Thermodynamic, kinetic, and conformational properties of a parallel intermolecular DNA triplex containing 5' and 3' junctions

The interaction of the 11-mer oligodeoxypyrimidine d(TCTTCTUTCCT) with the 17 bp duplex d(CGCTAGAAGAAAGGACG).d(CGTCCUTTCTTCTAGCG) in forming an intermolecular DNA triplex has been examined in solution by surface plasmon resonance (SPR), UV thermal denaturation, circular dichroism (CD), and NMR methods. Thermodynamic data were also acquired for the shorter 15 bp target duplex d(CGCTAGAAGAAAGGA). d(TCCUTTCTTCTAGCG), which forms a 3' flush-ended parallel triplex. CD titrations at pH 5 gave a triplex --> (duplex + strand) dissociation constant Kd of 0.5 microM at 15 degreesC and approximately 2 microM at 25 degreesC for both the 11-15.15 and 11-17.17 systems, in agreement with analysis of the UV melting data and a direct calorimetric measurement. In contrast, the "apparent" Kd value determined by SPR was 10-20-fold smaller. The rate constant for dissociation (kd) of the third strand from the triplex was found to be approximately 0.0002 s-1 at 25 degreesC by SPR. The rate constant for exchange between the triplex and duplex states determined by NMR was approximately 2 s-1 at 40 degreesC. The dissociation kinetics measured by SPR are considerably underestimated, which largely accounts for the poor estimation of Kd using this technique. Extensive 1H NMR assignments were obtained for both the 17 bp DNA duplex and the triplex. Large changes in chemical shifts were observed in the purine strand of the host duplex, but only small shift changes were induced in the complementary pyrimidine strand. Dramatic differences in shifts were observed for the G and A residues, especially in the minor groove, consistent with only small, localized conformational changes in the underlying duplex. The magnitude of the shift changes decreased to baseline within one base of the 3' triplex-duplex junction and over two to three bases at the 5' junction. Chemical shift changes at the 5' junction suggest small conformational anomalies at this site. COSY and NOESY spectra indicate that the nucleotides are in the "S" domain in both the triplex and duplex states. These data rule out major conformation changes at the triplex-duplex boundaries. NOEs between pyrimidines in the third strand and those in the duplex showed proximity for these bases in the major groove, which could be ascribed to buckling of the Hoogsteen bases out of the plane of the Watson-Crick base pairs.     

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.     

The role of 3'-5' exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance

In the D171G/D230A mutant generated at conserved aspartate residues in the Exo1 and Exo2 sites of the 3'-5' exonuclease domain of the yeast mitochondrial DNA (mtDNA) polymerase (pol-gamma), the mitochondrial genome is unstable and the frequency of mtDNA point mutations is 1500 times higher than in the wild-type strain and 10 times higher than in single substitution mutants. The 10(4)-fold decrease in the 3'-5' exonuclease activity of the purified mtDNA polymerase is associated with mismatch extension and high rates of base misincorporation. Processivity of the purified polymerase on primed single-stranded DNA is decreased and the Km for dNTP is increased. The sequencing of mtDNA point mutations in the wild-type strain and in proofreading and mismatch-repair deficient mutants shows that mismatch repair contributes to elimination of the transitions while exonucleolytic proofreading preferentially repairs transversions, and more specifically A to T (or T to A) transversions. However, even in the wild-type strain, A to T (or T to A) transversions are the most frequent substitutions, suggesting that they are imperfectly repaired. The combination of both mismatch repair and proofreading deficiencies elicits a mitochondrial error catastrophe. These data show that the faithful replication of yeast mtDNA requires both exonucleolytic proofreading and mismatch repair.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2'-5' oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range. Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2'-5' oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel features of the structure-function relationships involving this enzyme.     

