Introduction

Top Abstract Introduction Materials and methods Results Discussion References

It is well recognized that sugars play an important role in many specific interactions in living systems (Varki, 1993), including adhesion of pathogens to cells, regulation of the half-life of proteins, and cell-matrix or cell-cell interactions (Karlsson, 1991). Associated with these processes, some tissues and cells have sugar recognition molecules on their surface. Accordingly, it is an attractive idea to use a sugar as a "ligand" to deliver drugs to selected sites or cells in the body. A number of studies have been carried out with this end in view (Chiu et al., 1994; Monsigny et al., 1994; Palomino, 1994).

Lectins are well known sugar recognition molecules in the body and, in this context, the asialoglycoprotein receptor and macrophage lectin have been studied in detail. Such lectins are classified as C-type lectins that characterize Ca⁺⁺-dependent binding to sugars (Quiocho, 1989). In particular, many studies have been carried out looking at delivery to the liver using the asialoglycoprotein receptor system because this receptor exhibits liver-specific expression (Monsigny et al., 1994). However, there are few reports about delivery using specific sugar uptake mechanisms to organs other than the liver.

Until now, large molecular weight carriers, e.g., proteins or liposomes, have been modified with sugars and used to study the factors controlling distribution in vivo. Among the agents that have been proposed for delivering drug molecules to the liver using the asialoglycoprotein receptor system are natural asialoglycoprotein, a synthetic sugar-modified protein the delivery of which can be controlled by changing the number of attached sugar residues (Monsigny et al., 1994). It is also well known that clustering of galactose residues increases affinity for the receptor (Sasaki et al., 1995). Our purpose is to develop a new system of delivery to the kidney using sugar modification. First, we prepared novel peptides by introducing different sugars containing an octamethylene spacer arm and then studied their tissue distribution in rats. Because we had previously studied the biological activity of carbohydrate-modified Arg-vasopressin (AVP) (Susaki et al., 1994), we selected AVP as a model low-molecular-weight peptide.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Materials

AVP and oxytocin (Oxy) were purchased from Peptide Institute (Osaka, Japan). [³H]AVP (vasopressin, 8-L-arginine, [Phenylalanyl-3,4,5-³H]-, 2890 GBq/mmol), [³H]Oxy (oxytocin, [tyrosyl-2,6-³H]-, 1443 GBq/mmol), [¹⁴C]p-aminohippuric acid (PAH), and [¹⁴C]inulin were obtained from New England Nuclear. All other reagents were of the highest grade available commercially.

Synthesis, Purification, and Characterization of Glycosylated Peptides

General Procedure. Melting points were determined on a Yanagimoto melting point apparatus. Proton nuclear magnetic resonance (¹H NMR) spectra were obtained on a Varian VXR-500 spectrometer at 25°C. Tetramethylsilane was used as an internal standard, except for spectra taken in D₂O where no internal standard was used; the HOD peak is assigned at 4.80 ppm. Optical rotations were measured with a Perkin-Elmer 241 polarimeter. Infrared spectra were obtained using a Hitachi 270-30 infrared spectrophotometer. Fast atom bombardment mass spectrometry (FAB-MS) spectra were obtained on a JEOL LMS-HX110 mass spectrometer. Column chromatography was performed using Merck silica gel 60 (230-430 mesh) or Silica Gel 60 from Nacalai Tesque (230-430 mesh). Preparative high-performance liquid chromatography (HPLC) was performed using a column of YMC SH 345-5 S5 120A ODS (020 × 300 mm) and developed with a mixture of 0.05% trifluoroacetic acid (TFA)-containing acetonitrile/water at a flow rate of 10 ml/min. Amino acid analysis was performed on a JEOL JLC-300 amino acid analyzer after hydrolysis of the glycoprotein with 6 N HCl at 110°C for 22 h.

Structure of Peptides and Glycosylated Derivatives. Structure of peptides in this paper, namely, AVP, Glc-O-C8-AVP, Gal-O-C8-AVP, Man-O-C8-AVP, Man()-O-C8-AVP, 2dGlc-O-C8-AVP, Oxy, Glc-O-C8-Oxy, and Man-O-C8-Oxy are shown in Fig. 1. The synthesis of Glc-O-C8-AVP, Gal-O-C8-AVP, Man-O-C8-AVP, and Man()-O-C8-AVP has been reported elsewhere (Susaki et al., 1998).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Chemical structure of carbohydrate-modified AVP and Oxy derivatives. Position of the 3H label is denoted by the asterisk.

