Comparison of the antiviral activity of hydrophobic amino acid phosphoramidate monoesters of 2'3'-dideoxyadenosine (DDA) and 3'-azido-3'-deoxythymidine (AZT)

A series of hydrophobic, water soluble and non-toxic amino acid phosphoramidate monoesters of dideoxyadenosine (ddA) and 3'-azido-3'-deoxythymidine were shown to inhibit the replication of HIV-1 in human peripheral blood mononuclear cells (PBMC) from two donors. The tryptophan methyl ester phosphoramidates of AZT and ddA were equally potent (EC50S = 0.3-0.4 microM), while the phenyl methyl ester of ddA was 40- to 100- fold more potent than the AZT derivatives. The alaninyl methyl ester of AZT was found to be 70- fold more potent than the ddA derivative. The methyl amide derivatives were found to be 5-20 fold less active than the methyl esters for the ddA series, while for AZT the derivatives were found to be of similar potency or 60- to 166- fold more potent than the methylesters.     

New phosphonoformic acid derivatives of 3'-azido-3'-deoxythymidine

New 5'-alkyl ethoxy- and aminocarbonylphosphonates of 3'-azido-3'-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5'-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Sensitivity of CFU-GM from normal human bone marrow and leukaemic clonogenic cells (CFU-L) from blood of patients with myelogenous leukaemia to 3'-deoxy-3'-fluorothymidine in comparison to 3'-azido-3'-deoxythymidine

3'-Deoxy-3'-fluorothymidine (FT), a thymidine analogue highly effective against HIV 1 in vitro was investigated as to its in vitro effect on normal human bone marrow CFU-GM (agar colony assay) and on human peripheral myeloid leukaemic clonogenic cells (CFU-L, colony assay in methylcellulose). For comparison, 3'-azido-3'-deoxythymidine (AZT), structurally related and used in AIDS treatment, was included in the study. Both compounds inhibit the formation of clusters and colonies from bone marrow stem cells with an [IC]50 between 10(-6) and 10(-5)M. In concentrations only 5-10 times lower than the [IC]50, FT begins to stimulate cluster and colony formation. AZT and FT also inhibit the formation of clusters and colonies from CFU-L. Compared to CFU-GM, CFU-L were more sensitive to FT, and a stimulation was not seen. It is concluded that similar side effects on bone marrow could be expected for possible use of FT against AIDS as have been found for AZT and that both compounds are potential candidates for anti-leukaemic drugs.     

In vivo toxicity of 3'-azido-3'-deoxythymidine (AZT) on CBA/Ca mice

CBA/Ca male mice were given 3'-azido-3'-deoxythymidine (AZT) in drinking water (1 mg/ml) for up to 7 weeks. Water consumption and body weight decreased significantly. Neutropenia and lymphopenia were observed during and after exposure. Significant macrocytic anemia developed and disappeared as a function of red cell life span after stopping AZT intake. A microthrombocytosis was seen. Bone marrow cellularity and spleen colony-forming unit (CFU-s) content fell, but recovered completely and quickly after terminating AZT intake. Hemopoietic stem cell function measured by 2 different methods of rescuing fatally irradiated mice was normal 4 weeks after AZT exposure, suggesting that AZT treatment does not induce a long-lasting effect in genetic control of mitotic potential of stem cells. This is in marked contrast to exposure of CBA/Ca mice to benzene and ionizing radiation.     

Glucuronidation of 3'-azido-3'-deoxythymidine in human liver microsomes: enzyme inhibition by drugs and steroid hormones.

