Antisense hairpin loop oligonucleotides as inhibitors of expression of multidrug resistance-associated protein 1: their stability in fetal calf serum and human plasma.

Multidrug resistance-associated protein (MRP1) is a transmembrane pump protein responsible for the efflux of chemotherapeutic drugs, an important cause of anticancer treatment failure. Trying to circumvent MRP-mediated resistance we designed and synthesized hairpin loops forming antisense oligodeoxyribonucleotides (ODNs), both phosphodiesters (PO-ODNs) and their phosphorothioate analogues (PS-ODNs), to reduce the protein expression by targeting its mRNA in a sequence specific manner. Melting temperature measurements as well as polyacrylamide gel electrophoresis supported the preferential formation of a secondary structure, which was expected to protect ODNs against 3'-exonuclease degradation. ODNs and PS-ODNs designed in this work were successfully tested as antisense inhibitors of the expression of MRP1 in the leukaemia HL60/ADR cell line. Foreseeing the necessity to perform clinical studies with such ODNs we investigated their stability against the 3'-exonuclease activity of fetal calf serum and human plasma. Under the conditions, corresponding to physiological ones, we observed high stability of hairpin loop forming ODNs, especially those containing longer (e.g. 7 base pair) stems. Comparative studies on the stability of chemically unmodified hairpin loop forming ODNs and their PS-counterparts indicated that endonuclease activity did not play any important role in the process of their nucleolytic degradation. Our studies provide strong evidence for high stability of chemically unmodified hairpin loop ODNs, making them an attractive alternative to phosphorothioate analogues commonly used in antisense strategy.

Multidrug resistance-associated protein--reduction of expression in human leukaemia cells by antisense phosphorothioate olignucleotides.

Multidrug resistance-associated protein (MRP1) causes cellular drug resistance in several cancer cell lines. In this paper we show that antisense oligonucleotides decrease MRP1 expression in human leukaemia cells. We investigated biological activity of a series of 12 linear phosphorothioate oligonucleotides, complementary to several regions of MRP1 mRNA. The oligonucleotides were administered to leukaemia HL60/ADR cells overexpressing MRP1 protein. Then, the level of MRP1 mRNA was determined by means of semiquantitative RT-PCR and the protein level by reaction with specific monoclonal antibodies. Some of the investigated antisense oligonucleotides decrease the expression level of the MRP1 protein by 46% and its mRNA level by 76%.

Comparison of acetylation phenotype with genotype coding for N-acetyltransferase (NAT2) in children.

The present study focused on evaluation of the extent to which genotype coding for N-acetyltransferase agrees with acetylation phenotype in children at various ages. In 82 Caucasian children aged from 1 mo to 17 y (57 boys and 25 girls) and including 37 infants, the acetylation phenotype was evaluated from the urinary metabolic ratio of 5-acetylamino-6-formylamino-3-methyluracil (AFMU) to 1-methylxanthine (1X) after oral administration of caffeine. At the same time, by use of PCR and restriction analysis of amplified fragments of the N-acetyltransferase gene, four nucleotide transitions were identified: 481C-->T (KpnI), 590 G-->A (TaqI), 803 A-->G (DdeI), and 857 G-->A (BamHI). The wild-type allele was detected in 27 (33%) children, and the slow acetylation genotype was found in 55 (67%) children. The results of the study show that the metabolic ratio AFMU/1X could be calculated only in 72 children, because in 10 (14%) infants <20 wk of age, AFMU was not detected. Determination of the relation between the acetylation phenotype and genotype revealed that 18 children (23%) containing at least one wild-type allele had AFMU/1X <0.4 (slow acetylation activity) and 7 (8%) of genotypically slow acetylators presented high metabolic ratio (high acetylation activity). We concluded that the disagreement between the acetylation phenotype and genotype is more often found in the group of children characterized by low AFMU/1X and that in small children only N-acetyltransferase genotype studies enable the detection of genetic acetylation defect.

Genotyping of the arylamine N-acetyltransferase polymorphism in the prediction of idiosyncratic reactions to trimethoprim-sulfamethoxazole in infants.