Synthesis of 2dGlc-O-C8-AVP. Iodoalkoxylation (Horton et al., 1990) of 3,4,6-tri-O-acetyl-D-glucal (12.17 g, 44.7 nmol) with 8-(methoxycarbonyl) octanol (Kann et al., 1990) (15.53 g, 82.7 nmol) and N-iodosuccinicimide (16.07 g, 71.5 mmol) in acetonitrile (100 ml) was performed for 6 h at room temperature. The reaction mixture was diluted with ethyl acetate and washed with water and 10% Na₂S₂O₃ solution. The organic phase was dried over MgSO₄ and evaporated. Chromatography of the residue on a column of silica gel using toluene gave a 1:5 mixture of 8-(methoxycarbonyl)octyl 3,4,6-tri-O-acetyl-2-deoxy-2-iodo--D-glucopyranoside and 8-(methoxycarbonyl) octyl 3,4,6-tri-O-acetyl-2-deoxy-2-iodo--D-mannopyranoside as an oil. The mixture was separated by repeated silica gel chromatography. Tributyltin hydride (0.583 ml, 2.17 mmol) was added to a solution of 8-(methoxycarbonyl)octyl 3,4,6-tri-O-acetyl-2-deoxy-2-iodo--D-glucopyranoside (909 mg, 1.55 mmol) in benzene (15 ml). The mixture was stirred at 60°C for 1.75 h and then concentrated under reduced pressure. A solution of the residue in acetonitrile was washed with hexane and concentrated. Chromatography of the residue on a column of silica gel using toluene/acetone (20:1) gave 8-(methoxycarbonyl)octyl 3,4,6-tri-O-acetyl-2-deoxy-2--D-glucopyranoside as an oil. A mixture of 8-(methoxycarbonyl)octyl 3,4,6-tri-O-acetyl-2-deoxy-2--D-glucopyranoside in methanol (10 ml) and 28% sodium methoxide in methanol (0.17 ml) was stirred for 260 min. After addition of acetic acid (0.05 ml), the mixture was evaporated to dryness. Chromatography of the residue on a column of silica gel using chloroform/methanol (9:1) gave 8-(methoxycarbonyl)octyl 2-deoxy-2--D-glucopyranoside (446 mg, 95%) as a colorless solid. To a solution of 8-(methoxycarbonyl)octyl 2-deoxy-2--D-glucopyranoside (393 mg, 1.17 mmol) in methanol (3 ml), sodium hydroxide (93 mg) in water (3 ml) was added. The mixture was stirred at room temperature for 6 h and then 2 N HCl was added to bring the pH to 3.0. The resulting precipitate was separated from the solution by filtration, washed with water, and dried under vacuum to give 9-(2-deoxy--D-glucoxy)nonanoic acid (249 mg, 66%) as a colorless solid. Triethylamine (0.020 ml) was added to a solution of AVP (40 mg, 0.04 mmol) in methanol (2 ml) and the mixture evaporated to dryness. To the residue, N,N-dimethylformamide (DMF) (2 ml), 9-(2-deoxy--D-glucoxy)nonanoic acid (40 mg, 0.12 mmol), N-hydroxysuccinimide (NOBt) (22 mg, 0.16 mmol), and dicyclohexylcarbodiimide (DCC) (33 mg, 0.16 mmol) were added and the mixture was stirred for 18 h. After addition of 10% acqueous TFA solution to bring the pH to 2.0, the reaction mixture was purified by preparative HPLC to give 2dGlc-O-C8-AVP (15 mg). []_D 77.8° (c = 0.23, water). ¹H NMR (D₂O) : 1.20-1.34 (8H, m), 1.44 to 2.00 (8H, m), 2.04 to 2.36 (7H, m) 2.66 to 2.72 (1H, m), 2.85 (2H, d, J = 6.1 Hz), 2.89 to 3.08 (4H, m), 3.13 to 3.38 (6H, m), 3.69 to 3.97 (9H, m), 4.13 to 4.17 (1H, m), 4.32 to 4.36 (5H, m), 4.44 to 4.47 (1H, m), 6.79 (2H, d, J = 8.0 Hz), 7.02 (2H, d, J = 8.0 Hz), 7.27 (2H, d, J = 7.3 Hz), 7.37 to 7.44 (3H, m). FAB-MS m/z: 1386 (MH⁺). Amino acid ratios in 6 N HCl hydrolysate; Gly, 0.95; Arg, 0.97; Pro, 0.98; Cys, 1.57; Asp, 1.00; Glu, 0.99; Phe, 1.02; Tyr, 0.86; and ammonia, 3.05 (recovery of Asp 81%).

Synthesis of Glc-O-C8-Oxy. A mixture of 9-(-D-glucoxy)nonanoic acid (150 mg, 0.45 mmol), DCC (78 mg, 0.68 mmol), and N-hydroxysuccimimide (HOSu; 110 mg, 0.53 mmol) in 1,4-dioxane (8 ml) was stirred for 20 h. The reaction mixture was evaporated to dryness and chromatography of the residue on a column of silica gel using chloroform/methanol (9:1) gave 9-(-D-glucoxy)nonanoic acid N-hydroxysuccinimide ester (105 mg, 54%). Triethylamine (0.020 ml) was added to a solution of Oxy (45 mg, 0.04 mmol) in methanol (2 ml) and the mixture evaporated to dryness. To the residue, DMF (2 ml) and 9-(-D-glucoxy) nonanoic acid HOSu ester (60 mg, 0.14 mmol) were added and the mixture was stirred for 20 h. After addition of 10% aqueous TFA solution to bring the pH to 2.0, the reaction mixture was subjected to preparative HPLC to give Glc-O-C8-Oxy (30 mg, 61%). []_D 80.0° (c = 0.19, methanol). ¹H NMR (D₂O) : 0.93 (6H, d, J = 6.3 Hz), 0.96 (3H, d, J = 8.3 Hz), 0.97 (3H, t, J = 6.3 Hz), 1.08 to 1.16 (1H, m), 1.27 to 1.39 (9H, m), 1.51 to 1.59 (2H, m), 1.60 to 1.75 (5H, m), 1.95 to 2.17 (6H, m), 2.24 (2H, t, J = 7.3 Hz), 2.30 to 2.38 (1H, m), 2.40 to 2.45 (2H, m), 2.81 to 2.90 (2H, m), 2.95 (1H, dd, J = 9.0, 14.2 Hz), 3.01 (1H, dd, J = 6.4, 13.9 Hz), 3.06 (1H, dd, J = 9.0, 13.9 Hz), 3.21 (1H, dd, J = 7.8, 13.9 Hz), 3.25 to 3.31 (2H, m), 3.32 (1H, dd, J = 3.9, 13.9 Hz), 3.41 (1H, dd, J = 9.0, 9.8 Hz), 3.46 (1H, dd, J = 2.2, 5.9, 9.8 Hz), 3.51 (1H, dd, J = 9.0, 9.0 Hz), 3.69 (1H, dt, J = 9.8, 6.8 Hz), 3.72 to 3.81 (3H, m), 3.91 (1H, d, J = 17.3 Hz), 3.91 to 3.98 (2H, m), 3.98 (1H, d, J = 17.3 Hz), 4.19 (1H, dd, J = 5.9, 8.3 Hz), 4.32 to 4.38 (2H, m), 4.46 (1H, d, J = 8.1 Hz), 4.49 (1H, dd, J = 5.4, 8.3 Hz), 4.68 to 4.78 (3H, m), 4.82 to 4.85 (1H, m), 6.87 (1H, d, J = 8.5 Hz), 7.20 (1H, d, J = 8.5 Hz). FAB-MS m/z: 1326 (MH⁺). Amino acid ratios in 6 N HCl hydrolysate; Asp, 1.00; Cys, 1.86; Glu, 0.99; Gly, 1.00; Ile, 0.97; Leu, 1.03; Pro, 1.00; Tyr, 0.91; ammonia, 2.94 (recovery of Asp 89%).

