (r=0.931, CLyfract=1.21 CNafract-1.18). In a patient with unique ARF, who developed a period of oliguria followed by a massive diuresis- and saluresis during the diuretic phase, significant correlation was found about the increases in CLyfract and CNafract. In the rejection of a renal homotransplant, moreover, lysozymuria appeared with the elevated urinary Na/K ratios. The results of the present study suggest that the lysozyme reabsorption, which is essentially confined to the proximal tubule, may depend upon sodium reabsorption. Therefore, CLyfract may be useful as a clinical sign of proximal tubular dysfunction.