The molecular form of UDP-glucuronosyltransferase involved in the catalysis of 3'-azido-3'-deoxythymidine (AZT)-5'-O-glucuronide was characterized in human liver microsomes. The specific activity (1.3 nmol/min per mg protein) in transplantable liver was more than 2-times higher than in post-mortem fragments. Liver microsomes from patients suffering Crigler-Najjar syndrome, who are genetically deficient in bilirubin UDP-glucuronosyltransferase, could also glucuronidate AZT to a similar extent, thus indicating that this protein was not involved in that process. A genetically engineered V79 cell line stably expressing a cDNA which encodes a human isozyme active towards 1-naphthol was unable to glucuronidate AZT. Clinically used drugs, most of them being glucuronidated, were tested as potential inhibitors of the glucuronidation of AZT in human liver microsomes. The drugs chemically related to 2-phenylpropionic acid, naproxen and flurbiprofen, and the steroid compounds testosterone, estrone and ethynylestradiol strongly inhibited AZT glucuronidation. Codeine and morphine also decreased the reaction rate although to a lower extent. Except estrone which elicited a partial competitive inhibition, ethynylestradiol, flurbiprofen naproxen and testosterone could competitively inhibit AZT glucuronidation with an apparent Ki of 38, 50, 172 and 250 microM, respectively. The results suggest that these drugs were substrates of the same isozyme(s) involved in AZT glucuronidation. Probenecid was a weak inhibitor of the reaction (Ki 900 microM), only when non-disrupted microsomes were used. This drug may compete with the anion carrier system involved in the microsomal uptake of UDP-glucuronic acid.     

Synthesis, characterization, and in vitro assay of folic acid conjugates of 3'-azido-3'-deoxythymidine (AZT): toward targeted AZT based anticancer therapeutics

Conjugates of three components namely folic acid, poly(ethyleneglycol) and 3 '-azido-3 '-deoxythymidine (AZT) are presented. Folate-PEG units were coupled to AZT to facilitate delivery of the nucleoside into the cell. A convenient separation of the polydisperse PEGylated-folic acid regioisomers produced upon conjugation is described. This is to select for the active gamma-regioisomer over the inactive alpha-regioisomer. In vitro cytotoxicity assays were conducted against an ovarian cell line (A2780/AD) that overexpresses the folate receptor (FR) and compared to a FR free control cell line. Compared to AZT a approximately 20-fold greater potency against the resistant ovarian line was observed for the conjugates.     

HPLC and mass spectrometry analysis of the enzymatic hydrolysis of anti-HIV pronucleotide diastereomers

In one current strategy to develop membrane-soluble pronucleotides, the phosphoramidate derivatives of the approved anti-HIV nucleosides 2',3'-didehydro-3'-deoxythymidine (d4T), 3'-azido-3'-deoxythymidine (AZT), (-)-beta-L-2',3'-dideoxy-3'- thiacytidine (3TC), and 2',3'-dideoxyadenosine (ddA) exhibit promising antiviral activity. However, the non-stereoselective synthetic route results in a mixture of diastereoisomers, which differ in the configuration of the phosphorus chiral center. Since it is believed that enzymatic ester hydrolysis is the first step in the intracellular activation of these prodrugs and that this process could be dependent on the stereochemistry at the phosphorus center, analytical methods must be developed. In the present work, in vitro evaluation of the selectivity of pig liver esterase (PLE) towards each diastereomer of d4T, AZT, 3TC, and ddA prodrugs has been investigated, applying our recently published HPLC-MS procedure using a polysaccharide-type chiral stationary phase. This method has been used to analyze the products of the PLE-catalyzed hydrolysis of the pronucleotides. It was found that both diastereomers of the four prodrugs were substrates for PLE.     

Phenobarbital inducible UDP-glucuronosyltransferase is responsible for glucuronidation of 3'-azido-3'-deoxythymidine: characterization of the enzyme in human and rat liver microsomes