Anti-hepatitis B virus activity and metabolism of 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine

2',3'-Dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine [L(-)Fd4C] was found to be at least 10 times more potent than beta-L-2',3'-dideoxy-3'-thiacytidine [L(-)SddC; also called 3TC, or lamivudine]against hepatitis B virus (HBV) in culture. Its cytotoxicity against HepG2 growth in culture was also greater than that of L(-)SddC (3TC). There was no activity of this compound against mitochondrial DNA synthesis in cells at concentrations upto 10 microM. The dynamics of recovery of virus from the medium of cells pretreated with equal drug concentrations were slower with L(-)Fd4C than with L(-)SddC (3TC). L(-)Fd4C could be metabolized to mono-, di-, and triphosphate forms. The degree of L(-)Fd4C phosphorylation to the 5'-triphosphate metabolite was higher than the degree of L(-)SddC (3TC) phosphorylation when equal extracellular concentrations of the two drugs were used. The apparent K(m) of L(-)Fd4C phosphorylated metabolites formed intracellularly was higher than that for L(-)SddC (3TC). This may be due in part to a difference in the behavior of L(-)Fd4C and L(-)SddC (3TC) towards cytosolic deoxycytidine kinase. Furthermore, L(-)Fd4C 5'-triphosphate was retained longer within cells than L(-)SddC (3TC) 5-triphosphate. L(-)Fd4C 5'-triphosphate inhibited HBV DNA polymerase in competition with dCTP with a Ki of 0.069 +/- 0.015 microM. Given the antiviral potency and unique pharmacodynamic properties of L(-)Fd4C, this compound should be considered for development as an expanded-spectrum anti-HBV drug.     

Synthesis and biological properties of the four optical isomers of 5-o-carboranyl-2',3'-didehydro-2',3'-dideoxyuridine

The four isomers of the 5-o-carboranyl-2',3'-didehydro-2',3'-dideoxyuridine (d4CU) were synthesized and their antiviral activity and cytotoxicity in normal and cancer human cells determined. Coupling of silylated 5-o-carboranyluracil with the protected D/L 2,3-dideoxy-2-phenylselenenylribosylacetates provided after oxidative elimination and deprotection, the desired compounds. The presence of the electron deficient 5-o-carboranyl moiety on uracil influenced the yield of the various isomers. In general, the compounds demonstrated weak anti-human immunodeficiency virus activity in primary human lymphocytes. No marked difference in the biological profile was noted for the various optical isomers, suggesting that the high lipophilicity of these nucleosides imparted by the carboranyl moiety overrides stereochemical considerations in the 2',3'-didehydro-2',3'-dideoxyaglycon moiety.     

Toxicity of liposomal 3'-5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine in mice

Toxicities of 5-fluoro-2'-deoxyuridine (FUdR) and its liposome incorporated dipalmitoyl derivative (FUdR-dipalmitate) to mouse bone marrow, spleen, liver and ileum were compared after treatment for 6 consecutive days. The applied doses of the two formulations, which were shown earlier to have equal antitumor activity in mouse tumor models, were 600 and 2 mumol/kg respectively. When applied in these doses, toxicity to the hemopoietic system, measured as a decreases in progenitor and precursor cells of the erythroid and granuloid/macrophage lineage in bone marrow and spleen, was more severe for FUdR than for liposomal FUdR-dipalmitate. In the liver, mitotic figures, as indicators of cell division, were absent for both drugs while in control livers the number of cells in mitosis was approximately 2%. Toxicity to the ileum was more severe for liposomal FUdR-dipalmitate than for FUdR and was manifested by granulocyte infiltration, the presence of cell debris, loss of columnar epithelial cells and enlarged nuclei with prominent nucleoli in these cells. Thus, by prolonging the retention time of FUdR in vivo, using liposomes as a vehicle and FUdR-dipalmitate as a lipophilic prodrug, the dose-limiting toxicity appears to shift from bone marrow to the gastrointestinal tract.     

