Analysis of Intestinal Perfusion Data for Highly Permeable Drugs Using a Numerical Aqueous Resistance—Nonlinear Regression Method

Purpose. To develop, validate and apply a method for analyzing the intestinal perfusion data of highly permeable compounds using the Numerical Aqueous Resistance (NAR) theory and nonlinear regression (NAR-NLR) and to compare the results with the well-established Modified Boundary Layer (MBL) Analysis. Methods. The NAR-NLR method was validated and the results were compared to the MBL analysis results using previously reported cephradine jejunal perfusion data. Using the Single Pass Intestinal Perfusion (SPIP) method, the concentration dependence of intestinal permeability was investigated for formycin B, proline, and thymidine, three compounds reported to be absorbed by carrier-mediated transport processes. The MBL and NAR-NLR analyses were then applied to the three sets of SPIP data. Results. The results demonstrate that the intrinsic MBL transport parameters were highly variable and, in one case, the analyses failed to give a statistically significant Michaelis constant. The MBL mean dimensionless wall permeabilities (P*_w) were greater than the NAR-NLR P*_w and were also highly variable. In all cases, the NAR-NLR variability was significantly lower than the MBL variability. The extreme variability in the MBL-calculated P*_w is due to the sensitivity of P*_w when the fraction of unabsorbed drug (C_m/C_o) is low or, alternatively, when P*_w approached the aqueous permeability, P*_aq. Conclusions. The NAR-NLR method facilitates the analysis of intestinal perfusion data for highly permeable compounds such as those absorbed by carrier-mediated processes at concentrations below their K_m. The method also allows for the use of a wider range of flow conditions than the MBL analysis resulting in more reliable and less variable estimates of intestinal transport parameters as well as intestinal wall permeabilities.     

Differential effects of inhibitors of purine metabolism on two trichomonad species

Tritrichomonas foetus and Trichomonas vaginalis are both incapable of de novo purine nucleotide synthesis. Previous studies indicated that T. foetus relies mainly on the salvage of hypoxanthine and subsequent conversion of IMP to AMP and GMP, whereas T. vaginalis depends on direct conversions of exogenous adenosine to AMP and guanosine to GMP without much interconversion between the two nucleotides. These two different types of purine salvage suggest the possibility of differential sensitivities between the two species of trichomonad flagellates toward different purine antimetabolites. Mycophenolic acid, hadacidin, 8-azaguanine, and formycin B inhibited the growth of T. foetus but had no effect on T. vaginalis. Mycophenolic acid acted by blocking conversion of IMP to GMP, hadacidin inhibited conversion of IMP to AMP, and 8-azaguanine was incorporated into the T. foetus nucleotide pool, likely via hypoxanthine phosphoribosyl transferase. Formycin B was converted to 5'-monophosphate in T. foetus and inhibited the conversion of IMP to AMP. Its precise mechanism of action on T. foetus remains, however, to be elucidated. Alanosine, whose ribonucleotide derivative is a potent inhibitor of adenylosuccinate synthetase, had no effect on the growth or hypoxanthine incorporation in T. foetus, which may be due to the lack of conversion of alanosine to the ribonucleotide because of the absence of de novo purine nucleotide synthesis in parasites. Four adenosine analogs, adenine arabinoside, tubercidin, sangivamycin, and toyocamycin, were found inhibitory to the growth of T. vaginalis but showed little effect on T. foetus growth. Further investigations suggested that these four compounds acted on T. vaginalis by blocking incorporation of adenosine into the adenine nucleotide pool.     