Synthesis of Man-O-C8-Oxy. Condensation of 9-(-D-mannoxy)nonanoic acid (237 mg, 0.71 mmol) with HOSu (122 mg, 1.06 mmol) using DCC (176 mg, 0.85 mmol) as described for the synthesis of 9-(-D-glucoxy)nonanoic acid HOSu ester gave 9-(-D-mannoxy)nonanoic acid HOSu ester (176 mg, 57%) as a colorless oil. Condensation of 9-(-D-mannoxy)nonanoic acid HOSu ester (60 mg, 0.14 mmol) with Oxy (45 mg, 0.04 mmol) as described for the synthesis of Glc-O-C8-Oxy gave Man-O-C8-Oxy (30 mg, 61%) as a colorless oil. []_D 80.0° (c = 0.19, methanol). ¹H NMR (D₂O) : 0.91 (6H, d, J = 6.1 Hz), 0.94 (3H, d, J = 8.3 Hz), 0.96 (3H, t, J = 7.1 Hz), 1.06 to 1.13 (1H, m), 1.22 to 1.36 (9H, m), 1.49 to 1.56 (2H, m), 1.57 to 1.72 (5H, m), 1.92 to 2.13 (6H, m), 2.21 (2H, t, J = 7.6 Hz), 2.28 to 2.33 (1H, m), 2.40 (2H, t, J = 7.6 Hz), 2.82 (H, dd, J = 5.4, 6.8 Hz), 2.78 to 2.87 (1H, m), 2.93 (1H, dd, J = 10.0, 13.4 Hz), 2.99 (1H, dd, J = 7.6, 13.4 Hz), 3.06 (1H, dd, J = 9.3, 10.0 Hz), 3.15 to 3.39 (4H, m), 3.58 (1H, dd, J = 9.5, 9.8 Hz), 3.61 to 3.68 (2H, m), 3.72 to 3.79 (3H, m), 3.88 (1H, d, J = 17.3 Hz), 3.85 to 3.92 (2H, m), 3.96 (1H, d, J = 17.3 Hz), 4.16 (1H, dd, J = 7.3, 14.4 Hz), 4.30 to 4.35 (2H, m), 4.45 to 4.48 (1H, m), 4.63 (6H, m), 6.84 (1H, d, J = 7.8 Hz), 7.18 (1H, d, J = 7.8 Hz). FAB-MS m/z: 1326 (MH⁺). Amino acid ratios in 6 N HCl hydrolysate; Asp, 1.00; Cys, 1.86; Glu, 0.99; Gly, 1.00; Ile, 0.97; Leu, 1.03; Pro, 1.00; Tyr, 0.91; ammonia, 2.94 (recovery of Asp 89%).

Radiolabeled Peptides. ³H-Labeled Glc-O-C8-AVP, Gal-O-C8-AVP, Man-O-C8-AVP, and Man()-C8-AVP were obtained from Daiichi Pure Chemical Co., Ltd. (Tokai, Japan). [³H]2dGlc-O-C8-AVP, [³H]Glc-O-C8-Oxy, and [³H]Man-O-C8-Oxy were synthesized from [³H]AVP or [³H]Oxy by a procedure similar to that for cold glycopeptides in our laboratory. The specific radioactivities are as follows: [³H]AVP (2800 GBq/mmol), [³H]Glc-O-C8-AVP (140 GBq/mmol), [³H]Gal-O-C8-AVP (129 GBq/mmol), [³H]Man-O-C8-AVP (134 GBq/mmol), [³H]Man()-O-C8-AVP (80.7 GBq/mmol), [³H]2dGlc-O-C8-AVP (235 GBq/mmol), [³H]Oxy (1440 GBq/mmol), [³H]Glc-O-C8-Oxy (334 GBq/mmol), and [³H]Man-O-C8-Oxy (168 GBq/mmol).

Animals

Male Sprague-Dawley rats were purchased from Sankyo Labo Service Co., Ltd. (Tokyo, Japan) and were used when they were 6 to 7 weeks old and weighed 180 to 230 g. Food and water were available ad libitum. Each group consisted of three to four rats.

Early-Phase Tissue Uptake of Glycosylated AVP

The plasma concentration-time profiles of radioactivity over a 5-min period and the tissue concentration of radioactivity after i.v. administration of ³H-glycosylated AVP were determined as follows. Rats anesthetized with diethyl ether received i.v. injections of 1 nmol/kg [³H]AVP (adjusted to 185 GBq/mmol with unlabeled AVP) or ³H-labeled carbohydrate-conjugated derivatives. Blood samples (0.2 ml) were collected from the jugular vein by syringe at 1, 2, 3, and 5 min and plasma was quickly separated by centrifugation. Immediately after the last blood sample, the rats were sacrificed, and liver, kidney, heart, lung, small intestine, bone marrow, brain, muscle, and spleen were excised and rinsed with saline. Plasma samples, portions of tissues, or entire tissues were weighed and combusted using an automatic sample combustion system (Aloka ASC-113; Tokyo, Japan) to determine the radioactivity using a liquid scintillation counter (Aloka LSC-602). The apparent uptake clearance (CL_up,app) for each tissue was calculated using the following equation:

(1)

where X(5min) is the tissue concentration at 5 min and AUC_0-5min is the area under the plasma concentration-time curve from 0 to 5 min. The CL_up,app of ³H-labeled Oxy and glycosylated Oxy derivatives was determined with the method used for AVP.