Glucuronidation by liver microsomes of 3'-azido-3'-deoxythymidine (AZT) was characterized in human and in various animal species. The glucuronide isolated by HPLC, was identified by mass spectrometry (fast atom bombardment, desorption in chemical ionization), and beta-glucuronidase hydrolysis. AZT glucuronidation reaction in liver microsomes of human and monkey proceeded similarly with an apparent Vmax of 0.98 nmol/min/mg protein and apparent Km of 13 mM. Oleoyl lysophosphatidylcholine activated more than twofold the formation of the glucuronide. Human kidney microsomes could also biosynthesize AZT glucuronide, although to a lower extent (six times less than the corresponding liver). Probenecid, which is administered to AIDS patients, decreased hepatic AZT glucuronidation in vitro (I50 = 1.5 mM), whereas paracetamol did not exert any effect at concentrations up to 21.5 mM. Morphine also inhibited the reaction (I50 = 2.7 mM). AZT glucuronidation presented the highest rate in human and in monkey (0.50 nmol/min/mg protein); pig and rat glucuronidated the drug two and three times less, respectively. In Gunn rat, the specific activity in liver microsomes was similar (0.18 nmol/min/mg protein) to that of the congenic normal strain; this suggests that an isozyme other than bilirubin UDP-glucuronosyltransferase catalyzed the reaction. In rats, AZT glucuronidation was stimulated fourfold by phenobarbital; 3-methylcholanthrene or clofibrate failed to increase this activity. This result was consistent with the bulkiness of the AZT molecule (thickness 6.7 A), which is a critical structural factor for glucuronidation of the drug by phenobarbital-induced isozymes. Altogether, the results strongly indicate that UDP-glucuronosyltransferase (phenobarbital inducible forms) is responsible for AZT glucuronidation.     

Thymidine and 3'-azido-3'-deoxythymidine metabolism in human peripheral blood lymphocytes and monocyte-derived macrophages. A study of both anabolic and catabolic pathways.

3'-Azido-3'-deoxythymidine (AZT) is HIV-inhibitory in human macrophages, which is surprising in view of the low AZT phosphorylation reported in macrophage extracts. To elucidate the mechanism of AZT activation, we studied AZT anabolism as well as catabolism in human lymphocytes and macrophages, and compared it to that of thymidine. Thymidine kinase (TK)-specific activity in mitogen-stimulated lymphocytes was 15 times higher than in macrophages. However, the TK activity per cell was only 1.3 times higher, because of the large macrophage cell volume. Total cellular TK activity, but not specific activity, matched the level of intracellular AZT anabolism. The substrate specificity of TK in macrophages strongly suggests that mitochondrial TK2 was the enzyme phosphorylating thymidine and AZT in these cells, whereas it was cytosolic TK1 in stimulated lymphocytes. In vivo thymidine catabolism was extensive, forming thymine and dihydrothymine. In macrophages more than 95% of the added thymidine (0.5 microM) was degraded within 60 min. AZT, in contrast, was not catabolized, which explains the high AZT nucleotide accumulation, a process opposed only by AZTMP excretion. The lack of catabolism together with phosphorylation by TK2 clarifies how AZT can inhibit human immunodeficiency virus in macrophages. The fact that TK2 and not TK1 phosphorylates AZT in macrophages should have important implications for combination chemotherapy.     

Synthesis and anticancer activity of 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (AZT)

A series of novel N-alkyl 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (6-15) was synthesized by means of phosphonylation of 3'-azido-3'-deoxythymidine (4) with P-chloromethylphosphonic ditriazolide (3) followed by a reaction with the appropriate amine. The synthesized phosphonamidates 6-15 were evaluated for their cytotoxic activity in two human cancer cell lines: oral (KB) and breast (MCF-7) using the sulforhodamine B (SRB) assay. The highest activity in KB human cancer cells was displayed by phosphonamidate 8 (IC(50)=5.8 μg/mL), however, this compound was less potent than the parent AZT (IC(50)=3.1 μg/mL). Phosphonamidate 10 showed only moderate activity (IC(50)=12.1 μg/mL) whereas the other phosphonamidates proved inactive. Similarly, the highest activity in MCF-7 human cancer cells was displayed by phosphonamidate 8 (IC(50)=3.7 μg/mL) but it proved somewhat less active than AZT (IC(50)=2.6 μg/mL). Some activity was also displayed by phosphonamidate 10 (IC(50)=12.8 μg/mL) but the other phosphonamidates were found inactive. Hydrolysis studies indicate that the synthesized phosphonamidates are likely to act as prodrugs of the parent nucleoside (AZT). Transport measurements showed that the most active phosphonamidates (8 and 10) were able to permeate across the intestinal epithelium in vitro. The apparent permeability coefficients determined in Caco-2 cell monolayers indicated that these compounds could be moderately absorbed in humans.     

Electronic interaction between 3'-azido-2'-deoxythymidine and nucleic acid bases by fluorescence spectroscopy.

