Fluorescent derivatization of nitrite ions with 2,3-diaminonaphthalene utilizing a pH gradient in a Y-shaped microchannel.

The on-chip derivatization of nitrite ions with 2,3-diaminonaphthalene (DAN) utilizing a pH gradient formed in a Y-shaped microchannel was investigated. Nitrite ions react with DAN at low pH, and strongly fluoresced at high pH. Therefore, a reaction at low pH followed by the addition of a strong alkaline solution is the usual procedure in a batch scheme. However, a strong alkaline solution, like an NaOH aqueous solution, erodes the wall of the microchannels in substrates made of glass or polymers, and has not been considered suitable for use in microchannels. We first investigated the derivatization reaction and fluorescent properties of nitrite ions with DAN. We found that the on-chip fluorescent derivatization reaction and detection without the addition of an alkaline solution is possible by controlling the pH values of the nitrite solution and the DAN solution to form a suitable pH gradient by utilizing a buffering effect of triethanolamine solution, which is used as an NO2 gas-absorption medium. These results have suggested the feasibility of novel reaction schemes which can provide the desired products due to a controlled pH gradient in the microchannels, as well as the possibility of an on-site monitoring microchip device for ambient NO2.

Renal targeting of arginine-vasopressin by modification with carbohydrates at the tyrosine side chain.

To extend the utility of a renal targeting system using carbohydrate derivatives, we investigated the in vivo tissue distribution in rats of arginine-vasopressin (AVP) derivatives modified at the phenolic hydroxy group of tyrosine by linking it to some sugars, namely D-glucose, D-galactose, D-mannose and L-fucose, via an octamethylene group. The glycosyl and mannosyl derivatives of AVP exhibit renal-selective distribution in vivo. In addition, the glucosyl and mannosyl derivatives exhibited specific binding to the kidney microsomal fraction in vitro. Modification with D-glucose or D-mannose at the tyrosine side chain is a suitable methodology for renal targeting, as well as at N-terminal amine.

Structural requirements for alkylglycoside-type renal targeting vector.

PURPOSE: We have previously shown Glc-S-C7-Me (octyl beta-D-thioglucoside) exhibits renal targeting potential in vivo in addition to its specific binding to the renal membrane fraction in vitro. Thus, "alkylglycoside" is considered to be a novel targeting vector for the kidney (1,2). The present study is designed to clarify the structural requirements for alkylglycoside as a renal targeting vector. METHODS: Inhibitory effects of various sugars and glycosides on 3H-Glc-S-C7-Me binding to the kidney membrane fraction were evaluated by a centrifugation method. RESULTS: As far as the sugar moiety is concerned, no other sugars except D-aldohexose and D-aldohexose derivatives (containing F, S, and N) showed greater inhibition than D-glucose. Therefore, octylthio derivatives of various D-aldohexose were prepared and their inhibitory effects were investigated. The following findings were obtained: Equatorial OH at 4 position is essential; OH at 2 position can have either orientation or be deleted. As far as the alkyl moiety is concerned, the length, branching and electrical environment in the region of the glycoside bond are important; aromatic structures can substitute for the alkyl portion; the preferred glycoside bonding atom is as follows: S > NH > O. CONCLUSIONS: The structural requirements for the renal targeting vector have been identified to be as follows: a hydrophobic group (alkyl chain or aromatic ring) should be introduced to a sugar (D-glucose, D-mannose, or 2-deoxy-D-glucose) via a beta-glycoside binding atom (S > NH > O).

Specific renal delivery of sugar-modified low-molecular-weight peptides.

To develop a novel delivery system for peptides involving sugar modification, Arg-vasopressin (AVP) was modified by linking it to a variety of sugars via an octamethylene group and the subsequent tissue uptake by rats was then monitored after administration by i.v. injection. The glucosyl, mannosyl, and 2-deoxyglucosyl derivatives of AVP exhibited selective renal uptake. These derivatives were found to be distributed in the proximal tubules of the renal cortex. In addition, they exhibited specific binding to the kidney microsomal fraction in vitro (Kd = approximately 60 nM), suggesting that they are taken up by a specific recognition mechanism located in the kidneys. From the results of the uptake study of glucosyl derivatives, the following points are clear: 1) renal uptake in vivo becomes saturated with increasing dose, and the Km from the uptake study is almost the same as the Kd obtained in the binding assay in vitro and 2) because the renal first-pass uptake extraction is about 70% at a low dose (10 nmol/kg), there is an effective mechanism for uptake from blood. Furthermore, glucosyl and mannosyl derivatives of oxytocin, a neutral peptide, unlike AVP that is basic, also have high renal uptake clearances. Thus, the renal uptake may not be dependent on derivatives having a cationic nature. We conclude that there is a novel transport mechanism in the kidneys that can be used for the specific renal delivery of glycosylated peptides.

Renal drug targeting using a vector "alkylglycoside".

