2'-O-Methyl derivatives of the common ribonucleosides except for guanosine were synthesized via the 2'-O-methylation of appropriately-protected nucleosides with CH3I in the presence of Ag2O. The 2'-O-methylguanosine derivative was prepared by the monomethylation of a 2',3'-cis-diol system with diazomethane. These derivatives were converted to protected 2'-O-methylribonucleoside 3'-phosphates and used for oligonucleotide synthesis on polymer supports. Thus, oligo(2'-O-methyl-ribonucleotides) having the sequence identical to the consensus sequence of the 5'-splice junction CAGGUAAGU and its complement were synthesized in a stepwise manner using the phosphotriester method. Thermal stabilities (Tm's) of the duplex of these 2'-O-methyl ribo-oligomers and eight related duplexes containing ribo- or deoxyribo-oligomers were examined. It was found that the 2'-O-methyl oligoribonucleotides can be utilized as an alternative to an oligoribonucleotide probe in RNA hybridizations as the hybrid formed has a high, or a higher Tm, the probe is much easier to synthesize and it is less likely to be enzymatically degraded.

Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides.

Publisher Summary A new type of drug molecule synthesized with a specific base sequence designed to interact with a target nucleic acid containing the complementary sequence are being developed. In the earliest and most developed example of this approach, oligodeoxynucleotides and their analogs have been applied as antisense inhibitors of gene expression to bring about translation arrest. This chapter discusses the use of antisense oligodeoxynucleotides as a therapeutic approach. Once this approach is recognized in principle, other examples can be seen to fit a pattern, such as the use of oligodeoxynucleotides to form a triple-strand helix with duplex DNA. However, with a different base triplet code to arrest transcription, the opening of duplex DNA is not considered facile enough to allow the access of an oligo to interact with one of the strands. The development of synthetic oligoribonucleotides as ribozymes is another example in which the information for the selectivity is encoded in the portions of the oligomer flanking the catalytic site. Another example is the use of oligodeoxynucleotides to bind to protein sites that recognize specific sequences of bases, thus, inhibiting polymerase action or bringing about transcription arrest by sequestering specific transcription cofactors. Thus, a new area of pharmacology is developing that is differentiated by the purposeful inclusion of information in the form of nucleic-acid bases in a putative oligonucleotide drug molecule.

Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression

The sulphonylureas act by triggering insulin release from the pancreatic β cell. A specific site on the adenosine triphosphate (ATP)-sensitive potassium channels is occupied by sulphonylureas leading to closure of the potassium channels and subsequent opening of calcium channels. This results in exocytosis of insulin. The meglitinides are not sulphonylureas but also occupy the sulphonylurea receptor unit coupled to the ATP-sensitive potassium channel. Glibenclamide (glyburide), gliclazide, glipizide and glimepiride are the primary sulphonylureas in current clinical use for type 2 diabetes mellitus. Glibenclamide has a higher frequency of hypoglycaemia than the other agents. With long-term use, there is a progressive decrease in the effectiveness of sulphonylureas. This loss of effect is the result of a reduction in insulin-producing capacity by the pancreatic β cell and is also seen with other antihyperglycaemic agents. The major adverse effect of sulphonylureas is hypoglycaemia. There is a theoretical concern that sulphonylureas may affect cardiac potassium channels resulting in a diminished response to ischaemia. There are now many choices for initial therapy of type 2 diabetes in addition to sulphonylureas. Metformin and thiazolidinediones affect insulin sensitivity by independent mechanisms. Disaccharidase inhibitors reduce rapid carbohydrate absorption. No single agent appears capable of achieving target glucose levels in the majority of patients with type 2 diabetes. Combinations of agents are successful in lowering glycosylated haemoglobin levels more than with a single agent. Sulphonylureas are particularly beneficial when combined with agents such as metformin that decrease insulin resistance. Sulphonylureas can also be given with a basal insulin injection to provide enhanced endogenous insulin secretion after meals. Sulphonylureas will continue to be used both primarily and as part of combined therapy for most patients with type 2 diabetes.

https://link.springer.com/content/pdf/10.2165%2F00003495-200464120-00006.pdf

The role of sulphonylureas in the management of type 2 diabetes mellitus.

Gliclazide is a second generation sulphonylurea oral hypoglycaemic agent used in the treatment of non-insulin-dependent diabetes mellitus (NIDDM). It improves defective insulin secretion and may reverse insulin resistance observed in patients with NIDDM. These actions are reflected in a reduction in blood glucose levels which is maintained during both short and long term administration, and is comparable with that achieved by other sulphonylurea agents. Gradually accumulating evidence suggests that gliclazide may be useful in patients with diabetic retinopathy, due to its haemobiological actions, and that addition of gliclazide to insulin therapy enables insulin dosage to be reduced. Thus, gliclazide is an effective agent for the treatment of the metabolic defects associated with NIDDM and may have the added advantage of potentially slowing the progression of diabetic retinopathy. These actions, together with its good general tolerability and low incidence of hypoglycaemia have allowed gliclazide to be well placed within the array of oral hypoglycaemic agents available for the control of NIDDM.

Gliclazide : an update of its pharmacological properties and therapeutic efficacy in non-insulin-dependent diabetes mellitus

Dehydrative condensation catalyses

Methylation of 3', 5'-di-O-trityluridine (I) gave a mixture of two compounds, which was detritylated and then separated to give N3, 2'-O-dimethyluridine (V) and 2'-O-methyluridine (VI). After treatment of the above mixture with ethanolic ammonia, the reaction products were detritylated and separated to give V and 2'-O-methyl-cytidine (VII). By a similar method 3'-O-methyl nucleosides were obtained from 2', 5'-di-O-trityluridine (II). The relative rates of acidic hydrolysis of uridine, VI, and 3'-O-methyluridine have been determined and were shown to obey first order kinetics. Introduction of a methoxyl function at the 2'- or 3'-position of uridine enhances the stability of the glycosyl linkage to acid hydrolysis.