The ultraviolet (UV) absorption and fluorescence nature of the mixtures of 3'-azido-3'-deoxythymidine (AZT), poly 1, N6-ethenoadenylic acid (poly, epsilon A) and mixtures of AZT and poly A (AZT+poly epsilon A) at various molar ratio has been studied. On the basis of the present results, it may be concluded that the azide group of AZT (N6', N7', and/or N8') may link to the phosphate groups of polynucleotide. Thus, results obtained suggest that there are electronic interaction between thymine and ethenoadenine lings at the first excited singlet state.     

Synthesis and in vitro antibacterial activity of novel 5'-O-analog derivatives of zidovudine as potential prodrugs

An efficient, short synthesis of four potential prodrugs of 3'-azido-3'-deoxythymidine (AZT) and their antibacterial activity are reported. The 5'-OH group of AZT was functionalized with oxalyl chloride obtaining an acyl chloride derivative (AZT-Ox), which by further transformation with leucine, isoleucine and valine amino acids led to the corresponding AZT analogs, namely AZT-Leu, AZT-iLeu and AZT-Val. A carboxyl acid derivative (AZT-Ac) was also obtained by hydrolysis of AZT-Ox. These compounds, which exhibit anti HIV activity, have killed collection and clinical strains of some opportunistic infectious agents in AIDS-related complex. Thus, the clinical strains, K. oxytoca, S. typhi and K. pneumoniae, and collection strain K. pneumoniae ATCC 10031 showed sensitivity to antibiotics. The activity order for the studied compounds against the most sensitive strain (K. pneumoniae ATCC 10031) was AZT-Leu > AZT-iLeu > AZT-Val > AZT-Ac > AZT. On the other hand, the activity order for the second most sensitive strain (K. oxytoca) was AZT-Leu > AZT-Val = AZT-Ac > AZT-iLeu > AZT. The most effective antibacterial drug AZT-Leu, M.I.C.=0.125 microgmL(-1)) was 16 times more active than AZT (AZT, M.I.C.=2 microg mL(-1)) against K.     

Human immunodeficiency virus I-induced expression of P-glycoprotein

Because prolonged treatment of HIV infection with 3'-azido-3'-deoxythymidine (AZT) is associated with in vitro resistance to AZT, we examined whether HIV could induce/amplify the expression of p-glycoprotein in infected cells resulting in reduced drug accumulation leading to reduced sensitivity to AZT. We show that both H9 (T cell line) and U937 (monocytic cell line) cells, upon infection with HIV, expressed increased levels of P-glycoprotein and accumulated significantly less AZT and daunorubicin as compared to uninfected cells. Sodium azide increased intracellular accumulation of daunorubicin in infected cells, suggesting a metabolically active drug efflux mechanism. Addition of cyclosporin A partially corrected intracellular drug accumulation in HIV infected cells. In addition, similar to multidrug resistant tumor cells, HIV-infected cells show depolarization of plasma membrane. Taken together, these data suggest that HIV-induced increased P-glycoprotein expression could be one of the mechanisms for reduced intracellular accumulation of antiviral agents and resistance to AZT and perhaps to other anti-retroviral agents.     

3'-azido-3'-deoxythymidine binding to ribonuclease A: model for drug-protein interaction

Ribonuclease A (RNase A) with several high affinity binding sites is a possible target for many organic and inorganic molecules. 3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus (HIV) infection. The drug interactions with protein and nucleic acids are associated with its mechanism of action in vivo. This study was designed to examine the interaction of AZT with RNase A under physiological conditions. Reaction mixtures of constant protein concentration (2%) and different drug contents (0.0001-0.1 mM) are studied by UV-visible, FTIR, and circular dichroism spectroscopic methods in order to determine the drug binding mode, the drug binding constant, and the effects of drug complexation on the protein and AZT conformations in aqueous solution. The spectroscopic results showed one major binding for the AZT-RNase complexes with an overall binding constant of 5.29 x 10(5) M(-1). An increase in the protein alpha helicity was observed upon AZT interaction, whereas drug sugar pucker remained in the C2'-endo/anti conformation in the AZT-RNase complexes.