Pharmacokinetics and tissue distribution of (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine, a prodrug of 3'-azido-2',3'-dideoxythymidine (AZT) in mice

The in-vivo biodistribution and pharmacokinetics in mice of 3'-azido-2',3'-dideoxythymidine (1, AZT), 2-bromomyristic acid (2) and their common prodrug, (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine (3) are reported. The objectives of the work were to enhance the anti-human immunodeficiency virus and anti-fungal effects of 1 and 2 by improving their delivery to the brain and liver. The pharmacokinetics of AZT (beta t1/2 (elimination, or beta-phase, half-life) = 112.5 min; AUC (area under the plot of concentration against time) = 29.1 +/- 2.9 micromol g(-1) min; CL (blood clearance) = 10.5 +/- 1.1 mL min(-1) kg(-1)) and its ester prodrug (3, beta t1/2 = 428.5 min; AUC = 17.3 +/- 4.7 micromol g(-1) min; CL = 17.6 +/- 4.8 mL min(-1) kg(-1) were compared after intravenous injection of equimolar doses (0.3 mmol kg(-1)) via the tail vein of Balb/c mice (25-30 g). The prodrug was rapidly converted to AZT in-vivo, but plasma levels of AZT (peak concentration 0.17 micromol g(-1)) and AUC (12.3 micromol min g(-1)) were lower than observed after AZT administration (peak concentration 0.36 micromol g(-1); AUC 29.1 micromol min g(-1). The prodrug also accumulated rapidly in the liver immediately after injection, resulting in higher concentrations of AZT than observed after administration of AZT itself (respective peak concentrations 1.11 and 0.81 micromol g(-1); respective AUCs 42.5 and 12.7 micromol min g(-1)). Compared with doses of AZT itself, 3 also led to significantly higher brain concentration of AZT (25.7 compared with 9.8 nmol g(-1)) and AUCs (2.8 compared with 1.4 micromol min g(-1)). At the doses used in this study the antifungal agent 2-bromomyristic acid was measurable in plasma and brain within only 2 min of injection. Hepatic concentrations of 2-bromomyristic acid were higher for at least 2 h after dosing with 3 than after dosing with the acid itself. In summary, comparative biodistribution studies of AZT and its prodrug showed that the prodrug led to higher concentrations of AZT in the brain and liver. Although the prodrug did not result in measurably different concentrations of 2-bromomyristic acid in the blood and brain, it did lead to levels in the liver which were higher than those achieved by dosing with the acid itself.     

Enzymatic properties of the unnatural beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine

The beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine have been stereospecifically synthesized. In an attempt to explain the previously reported antiviral activities of these compounds, their enzymatic properties were studied with respect to adenosine kinase, deoxycytidine kinase, adenosine deaminase, and purine nucleoside phosphorylase. Adenosine deaminase was strictly enantioselective and favored beta-D-ddA and beta-D-d4A, whereas adenosine kinase and purine nucleoside phosphorylase had no apparent substrate properties for the D- or L-enantiomers of beta-ddA or beta-d4A. Human deoxycytidine kinase showed a remarkable inversion of the expected enantioselectivity, with beta-L-ddA and beta-L-d4A having better substrate efficiencies than their corresponding beta-D-enantiomers. Our results demonstrate the potential of beta-L-adenosine analogues as antiviral agents and suggest that deoxycytidine kinase has a strategic importance in their cellular activation.     

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3'-5' exonuclease proofreading function

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD'2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3'-5' exonuclease proofreading activity of the pol III epsilon-subunit also is disabled. TR was measured in isogenic uvrA6 DeltaumuDC strains carrying the dominant negative dnaQ allele, mutD5, or DeltadnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T-T dimer. As expected, little TR was observed in the DeltaumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated DeltaumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind-) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for approximately 70% of the TR, and another LexA-independent, responsible for the remaining approximately 30%. Curiously, the DeltaumuDC DeltadnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in DeltadnaQ strains, since introduction of spq-2 into the DeltaumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041-6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the DeltaumuDC mutD5 strain is unchanged by introduction of a DeltapolB mutation.     