A specific sugar-modified peptide has previously been shown to have renal targeting potential in vivo and to have a specific binding site which has been identified in the kidney membrane fraction. In this report, we studied the inhibitory effects of glycosylated derivatives on the binding of [3H]Glc-O-C8-AVP [a glucosylated derivative of Arg8-vasopressin (AVP), Kd = 55 nM] to clarify the structural requirements necessary for renal recognition. Glc-S-C7-Me (octyl beta-D-thioglucoside) markedly inhibited the binding, to a much greater extent than Glc-O-C7-Me (octyl beta-D-glucoside) and Gal-S-C7-Me (octyl beta-D-thiogalactoside). Also, [3H]Glc-S-C7-Me was shown to have a specific binding site on the kidney membrane (Kd = 17 nM, Bmax = 24 pmol/mg protein) rather than the liver membrane and, in addition, Glc-S-C7-Me exhibited effective and selective renal uptake in vivo. To examine the possibility that Glc-S-C7-Me might be of practical use as a renal targeting vector, AVP, tryptamine and 4-nitrobenz-2-oxa-1,3-diazole were modified with Glc-S-C8- and the tissue uptake of the resulting derivatives was evaluated. All of these derivatives showed clear renal targeting potential because the apparent uptake clearance by the kidney was greater than 3 ml/min/g kidney in each case. As far as the AVP derivatives were concerned, derivatives having different numbers of methylene groups were compared with Glc-S-C8-AVP. Glc-S-C11-AVP exhibited increased kidney targeting potential, whereas that of Glc-S-C5-AVP was reduced. These differences suggest that the "alkylglycoside" moiety is important for renal uptake. In addition, these renally targeted derivatives inhibited the binding of [3H]Glc-S-C7-Me to the kidney membrane fraction. Our findings allow us to conclude that the alkylglycoside is a suitable candidate vector for renal targeting.

Synthesis and antidiuretic activities of novel glycoconjugates of arginine-vasopressin.

Arginine-vasopressin (AVP) was acylated with various acyl azides (2a-j) in pH 9.1 buffer to give AVP derivatives (11a-j) modified at the tyrosine side chain with a carbohydrate via a spacer arm. Glycoconjugates of AVP modified at the N-terminal amide (12a-e) were also synthesized from AVP and carboxylic acids (3a-e) using dicyclohexylcarbodiimide and 1-hydroxybenzotriazole as coupling agent. Analogues (11a-j) exhibited greater in vivo antidiuretic activity than AVP. AVP and glycoconjugates (12a-e) were stable in rat plasma. On the other hand, glycoconjugates (11a-i) were found to readily convert to AVP according to first order kinetics. Hence, 11a-j are considered to be prodrugs of AVP.

Synthesis of artificial glycoconjugates of arginine-vasopressin and their antidiuretic activities.

Arginine-vasopressin (AVP) derivatives modified at the glutamine side chain amide with carbohydrate via an alkylene spacer (1a--d) were synthesized from new glycosylated glutamine derivatives (3a--d) by solid-phase synthesis. Glycoconjugates of AVP modified at the C-terminal amide (2a--d) were also synthesized from vasopressionic acid. All of them exhibited antidiuretic activity.

Tissue-targeting ability of saccharide-poly(L-lysine) conjugates.

To evaluate the effect of introducing a saccharide moiety to poly(amino acids) on tissue distribution, several glycoconjugates of epsilon-(2-methoxyethoxyacetyl)-poly(L-lysine) of three molecular weights were synthesized using an octylene spacer between the sugar and polymer chain. Methoxyethoxyacetylation of the epsilon-amino group of the lysine unit in poly(L-lysine) was useful for avoiding nonspecific distribution to many tissues as the result of cationic charges. The tissue-targeting ability of each saccharide moiety was considered as the actual amount changed in each tissue caused by saccharide modification. Galactose terminated saccharides such as galactose, lactose and N-acetylgalactosamine accumulated exclusively in the liver, probably by the hepatic receptor. These conjugates could therefore be good carriers for a drug delivery system to the liver. On the other hand, the mannosyl and fucosyl conjugates were preferentially delivered to the reticuloendothelial systems such as those in the liver, spleen and bone marrow. In particular, fucosyl conjugates accumulated more in the bone marrow than in the spleen. Xylosyl conjugates accumulated mostly in the liver and lung. Generally, the accumulated amount in the target tissue increased with increasing molecular weight and an increased number of saccharides on one molecule of polymer.

[Influence of OK-432 on intratumor 5-fluorouracil concentration in cases given UFT].

The influence of OK-432 on the activation of UFT, consisting of tegafur and Uracil, was examined in patients with gastric cancer and colonic cancer. In 14 gastric cancer and 15 colonic cancer cases, to which UFT 400 mg/day and OK-432 2KE 2/W were administered orally and intra-muscularly for 2 weeks preoperatively until surgical treatment, intratumor 5-fluorouracil (5-FU) concentration was measured and compared with that of patients who given UFT 400 mg/day orally for 2 weeks (15 gastric cancer and 15 colonic cancer cases). As the results, in the groups of patients given OK-432, the concentration in gastric cancer tissue was 0.093 +/- 0.067 microgram/g and that in colonic cancer was 0.098 +/- 0.058 microgram/g. Both values exceeded 0.05 microgram/g which is considered to be the effective intratumor 5-FU concentration. No difference was observed in these cases given UFT alone. The ratio of intratumor 5-FU concentration vs. that of normal tissue was 2.5 for gastric cancer and 2.7 for colonic cancer and the ratio of tumor vs. serum 5-FU concentrations was 8.5 for gastric cancer and 10.9 for colonic cancer. No difference was also observed in these values in cases given UFT alone. From above results, it seemed that the clinical dose of OK-432 2KE 2/W had no influence on the activation of UFT, so that the combination therapy of UFT and OK-432 was found to be clinically useful.