The pathogenesis of hypersensitivity to trimethoprim-sulfamethoxazole (TMP-SMX) is supposed to be associated with the slow acetylation phenotype. This pharmacogenetic defect is associated with the mutations of the arylamine N-acetyltransferase (NAT2) encoding gene. The aim of the study was to compare the usefulness of the acetylation phenotype and NAT2 coding genotype in the prediction of idiosyncratic reaction to Cotrimoxazole in infants. The study was carried out in the group of 20 infants, aged 2-12 months (mean age 6.3 months) treated with Cotrimoxazole, administered at 100 mg/kg b.w./24 h doses. In seven children (35%) no adverse effects of the treatment have been observed, whereas in 13 (65%) children various adverse effects occurred as a result of the therapy, such as rash (4 children), granulocytopenia with anemization (5 children) or liver impairment (4 children). The acetylation phenotype of each child was determined on the basis of urine of N-acetyl isoniazid/isoniazid ratio, after ingestion of isoniazid as a model drug. Furthermore we used polymerase chain reaction (PCR) followed by the analysis of restriction fragments length polymorphism (RFLP) technique to identify the known mutant alleles of the NAT2 gene. It has been presumed that the genotype determining fast acetylation contains at least one of wild-type allele. No correlation has been found between the observed adverse effects of Cotrimoxazole and age, gender and acetylation phenotype. However, it has been demonstrated that the risk of adverse effects of Cotrimoxazole is considerably higher in children with mutations of the NAT2 encoding gene. The comparison of the results from PCR-RFLP genotyping with phenotyping suggested that in infants, the NAT2 genotype rather than phenotype provides the basis for the detection of hypersensitivity to TMP-SMX.

The arylamine N-acetyltransferase (NAT2) polymorphism and the risk of adverse reactions to co-trimoxazole in children.

OBJECTIVE: The evaluation of the importance of the genetically determined acetylation defect for the development of adverse reactions to co-trimoxazole in children. METHODS: The study comprised 48 children aged 3 months to 3 years, who were being treated for interstitial pneumonia with co-trimoxazole. During the treatment, daily clinical examinations and biochemical tests to monitor the functions in various organs enabled us to detect adverse reactions to the drug. The therapy was continued or discontinued according to the results of these examinations. In all children we identified the genotype coding for N-acetyltransferase (NAT2). For this purpose, DNA was isolated from peripheral blood. Polymerase chain reaction (PCR) was then carried out, followed by restriction mapping with the KpnI, Ddel, TaqI, and BamHI endonucleases in order to identify the four mutations at the NAT2 gene locus: 481C-->T; 803A-->G; 590G-->A and 857G-->A, respectively. RESULTS: In 29 children (60%) various adverse effects occurred and in 19 children (40%) no adverse reactions to treatment occurred. We found statistically significant differences in the occurrence of the identified wt alleles, and alleles with 590A and 857A mutations between the two groups of children studied. In the group with adverse effects, 87% of children had genotype coding for slow acetylation and only 13% had genotypes containing the wt allele. In the group without adverse effects the results were reversed: 89% had genotypes with the wt allele, and only 2 children (10%) were found to have the homozygotic mutation (slow acetylation). CONCLUSION: The results show that the occurrence of adverse effects from co-trimoxazole is closely connected with the genotype coding for slow acetylation.

Novel cost-effective methanephosphonoanilidothioate approach to the stereoselective synthesis of dinucleoside (3',5')-methanephosphonates.

A new method of stereoselective preparation of di(2'-deoxy or 2'-OMe)ribonucleoside (3',5')-methanephosphonate 5 is presented. The DBU/LiCl-assisted reaction of 5'-O-DMT-(2'-deoxy or 2'-OMe)ribonucleoside 3'-O-(S-alkyl methanephosphonothioate) 9 with 5'-OH nucleosides proceeds with full stereospecificity, giving 5 in moderate to good yield. The conversion of 5'-O-DMT-(2'-deoxy or 2'-OMe) ribonucleoside 3'-methanephosphonoanilidothioates 8 and 3'-O-methanephosphonoanilidates 10 by means of NaH/CX2 (X = O,S) followed by S-alkylation leads to monomers 9, with the possibility of use of both separated diastereomers of 8 for the preparation of one selected diastereomer of 5. The relative configuration at the P atom in 2'-OMe and deoxynucleoside derivatives of compounds 9 was established by means of stereoselective degradation of nucleoside 3'-O-methanephosphonothioates 11 (precursors of 9) with nuclease P1.

Arylamine N-acetyltransferase (NAT2) gene mutations in children with allergic diseases.