Integration Plots for [³H]Glc-O-C8-AVP

The uptake clearance (CL_up) by kidney was calculated using the following equation, as described previously (Kuwabara et al., 1995a):

(2)

where X(t) and Cp(t) are the kidney and plasma concentrations at time t, AUC_0-t is the area under the plasma concentration-time curve from time 0 to time t, and Ve,app is the initial extracellular distribution volume defined as X(0)/Cp(0). Rats anesthetized with diethyl ether received i.v. injections of 1 nmol/kg [³H]Glc-O-C8-AVP and 20 nmol/kg [¹⁴C]PAH, and their renal uptake was determined at 1, 3, and 5 min. In the same way, rats were injected with 300 nmol/kg [³H]Glc-O-C8-AVP along with 1 µmol/kg [¹⁴C]inulin and their renal uptake was measured.

Saturation of CL_up,app by Tissues

A fixed amount of [³H]Glc-O-C8-AVP and different amounts of unlabeled Glc-O-C8-AVP dissolved in saline were injected into the femoral vein. The doses of Glc-O-C8-AVP were 1, 10, 30, 60, 100, and 300 nmol/kg. Blood sampling and the calculation of CL_up,app were performed as described above. The early-phase (0-5 min) plasma clearance [CLtot(early)] was calculated from eq. 3 after fitting the plasma disappearance time profile (Cp(t)) to a single exponential equation (Cp(t) = Ae^t):

(3)

where A is the initial plasma concentration and  is the elimination rate constant (Yanai et al., 1990). Saturable tissue uptake clearance can be expressed by Michaelis-Menten kinetic parameters as:

(4)

where V_max and K_m,app represent the maximum velocity and apparent Michaelis-Menten constant for the saturable process, respectively, and CL_ns represents nonsaturable uptake clearance. The initial plasma concentration (CO) was estimated by fitting the plasma disappearance-time profile to single exponential equation (Cp(t) = Ae^t) described above. The CO and CL_up,app values were simultaneously fitted to eq. 4 using a SAMM II program (SAMM Institute).

Effect of Cold Glc-O-C8-AVP Pretreatment on Early-Phase Tissue Uptake of [³H]Glc-O-C8-AVP

Rats received i.v. injections of [³H]Glc-O-C8-AVP (1 nmol/kg) and the tissue uptake determined at 0.25, 0.5, 1, 2, and 4 h after the i.v. administration of unlabeled Glc-O-C8-AVP (300 nmol/kg) via the tail vein. CL_up,app values were determined as described above.

Plasma Concentration-Time Profiles of Glc-O-C8-AVP in Rats

A fixed amount of [³H]Glc-O-C8-AVP and unlabeled Glc-O-C8-AVP dissolved in saline were injected into the femoral vein. The dose of Glc-O-C8-AVP was 10 and 300 nmol/kg. Plasma samples were extracted with Sep-Pak C₁₈ (Waters) cartridges and intact [³H]Glc-C8-N-AVP was measured by reversed-phase HPLC. HPLC analyses were carried out on a Beckman HPLC system fitted with a GL Science Inertsil ODS-2 column (4.6 × 250 mm). The mobile phase was acetonitrile/water (23% v/v) containing 0.1% TFA at a flow rate of 1 ml/min. Radioactivity in the eluate was measured in a liquid scintillation counter. The typical retention time for [³H]Glc-O-C8-AVP was 8 min, and the recovery of [³H]Glc-O-C8-AVP from plasma was more than 95%.

Binding of Glycosylated Peptides to Membrane Fractions Prepared from Kidney and Liver

Rat kidney and liver microsomes were prepared by centrifugation (Stassen et al., 1982) and incubated (1 mg/ml as protein concentration) with the ³H-labeled glycosylated derivative of AVP (20 pmol/ml) in phosphate-buffered saline (pH 7.4) containing 0.1% (w/v) bovine serum albumin on ice for 10 min. After ultracentrifugation (50,000g for 5 min at 4°C), the supernatant was aspirated and the precipitate dissolved in 1% Tween 20, and the radioactivity was determined in a liquid scintillation counter. In the Scatchard plot analysis of the ³H-labeled AVP derivatives, a preliminary binding assay was performed to determine the incubation time for equilibration. This was found to be 10 min for Glc-O-C8-AVP and 1 h for Man-O-C8-AVP and 2dGlc-O-C8-AVP, whereas the other conditions were as described above. To correct for any supernatant remaining in the precipitate, [¹⁴C]sucrose was added to the incubation buffer.

Inhibition of [³H]Glc-O-C8-AVP Binding to Kidney Membrane Fraction by Glucose or Different Glycosides

[³H]Glc-O-C8-AVP (20 pmol/ml), inhibitors, and kidney microsomes (1 mg/ml as protein concentration) were incubated in phosphate-buffered saline (pH 7.4) containing 0.1% bovine serum albumin on ice for 1 h. The other conditions were as in the binding assay described above. All samples were examined in triplicate and IC₅₀ values were evaluated by log-logit analysis of the mean values from three to four points at a ligand concentration of approximately 50% inhibition.

Autoradiographic Study in Vivo

Under light ether anesthesia, rats were given [³H]AVP or ³H-labeled AVP derivatives via the femoral vein. Then, at fixed intervals, kidneys were excised quickly and immersed in liquid nitrogen. The sections prepared by microtome were dried under vacuum. The imaging plates (Fuji Film) were placed in close contact with the sections and the density of radioactivity in the autoradiographic images was measured using an image analyzer (BAS2000, Fuji Film). To study the binding sites of [³H]AVP and ³H-labeled glycosylated derivatives of AVP at the microscopic level, the sections were mounted on glass slides and dipped into nuclear track emulsion and exposed. After developing and fixing the preparations, they were stained with H&E.