Production and purification of recombinant 2'-5' oligoadenylate synthetase and its mutants using the baculovirus system

Investigation of the structure-function relationship of the 2'-5' oligoadenylate [2-5 (A)] synthetases has been hampered by the lack of an efficient expression system for a recombinant enzyme. Here, we report that the 9-2 isozyme of murine 2-5 (A) synthetase can be efficiently expressed in insect cells using the baculovirus system. The recombinant protein was purified to apparent homogeneity, and its enzymatic activity was characterized. It had a high specific activity, required double-stranded RNA as a cofactor, and synthesized dimers to hexamers of 2-5 (A). The utility of our expression system was demonstrated by studying the properties of two previously reported mutant proteins. Both of these mutants, when produced in bacteria, are enzymatically inactive, although similarly produced wild-type protein is active. Unexpectedly, when expressed in insect cells, both mutant proteins were enzymatically as active as the wild-type protein. These results suggest that in the eukaryotic expression system described here, the mutant proteins can undergo appropriate modifications or folding that is required for attaining an enzymatically active conformation.     

Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.     

Probing the nucleotide binding sites of axonemal dynein with the fluorescent nucleotide analogue 2'(3')-O-(-N-Methylanthraniloyl)-adenosine 5'-triphosphate

MantATP [2'(3')-O-(-N-methylanthraniloyl)-adenosine 5'-triphosphate] was employed as a fluorescence probe of the nucleotide-binding sites of dynein from sea urchin sperm flagella. MantATP binds specifically with enhanced fluorescence (approximately 2.2-fold), homogeneous lifetime (8.4 ns), and high anisotropy (r approximately 0.38) to dynein and can be displaced by ATP and ADP added to the medium. The association constants of mantATP complexed with dynein were determined from anisotropy titration data. Using a multiple stepwise equilibrium model, the average values of the first two association constants are K1 = 2.7 x 10(5) M-1 and K2 = 1.8 x 10(4) M-1. This value of K1 is 7-8 times higher than that found previously for unsubstituted ATP, whereas K2 is little changed [Mocz and Gibbons (1996) Biochemistry 35, 9204-9211]. The lower-affinity binding sites, K3 and K4, observed previously could not be studied with mantATP within the available protein concentrations. The alpha and beta heavy chain subfractions have binding parameters similar to those of intact dynein. Formation of the stable ternary complex of mantATP with dynein and monomeric vanadate is accompanied by only a moderate increase in the binding affinities. Oligomeric vanadate reduces the binding affinities by approximately 50%. Addition of TritonX-100, methanol, or various salts changes the binding affinities by up to 50%, suggesting that the microenvironment of the nucleotide-binding sites involves significant contributions from both polar and apolar interactions. The distinct affinities of the individual binding sites are consistent with a physiological role in regulating nucleotide binding.     

Non-enzymatic, template-directed ligation of 2'-5' oligoribonucleotides. Joining of a template and a ligator strand

Decauridylate containing exclusively a 2'-5' phospho-diester bond ([2'-5']U10) served as a template for the synthesis of oligoadenylates [oligo(A)s] from the 5'-phosphorimidazolide of 2'-5' diadenylate (ImpA-2'p5'A). Joining of [2'-5']U10and ImpA2'p5'A also took place in substantial amounts to yield long-chain oligoribonucleotides in the template-directed reaction. An unusual CD spectrum ascribed to helix formation between [2'-5']U10and [2'-5'](pA)2was observed under the same conditions as that of the template-directed reaction. The 3'-5' linked decauridylate ([3'-5']U10) also promoted the template-directed synthesis of oligo(A)s from ImpA2'p5'A, but more slowly compared with [2'-5']U10. The results indicate that short-chain RNA oligomers with a 2'-5' phosphodiester bond could lead to longer oligoribonucleotides by template-directed chain elongation.     