The overrepresentation of phenotypically slow acetylators among patients with atopic allergy has been reported in previous studies. The N-acetyltransferase coding gene has not yet been investigated in allergic diseases. This study was designed to determine the differences in the distribution of mutation frequency and genotypes that encode normal and defective activity of N-acetyltransferase in children with atopic allergies compared with healthy children. In 56 children with documented inhalational, food, or mixed allergies and in 100 healthy control children with no clinical or laboratory signs of allergy, the genotype coding for N-acetyltransferase was identified by means of the polymerase chain reaction followed by analysis of restriction fragment length polymorphism. Nucleotide transitions in the following positions were investigated: 481 C-->T, 590 G-->A, 803 A-->G, and 857 G-->A, which enabled the identification of six genotypes, including the wild-type (wt) allele, and 16 genotypes, including mutated alleles (homozygotic and herterozygotic). The statistical analysis showed significant differences in the distribution of the frequency of the occurrence of mutated alleles and genotypes between the two groups of children. In 51 children (91%) with allergy, genotypes that encode acetylation defect were found; genotypes that code for normal N-acetyltransferase were observed in only five allergic children (9%). In the control group the distribution of genotypes coding for normal and defective N-acetyltransferase activity is 38% and 62%, respectively. Thus study enabled the conclusion that the slow acetylation genotype is a genetic marker of predisposition to atopy.

Diastereomers of thymidine 3'-O-(methanephosphonothioate): synthesis, absolute configuration and reaction with 3'-methoxyacetylthymidine under conditions of triester approach to oligonucleotide synthesis.

Diastereomers of the title compound were obtained and absolute configuration was assigned by means of stereochemical correlation. Their reaction with 3'-O-methoxyacetylthymidine in the presence of triisopropylbenzenesulphonyl (4-nitro) triazole is neither chemo- nor stereo-selective and leads to diastereomeric pairs of dithymidyl (3',5')methanephosphonate and -methanephosphonothioate. Obtained results are discussed in terms of mechanism of activation of phosphodiesters under conditions known as "phosphotriester approach to oligonucleotide synthesis".

Activity of acid deoxyribonuclease towards diastereoisomers of thymidyl 3'-(4-nitrophenyl phosphorothioate). Stereochemistry of transnucleotidylation reaction.

The (Rp)- and (Sp)-diastereoisomers of thymidyl 3'-(4-nitrophenyl phosphorothioate) (1) were found to act as unusual substrates for acid deoxyribonuclease (DNase II). Instead of the expected thymidine 3'-phosphorothioate, the product resulting from the reaction of (Rp)-1 catalyzed by DNase II was identified as (Sp, Rp)-thymidyl (3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate), while that from (Sp)-1 has been recognized as a 10:1 mixture of (Sp, Rp)-thymidyl (3'-5')thymidyl phosphorothioate 5'-(4-nitrophenyl phosphorothioate) and (Rp, Sp)-thymidyl (3'-5')-thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate), respectively. Both types of transnucleotidylations were found to occur with retention of configuration at phosphorus. Stereochemical results may be interpreted in terms of two step mechanisms involving the formation of the intermediate, covalent substrate enzyme complexes.

Substrate specificity and stereospecificity of calf spleen phosphodiesterase towards deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates.

Phosphodiesterase from calf spleen exhibits nucleotidyltransferase activity when incubated with either the (PR) or the (PS) diastereomer of thymidyl 3'-(4-nitrophenyl phosphorothioate). Thymidylyl(3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate) was identified as the main product of the enzyme-catalyzed reaction and the absolute configuration at the internucleotide phosphorus atom of the product was determined. The nucleotidyltransferase reaction is shown to proceed with retention of configuration at phosphorus, implying involvement of a double displacement mechanism with the formation of a nucleotidylated enzyme intermediate. To study the substrate specificity of spleen phosphodiesterase a series of deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates were synthesized, and Km and V parameters for each substrate were measured. The results obtained show virtually no specificity for substrates with different nucleosidyl moieties, while about a 20 - 30-fold drop in V and a slight increase in Km values is observed for phosphorothioate analogues as compared with corresponding phosphates. The enzyme showed no significant stereoselectivity towards phosphorothioates of opposite configurations at phosphorus.

Further studies on the substrate specificity of calf spleen phosphodiesterase [EC 3.1.4.18].

The synthesis of 5'-deoxy-5'-chlorothymidine-3'-(4-nitrophenyl)phosphate (5) and 5'-deoxy-5'-chlorothymidine-3'-(4-nitrophenyl)phosphorothioate (6) via corresponding phosphoranilidodiester intermediate is described. The affinity of 5 and 6 towards SPDE in comparison with thymidine-3'-(4-nitrophenyl)phosphate is tested. These findings reveal that the presence of 5'-hydroxyl function in the substrate is not necessary for hydrolytic action of this enzyme.