Statistical Methods

Comparison of the CL_up,app values was performed using Dunnett's test. Statistical significance was taken as p < .05.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

Tissue Distribution of the Glycosylated Derivatives of AVP. Table 1 shows the CL_up,app (ml/min/g tissue) of AVP derivatives in rats. All glycosylated derivatives showed lower CL_up,app values than AVP in all tissues except the kidney, and there was no difference among the derivatives. In contrast to this, there were clear differences in the CL_up,app of the kidney as far as the different glycosylated derivatives were concerned. Although the CL_up,app of glucosyl, mannosyl, and 2-deoxyglucosyl derivatives was more than 2 ml/min/g tissue, the CL_up,app of Gal-O-C8-AVP and Man()-C8-AVP was almost the same as that of AVP, i.e., 0.36 ~ 0.67 ml/min/g tissue.

Binding of Glycosylated Peptides to Membrane Fractions. The binding of all derivatives to the liver microsomal fraction was less than that of AVP, and there were no clear differences among the glycosylated derivatives (0.15 ~ 0.48 pmol/mg protein). The binding of Glc-O-C8-AVP and Man-O-C8-AVP to the kidney microsomal fraction was 3.8 and 10 pmol/mg protein, respectively, whereas that of Gal-O-C8-AVP was 0.32 pmol/mg protein (Fig. 2). The ratio of the amount bound to the kidney microsomal fraction to that bound to the liver microsomal fraction (Bkid/Bliv) for Glc-O-C8-AVP and Man-O-C8-AVP was 25 and 42, respectively. In contrast, the Bkid/Bliv of AVP, Gal-O-C8-AVP, and Man()-O-C8-AVP was 1.8, 1.9, and 3.5, respectively. These results suggest that Glc-O-C8-AVP and Man-O-C8-AVP, which are taken up by the kidney in vivo, are bound specifically to the kidney membrane.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Binding of carbohydrate-modified AVP derivatives to liver (white bar) or kidney (black bar) microsomal fractions. The initial concentration of 3H-labeled derivatives was 20 pmol/ml, and the microsome concentration was 1 mg protein/ml. Values and vertical bars represent means ± S.E. (n = 3).

8

7

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Scatchard plots of 3H-labeled Glc-O-C8-AVP (), Man-O-C8-AVP (), and 2dGlc-O-C8-AVP () binding to rat kidney microsomal fraction.

Inhibition of [³H]Glc-O-C8-AVP Binding to Kidney Membrane by Glucose of Different Glycosides. Next, we studied the inhibition of [³H]Glc-O-C8-AVP binding to the kidney membrane by glucose or different glycosides. Although glucose and methyl -D-glucoside (Glc-O-Me) had no inhibitory effect on binding up to 1 mM (Fig. 4), hexyl -D-glucoside (Glc-O-C5-Me; IC₅₀ = 2.9 nM), or octyl -D-glucoside (Glc-O-C7-Me; IC₅₀ = 5.9 nM) did. These IC₅₀ values were almost 10 times that of Glc-O-C8-AVP (IC₅₀ = 0.075 nM) (Fig. 4). The glycoside structure was considered to play an important role in this recognition by the kidneys.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Inhibition of specific binding of Glc-O-C8-AVP to rat kidney membranes by glucosides. Inhibition of binding of [3H]Glc-O-C8-AVP (20 pmol/ml) to membrane (1 mg/ml) was measured in the presence of the glucosides as inhibitors, i.e., Glc-O-C8-AVP (), Glc-O-C7-Me (), Glc-O-C5-Me (), Glc-O-Me (), and glucose ().

Uptake Clearance of Glc-O-C8-AVP at Low and High Doses in Vivo. Fig. 5 shows the integration plots for Glc-O-C8-AVP to evaluate the CL_up by the kidneys. When Glc-O-C8-AVP was administered at a low dose (1 nmol/kg), the CL_up was 1.7 ml/min/g tissue, and the CL_up of PAH, which is known to be taken up by the kidney in a blood flow-limited manner (Chaudhuri et al., 1987), was 2.4 ml/min/g. Considering that neither Glc-O-C8-AVP nor PAH (Hori et al., 1991) distributes to red blood cells, we estimated the single-pass renal extraction ratio of Glc-O-C8-AVP to be 71%. The CL_up of Glc-O-C8-AVP at the high dose (300 nmol/kg) was 0.33 ml/min/g, i.e., about one-fifth that at the low dose (1 nmol/kg). In addition, the CL_up of coadministered inulin (0.18 ml/min/g) was comparable to the CL_up of Glc-O-C8-AVP at the high dose. This suggests that the renal uptake is fully saturated at the high dose (300 nmol/kg).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Integration plots for the early-phase uptake (and/or binding) of [3H]Glc-O-C8-AVP in rat kidney after administration via the femoral vein. A total of 1 nmol/kg Glc-O-C8-AVP () was coadministrated with 20 nmol/kg [14C]PAH (). A total of 300 nmol/kg Glc-O-C8-AVP () was coadministrated with 1 µmol/kg [14]Cinulin (). The line represents the regression line using the tissue uptake data at 1, 3, and 5 min. The slope of the line represents the CLup.