Chemical degradation kinetics of recombinant hirudin (HV1) in aqueous solution: effect of pH

PURPOSE: To gain information on the chemical stability pattern and the kinetics of the degradation of recombinant hirudin variant HV1 (rHir), a thrombin-specific inhibitor protein of 65 amino acids, in aqueous solution as a function of pH. METHODS: Stability of rHir was monitored at 50 degrees C in the framework of a classical pH-stability study in aqueous buffers pH 1-9.5. Two capillary electrophoresis (CE) protocols were used: one for the kinetics of succinimide formation at Asp53-Gly54 (C-terminal tail) and Asp33-Gly34 (loop section), the other for the kinetics of rHir degradation. To check for potential effects of conformational changes by thermal denaturation, circular dichroism (CD) measurements were performed between 25 and 80 degrees C. RESULTS: Throughout the pH range studied no effect of thermal denaturation on rHir confirmation at 50 degrees C was observed. rHir was most stable at a neutral pH whereas, at slightly acidic pH, an intermediate stability plateau was found. Both, strongly acidic and alkaline conditions led to fast rHir degradation. Depending on the pH of degradation, rHir was found to degrade in various combinations of multiple parallel and sequential degradation patterns. Special focus was on succinimide formation at Asp53-Gly54 (C-terminal tail) and Asp33-Gly34 (loop) and on the potential of isoAsp formation in position 53 and 33. CONCLUSIONS: Chemical rHir stability in the intermediate pH range depends strongly on succinimide formation. At slightly acidic conditions succinimides represent the major degradation product (up to 40%). Around neutral pH succinimides react further, presumably by isoAsp formation, and concentrations remain low. Relative preference of succinimide formation in the C-terminal tail domain versus the loop domain is explained by higher backbone flexibility in the tail.     

Pharmacokinetics and distribution over the blood-brain barrier of zalcitabine (2',3'-dideoxycytidine) and BEA005 (2', 3'-dideoxy-3'-hydroxymethylcytidine) in rats, studied by microdialysis

Microdialysis was applied to sample the unbound drug concentration in the extracellular fluid in brain and muscle of rats given zalcitabine (2',3'-dideoxycytidine; n = 4) or BEA005 (2', 3'-dideoxy-3'-hydroxymethylcytidine; n = 4) (50 mg/kg of body weight given subcutaneously). Zalcitabine and BEA005 were analyzed by high-pressure liquid chromatography with UV detection. The maximum concentration of zalcitabine in the dialysate (Cmax) was 31.4 +/- 5. 1 microM (mean +/- standard error of the mean) for the brain and 238. 3 +/- 48.1 microM for muscle. The time to Cmax was found to be from 30 to 45 min for the brain and from 15 to 30 min for muscle. Zalcitabine was eliminated from the brain and muscle with half-lives 1.28 +/- 0.64 and 0.85 +/- 0.13 h, respectively. The ratio of the area under the concentration-time curve (AUC) (from 0 to 180 min) for the brain and the AUC for muscle (AUC ratio) was 0.191 +/- 0.037. The concentrations of BEA005 attained in the brain and muscle were lower than those of zalcitabine, with Cmaxs of 5.7 +/- 1.4 microM in the brain and 61.3 +/- 12.0 microM in the muscle. The peak concentration in the brain was attained 50 to 70 min after injection, and that in muscle was achieved 30 to 50 min after injection. The half-lives of BEA005 in the brain and muscle were 5.51 +/- 1.45 and 0.64 +/- 0.06 h, respectively. The AUC ratio (from 0 to 180 min) between brain and muscle was 0.162 +/- 0.026. The log octanol/water partition coefficients were found to be -1.19 +/- 0.04 and -1.47 +/- 0.01 for zalcitabine and BEA005, respectively. The degrees of plasma protein binding of zalcitabine (11% +/- 4%) and BEA005 (18% +/- 2%) were measured by microdialysis in vitro. The differences between zalcitabine and BEA005 with respect to the AUC ratio (P = 0.481), half-life in muscle (P = 0.279), and level of protein binding (P = 0.174) were not statistically significant. The differences were statistically significant in the case of the half-life in the brain (P = 0.032), clearance (P = 0.046), volume of distribution (P = 0.027) in muscle, and octanol/water partition coefficient (P = 0.019).