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Synthesis of 2'-O-Methyluridine, 2'-O-Methylcytidine and Their Relating Compounds.

Both pharmacokinetics of total and free gliclazide, a potential hypoglycemic drug, were studied in the healthy (n = 12) and diabetic subjects (n = 11). The blood level of gliclazide was determined by a high-performance liquid chromatography, and the free gliclazide (unbound to proteins) in the serum separated by means of an ultrafiltration. The binding ratio of gliclazide to the blood proteins was about 96% during the periods of 24 hr after administration of the drug. Several pharmacokinetic parameters for the blood gliclazide were derived from the decay curves of the blood drug levels. Each pharmacokinetic parameter was not changed by differences between the healthy and diabetic subjects, the total and free drug levels, and method of administration of the drug; each mean parameter, the elimination rate (ke), the time to peak level (tmax), the elimination half-life (t1/2) and the volume of distribution (Vd beta) was 0.07 hr-1, 2.8 hr, 12.3 hr and 16.4 1 (total level), respectively. The serum from healthy subject receiving orally administered gliclazide was gel-filtered on a Sephadex G-150 column. Each fractionated serum protein of macroglobulin (IgM), gamma-globulin (IgG), albumin (A) and small molecular substances (F), contained gliclazide at average of 3.7, 0.7, 82.3 and 13.2%, respectively, during the periods of 24 hr after administration. In in vitro experiment, it was found that the ratio of gliclazide-albumin binding kept a constant level at 96.5% in the range of normal protein levels (3.3--4.8 g/100 ml). In conclusion, the present result shows that the pharmacokinetics of the total blood level of gliclazide reflect the free gliclazide level, moreover, gliclazide predominantly binds with albumin in the blood and its binding ratio is not constant, but variable according to the dose-relation between the drug and the serum protein.

Influence of blood proteins on biomedical analysis. III. Pharmacokinetics and protein binding of gliclazide.

In this sensitive, reproducible method for determination of D-3-hydroxybutyrate (3-OHB) in plasma, it is converted to acetone by use of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)/lactate dehydrogenase (EC 1.1.1.27) coupled with acetoacetate decarboxylase (EC 4.1.1.4). The resulting acetone is detected by head-space gas chromatography. The lowest concentration of 3-OHB detectable in plasma was 2 mumol/L. The calibration curve showed a linear relationship for 3-OHB concentration from 0 to 5 mmol/L (r = 0.999). Analytical recovery of 3-OHB (50 mumol/L) was 97.9 (SD 3.8)%. The method was developed for determination of the three ketone bodies in plasma. The ratio of acetone to acetoacetate was not significantly different (p greater than 0.2) between normals (n = 31) and diabetics (n = 86). In normal subjects, the ratio of 3-OHB to acetoacetate was 1.20 (SD 0.44). In diabetic patients, the ratio correlated with the logarithm of the total ketone body concentration (r = 0.828).

Head-space gas-chromatographic determination of 3-hydroxybutyrate in plasma after enzymic reactions, and the relationship among the three ketone bodies.

A high-performance liquid chromatography (HPLC) procedure was developed for the determination of plasma gliclazide, a new oral hypoglycemic drug. Gliclazide, tolbutamide (used as an internal standard) and plasma components in plasma were successfully separated as sharp peaks by reversed-phase HPLC on a Jasco SC-01 column with methyl alcohol containing 0.2% acetic acid (3 : 2, v/v) as the mobile phase (retention time ; gliclazide : 16 min, IS : 10 min, plasma components : 5.5 and 7.0 min). The relationship between the plasma levels of gliclazide and the peak height ratio (gliclazide/IS) was linear (correlation coefficient, γ=1.00) in the range of 0.3 to 10 μg/ml plasma. The detection limit and the reproducibility (CV) of the present method were 0.3μg/ml and 4.0% (n=8), respectively. The decay curve of plasma gliclazide in a diabetic patient given 80 mg of the drug orally was determined by the present method and GLC (ECD method). The two results agreed well. Reversed-phase HPLC was shown to be suitable for the routine clinical assay of gliclazide in plasma.

Reversed-Phase High-Performance Liquid Chromatographic Determination of Gliclazide in Human Plasma

Ring closure of 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICA-riboside) with phenyl isothiocyanate afforded 2-mercaptoinosine (I) in good yield. Similarly, the ring closure of AICA-riboside 5'-phosphate (AICAR) led to the formation of 2-mercaptoinosine 5'-phosphate (II). Various 2-substituted inosine 5'-phosphates were prepared from I and II or starting with AICA-riboside. It was found that 2-furfuryl-thioinosine 5'-phosphate possessed a flavor enhancing activity of about 17-times that of inosine 5'-phosphate. The chemical structure-flavor enhancing activity relationship was presented.

Kunio Kobayashi

Papers

Synthesis of 2'-O-Methyluridine, 2'-O-Methylcytidine and Their Relating Compounds.

Influence of blood proteins on biomedical analysis. III. Pharmacokinetics and protein binding of gliclazide.

Head-space gas-chromatographic determination of 3-hydroxybutyrate in plasma after enzymic reactions, and the relationship among the three ketone bodies.

Reversed-Phase High-Performance Liquid Chromatographic Determination of Gliclazide in Human Plasma

Synthesis of Compounds related to Inosine 5'-Phosphate and Their Flavor Enhancing Activity. IV. 2-Substituted Inosine 5'-Phosphates