Effect of Dose on Tissue Uptake Clearance. The changes in the early plasma disappearance clearance and tissue uptake clearance with increasing dose were evaluated to clarify the saturable renal uptake. The CLtot(early) of Glc-O-C8-AVP, which was estimated from the plasma disappearance, did not change until 10 nmol/kg, and was about 7 ml/min/rat. However, above that dose, it decreased clearly with increasing dose (Fig. 6). The CL_up,app by kidney also decreased with increasing dose, and the magnitude of this reduction was almost the same as that of CLtot(early). However, the CL_up,app of the other tissues, such as liver, spleen, lung, and muscle, did not show any clear change with increasing dose (Fig. 6B). From these results, the saturation of CLtot(early) could be explained by saturation of renal uptake clearance.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Dose-dependent change in apparent tissue uptake clearance (CLup,app) in several organs and early-phase plasma clearance [CLtot(early)] of [3H]Glc-O-C8-AVP (1 nmol/kg) with different doses of unlabeled Glc-O-C8-AVP. A, CLtot(early) () and CLup,app of kidney (). B, CLup,app of lung (), spleen (), muscle (), and liver (). Each point represents the mean ± S.E. for three rats. *Significantly different from the value at 1 nmol/kg (P < .05).

Recovery of Renal CL_up,app after Administration of Excess Glc-O-C8-AVP. At designated times after i.v. administration of unlabeled Glc-O-C8-AVP at a dose sufficient for saturation (300 nmol/kg), [³H]Glc-O-C8-AVP (1 nmol/kg) was administered i.v., and the tissue CL_up,app was measured at 5 min. The renal CL_up,app exhibited a clear reduction shortly after unlabeled Glc-O-C8-AVP was administered (Fig. 7). The CL_up,app recovered in a time-dependent fashion and had returned to the control level 2 h after administration of unlabeled Glc-O-C8-AVP. To evaluate the effect of the unlabeled Glc-O-C8-AVP remaining in the plasma, plasma concentrations of Glc-O-C8-AVP were determined for 4 h (Fig. 8). When the renal uptake was not saturated at 10 nmol/kg, the initial blood concentration was about 90 pmol/ml. This concentration was almost the same as the plasma concentration 60 min after Glc-O-C8-AVP administration at a saturating dose of 300 nmol/kg. This suggests that within 60 min of the administration of unlabeled Glc-O-C8-AVP, uptake of [³H]Glc-O-C8-AVP could be influenced by the unlabeled Glc-O-C8-AVP remaining in the plasma.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Time-dependent changes in tissue uptake clearance (CLup,app) of 3H-labeled Glc-O-C8-AVP (1 nmol/kg) after i.v. administration of unlabeled Glc-O-C8-AVP (300 nmol/kg) to Sprague-Dawley rats. Value at time 0 represents CLup,app of control (untreated) rats. Values and vertical bars represent means ± S.E. of three to four rats. *Significantly different from the control (P < .05)

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Comparison of plasma concentration-time profiles of Glc-O-C8-AVP in rats after low and high doses. Rats received Glc-O-C8-AVP (, 10 nmol/kg; , 300 nmol/kg) i.v. Each point represents the mean ± S.E. for three rats.

Influence of Plasma Concentration of Glc-O-C8-AVP on Renal CL_up,app. We have tried to distinguish between the two mechanisms (down-regulation of receptors or competition by unlabeled ligand) for reduction in the CL_up,app of [³H]Glc-O-C8-AVP shown in Fig. 7 as follows: 1) From the experiment on the effect of dose (1 ~ 300 nmol/kg) on tissue uptake clearance (Fig. 6), the initial CO and kidney CL_up,app were estimated. 2) From the experiment on the recovery of CL_up,app after administration of unlabeled Glc-O-C8-AVP sufficient to cause saturation, the CL_up,app was measured at selected intervals (15 ~ 240 min) (Fig. 7), and the plasma concentration at each interval was estimated from the plasma concentration-time profile after administration of Glc-O-C8-AVP at a dose of 300 nmol/kg (Fig. 8). We then compared the CL_up,app-plasma concentration profiles obtained by 1) and 2) (see above) at the same plasma concentrations. If receptor-mediated endocytosis is essential for renal uptake, then the CL_up,app obtained by 2) should be less than that obtained by 1). From the plots obtained (Fig. 9), the CL_up,app measured by 2) was somewhat less than that measured by 1) when the plasma concentration ranged from 100 to 400 pmol/ml (this was 15 ~ 60 min after administration of unlabeled Glc-O-C8-AVP). Therefore, RME may be partially related to the renal uptake of Glc-O-C8-AVP. However, the difference was small and, consequently, the reduction in CL_up,app after administration of Glc-O-C8-AVP, sufficient to cause saturation, could be largely explained by competition with unlabeled Glc-O-C8-AVP remaining in the plasma (Figs. 7-9).

View larger version (16K): [in this window] [in a new window]   Fig. 9.   The relationship between plasma concentration and renal CLup,app of Glc-O-C8-AVP. CLup,app-plasma concentration profiles were compared between the plot obtained using data from the experiment on the effects of dose (1 ~ 300 nmol/kg) on CLup,app () and the plot obtained using data from the experiment on the recovery of CLup,app after administration of unlabeled Glc-O-C8-AVP, at a concentration sufficient to cause saturation recovery () as described in Results. Each point represents the mean ± S.D. of the values in three or four rats.

Distribution of the Derivatives in the Kidney. The renal distribution of the glycosylated derivatives was evaluated by semimicro- (Fig. 10) and microautoradiography (Fig. 11). Unmodified AVP distributed in the medulla, especially in the renal calices although, in contrast, Glc-O-C8-AVP and Man-O-C8-AVP distributed mainly in the cortex (Fig. 10). In addition, Glc-O-C8-AVP did not distribute in the glomerulli but rather in the proximal convoluted tubules (Fig. 11).

View larger version (33K): [in this window] [in a new window]   Fig. 10.   Intrarenal distribution of 3H-labeled carbohydrate-modified AVP derivatives after i.v. administration for 5 min determined by autoradiography. A, Glc-O-C8-AVP, 10 nmol/kg. B, Gal-O-C8-AVP, 10 nmol/kg. C, Man-O-C8-AVP, 10 nmol/kg. D, AVP, 1 nmol/kg. E, Glc-O-C8-AVP, 300 nmol/kg.

View larger version (141K): [in this window] [in a new window]   Fig. 11.   Distribution of 3H radioactivity in cortex of the kidney after [3H]Glc-O-C8-AVP (10 nmol/kg) i.v. administration for 5 min determined by microautoradiography. G, glomerulus; Pc, proximal convolution.

Tissue Uptake of Glycosylated Derivatives of Oxy. The structures of the Oxy and glycosylated derivatives are shown in Fig. 1. In comparison to the renal CL_up,app of Oxy (0.39 ml/min/g), that of the glucosylated (1.05 ml/min/g) and mannosylated (3.59 ml/min/g) derivatives was clearly higher (Table 2), similar to the AVP and glycosylated derivatives. When distribution to tissues other than kidney was considered, the CL_up,app of liver and small intestine was higher than that of Oxy. These results differed from that for AVP and its derivatives (Table 1).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

Of the AVP derivatives modified with different sugars that were the subject of this investigation, some specific ones such as the glucosylated and mannosylated derivatives exhibited high renal uptake in vivo (Table 1). In comparison to this, galactosylated and -mannosylated derivatives exhibited relatively low renal uptake similar to unmodified AVP itself (Table 1; Fig. 10). These results suggest that there are distinct structural differences among these sugars as far as renal uptake is concerned. Only those derivatives exhibiting high renal uptake in vivo bound specifically to the kidney microsomal membrane (Figs. 2 and 3). As far as the specific binding of [³H]Glc-O-C8-AVP to the kidney microsomal membrane is concerned, glucose and methyl -D-glucoside produced no inhibition, despite the fact that hexyl -D-glucoside and octyl -D-glucoside inhibited binding (Fig. 4). This indicates that not only sugar moieties but also aglycones play an important role in such recognition by the kidney.

In the tissue uptake analysis of Glc-O-C8-AVP in vivo, we found that renal uptake was saturated with increasing dose (Fig. 6), suggesting that there is a specific uptake mechanism in kidneys. The K_m,app of the renal uptake of Glc-O-C8-AVP, estimated from the initial plasma concentration and CL_up,app, was 203 nM (Fig. 9). Because the K_m,app evaluated by this method is defined with respect to the circulating plasma concentration, the K_m defined in terms of the plasma concentration in renal capillaries needs to be measured to compare the K_m in vivo with the K_d obtained by binding analysis in vitro. If the initial plasma concentration is 203 nM, CL_up,app can be estimated to be 1.7 ml/min/g (eq. 4). Comparing the CL_up,app with the renal plasma flow (2.4 ml/min/g) evaluated by PAH uptake clearance (Fig. 5), we estimated the renal extraction ratio (ER) to be 0.71. When the K_m,app (203 nM) is transformed as described below to define the plasma concentration in the renal capillaries according to the well stirred and parallel-tube models (Pang and Rowland, 1977): K_m,app (well-stirred model) = 203 · (1  ER) = 57 nM; K_m,app (parallel tube model) = 203 · ER/ln(1/(1  ER)) = 116 nM.

In addition, considering that the unbound fraction of Glc-O-C8-AVP in plasma is 0.71, the true K_m can be estimated to be 40 ~ 82 nM. This value is comparable with the K_d (55 nM) obtained from the in vitro kidney membrane binding study (Fig. 3), which strongly suggests that the specific binding in vitro is critical for renal uptake in vivo.

There are two routes of renal uptake, one is from circulating blood through the basolateral membrane and the other is from the luminal side through the brush border membrane after glomerular filtration. Consequently, the latter uptake clearance cannot be greater than the glomerular filtration clearance. The CL_up of Glc-O-C8-AVP at a dose of 1 nmol/kg, estimated by integration plot, was 1.7 ml/min/g (Fig. 5), and the glomerular filtration clearance was estimated from the CL_up of inulin which was 0.18 ml/min/g (Fig. 5). In addition, the glomerular filtration clearance of Glc-O-C8-AVP was 0.13 ml/min/g, calculated from the glomerular filtration clearance of inulin (0.18, as described above) multiplied by the unbound fraction of Glc-O-C8-AVP in plasma (0.71). Because the apparent renal uptake clearance of Glc-O-C8-AVP (1.7 ml/min/g) is 13 times larger than the glomerular filtration clearance (0.13 ml/min/g), this suggests that renal uptake is mainly from blood. However, the CL_up of Glc-O-C8-AVP at a dose of 300 nmol/kg, when the specific renal uptake is saturated, was 0.33 ml/min/g, and the unbound clearance (0.46 ml/min/g), corrected for the unbound fraction in plasma, approached that of inulin (0.18 ml/min/g). Therefore, transport in the kidney can largely be explained by glomerular filtration at a high dose. Together, these results allow us to conclude that the saturable and specific uptake of Glc-O-C8-AVP in kidneys is mainly from blood via the basolateral membrane.

Two systems, receptor-mediated endocytosis (RME) and transporter-mediated active transport, have been identified as tissue-specific uptake mechanisms. In the case of bioactive peptides such as epidermal growth factor (Kim et al., 1988; Yanai et al., 1990; Kim et al., 1991), hepatocyte growth factor (Liu et al., 1992), and granulocyte-stimulating factor derivatives (Kuwabara et al., 1994, 1995a, b), these have been shown to be taken up by RME. When a dose sufficient to cause saturation is administered, the receptor is down-regulated, because of internalization of the receptor with the ligand. Therefore, the CL_up decreases. Previously, we reported that RME can be distinguished by an analysis of the recovery of CL_up with time (Kim et al., 1988; Yanai et al., 1990). Using this approach, we tried to show that the renal uptake mechanism reported here is due to either the receptor or transporter. The renal CL_up,app of [³H]Glc-O-C8-AVP exhibited a clear reduction shortly after exposure to excess unlabeled Glc-O-C8-AVP (300 nmol/kg), and this was then followed by a gradual recovery to the control level (Fig. 7). Possible reasons for the reduction in renal CL_up,app are 1) competition between [³H]Glc-O-C8-AVP and unlabeled Glc-O-C8-AVP remaining in blood and/or 2) down-regulation of the receptor. An analysis of the relationship between the plasma concentration of Glc-O-C8-AVP and renal CL_up,app is shown in Fig. 9. The CL_up,app obtained from the data under "Effect of Cold Glc-O-C8-AVP Pretreatment on Early-Phase Tissue Uptake of [³H]Glc-O-C8-AVP" was somewhat smaller than that obtained from the data under "Effect of Doses of Glc-O-C8-AVP on CL_up,app by Tissues." However, the difference was small and, consequently, the reduction in CL_up,app after administration of excess unlabeled Glc-O-C8-AVP, could be mainly explained by competition with unlabeled Glc-O-C8-AVP remaining in the plasma (Figs. 7-9). As yet, we have not been able to discover from these results if the renal mechanism is due to receptor or transporter.

As far as the structural requirements of glycosylated peptides for this renal uptake are concerned, we began by considering the structure of the individual sugars involved. The renal recognition of the D-galactosyl derivative was markedly lower than for the D-glucosyl or D-mannosyl derivative. Therefore, we suggest that in this carbohydrate recognition system it is important that the hydroxyl group at the C-4 position is not axial. Also, the hydroxyl group at the C-2 position is unimportant and it can be substituted for hydrogen (Fig. 1; Table 1). In addition, from the results involving the mannosyl derivatives, we suggest that the -anomer is recognized much more easily than the corresponding -anomer (Table 1). In the case of lectins or sugar transporters, which have been the subject of detailed studies as sugar-binding molecules, hydrogen and hydrophobic bonds are both important for specific sugar binding (Quiocho, 1989; Bourne et al., 1993; Elgavish and Shaanan, 1997). Because the pattern of sugar recognition described above is glucose/mannose, this suggests a similarity to concanavalin A (Vyas, 1991), a plant lectin. The mechanism for the renal uptake of glycosylated peptides remains unsolved.

What about the structure of the peptide? The derivatives of Oxy exhibited a high renal CL_up,app like the AVP derivatives (Tables 1 and 2). As far as the structures of AVP and Oxy are concerned, AVP containing Arg⁸ is basic whereas Oxy containing Leu⁸ is neutral (Fig. 1). Because both derivatives appear capable of renal targeting, we suggest that the peptide need not necessarily be cationic. Considering the distribution of the Oxy derivatives to other tissues, the CL_up,app of liver and small intestine increased following modification with sugars, unlike the AVP derivatives (Table 1). Kidney, liver, and small intestine have epithelial cells that have many transport systems, and some common transporters have been reported in these tissues (Sadee et al., 1995; Thorens, 1996). More detailed studies are needed to explain the differences among these derivatives as far as uptake by various tissues is concerned.

Glycosylated AVP was shown by autoradiography to be mainly located in the proximal tubules of the renal cortex and outer medulla (Figs. 10 and 11). The proximal tubule has a high capacity for the transepithelial transport of organic compounds and peptides including tubular secretion and reabsorption, and many transport mechanisms have been reported (Sadee et al., 1995; Pager, 1996; Saito et al., 1996). We have examined the relationship between the transporters in the proximal tubule identified up to now in terms of the structures of the glycosylated peptide substrates investigated in this report. Renal uptake clearly depends on the sugar involved and is independent of the substrate basicity (Tables 1 and 2; Fig. 2). Thus, this differs from mechanisms such as the organic cation transporter, organic anion transporter, amino acid transporters, and peptide transporters. Sugar transporters can be classified as facilitated sugar transporters and Na⁺/glucose cotransporters (Hediger and Rhoads, 1994; Thorens, 1996). Transport by the former depends on a concentration gradient of the substrate and, therefore, cannot produce a concentration of the substrate in tissues. The latter, related to glucose reabsorption from urinary tubules (Sadee et al., 1995; Thorens, 1996), is located on the brush border membrane of epithelial cells of the urinary tract. However, renal uptake of glycosylated peptides is mainly from blood and glucose at 10 mM does not inhibit the specific binding of [³H]Glc-O-C8-AVP to the kidney membrane (Fig. 4). These results indicate that the sugar transporters identified so far cannot explain the renal uptake of glycosylated peptides.

Kidney lectin has been studied as a sugar-binding molecule in mammalian kidneys (Matsumoto et al., 1986), and this compound has been shown to belong to the annexin family (Kojima et al., 1992), members of which distribute to many organs in addition to the kidney (Kojima et al., 1996). Because kidney lectin recognizes acidic sugars and is found in many tissues, it may not play a significant role in the renal uptake of glycosylated peptides investigated in our study. Therefore, it is difficult to explain the renal uptake of glycosylated peptides by transporters and sugar recognition molecules reported so far.

It is very important to develop tissue-specific targeting vectors to achieve highly efficient drug delivery systems. In the case of kidney targeting, there have been reports of some methods such as peptide reabsorption after glomerular filtration through the brush border membrane (Franssen et al., 1992) and prodrug activation by kidney-specific enzymes (Elfarra et al., 1995). However, renal delivery of drugs from blood via the basolateral membrane has not been reported until now. In this report, we have demonstrated a novel method of delivering peptides from blood to the kidney. The most important application of this renal uptake mechanism will be delivery of drugs to the kidney, especially in the case of proximal tubule disorders. In addition, from a basic physiological point of view, the role of the renal uptake of glycosylated peptides is interesting and deserves further study.