Structure-activity relationship for adenosine kinase from Mycobacterium tuberculosis II. Modifications to the ribofuranosyl moiety

Adenosine kinase (Ado kinase) from Mycobacterium tuberculosis is structurally and biochemically unique from other known Ado kinases. This purine salvage enzyme catalyzes the first step in the conversion of the adenosine analog, 2-methyl-Ado (methyl-Ado), into a metabolite with antitubercular activity. Methyl-Ado has provided proof of concept that the purine salvage pathway from M. tuberculosis may be utilized for the development of antitubercular compounds with novel mechanisms of action. In order to utilize this enzyme, it is necessary to understand the topography of the active site to rationally design compounds that are more potent and selective substrates for Ado kinase. A previous structure-activity relationship identified modifications to the base moiety of adenosine (Ado) that result in substrate and inhibitor activity. In an extension of that work, 62 Ado analogs with modifications to the ribofuranosyl moiety, modifications to the base and ribofuranosyl moiety, or modifications to the glycosidic bond position have been analyzed as substrates and inhibitors of M. tuberculosis Ado kinase. A subset of these compounds was further analyzed in human Ado kinase for the sake of comparison. Although no modifications to the ribose moiety resulted in compounds as active as Ado, the best substrates identified were carbocyclic-Ado, 8-aza-carbocyclic-Ado, and 9-[alpha-l-lyxofuranosyl]-adenine with 38%, 4.3%, and 3.8% of the activity of Ado, respectively. The most potent inhibitor identified, 5'-amino-5'-deoxy-Ado, had a K(i)=0.8muM and a competitive mode of inhibition. MIC studies demonstrated that poor substrates could still have potent antitubercular activity.     

Structure-activity relationship for nucleoside analogs as inhibitors or substrates of adenosine kinase from Mycobacterium tuberculosis. I. Modifications to the adenine moiety

Adenosine kinase (Ado kinase, EC 2.7.1.20) is a purine salvage enzyme that phosphorylates adenosine (Ado) to AMP. Ado kinase from Mycobacterium tuberculosis also catalyzes an essential step in the conversion of 2-methyl-Ado to a compound with selective antimycobacterial activity. In order to aid in the design of more potent and selective Ado analogs, eighty nucleoside analogs with modifications to the adenine (Ade) moiety of Ado were evaluated as both substrates and inhibitors of Ado kinase from M. tuberculosis, and a subset was further tested with human Ado kinase for the sake of comparison. The best substrates were 2-aza-Ado, 8-aza-9-deaza-Ado, and 2-fluoro-Ado and the most potent inhibitors were N1-benzyl-Ado (Ki=0.19 microM), 2-fluoro-Ado (Ki=0.5 microM), 6-cyclopentyloxy-purine riboside (Ki=0.15 microM), and 7-iodo-7-deaza-Ado (Ki=0.21 microM). These studies revealed the presence of a hydrophobic pocket near the N6- and N1-positions that can accommodate substitutions at least as large as a benzyl group. The ability to fit into this pocket increased the likelihood that a compound would be an inhibitor and not a substrate. The 2-position was able to accommodate exocyclic substitutions as large as a methoxy group, although substrate activity was low. Similarly, the 7-position could bind an exocyclic group as large as a carboxamido moiety. However, all of the compounds tested with modifications at the 7-position were much better inhibitors than substrates. MIC studies performed with selected compounds have yielded several Ado analogs with promising antitubercular activity. Future studies will utilize this information for the design of new analogs that may be selective antitubercular agents.     

Inhibition of methylation of nuclear ribonucleic acid in L1210 cells by tubercidin, 8-azaadenosine and formycin

The adenosine analogs tubercidin (7-deazaadenosine), formycin (7-amino-3-[β-d-ribofuranosyl] pyrazolo[4,3-d]pyrimidine) and 8-azaadenosine were examined for their effects on the synthesis and methylation of nuclear RNA in L1210 cells in vitro. Total RNA and DNA synthesis was affected to the greatest extent by tubercidin (IC₅₀ = 7 × 10^?6M) and to an insignificant degree by 8-azaadenosine and formycin; however, the effects of the latter two drugs, but not of tubercidin, were potentiated by 2'-deoxycoformycin, an inhibitor of adenosine deaminase. In the presence of 2'-deoxycoformycin, RNA synthesis was inhibited by 40 per cent at 1 × 10^?4 M 8-azaadenosine and by 50 per cent at 2 × 10^?4 M formycin, while DNA synthesis was inhibited less extensively. Alkaline hydrolysis of nuclear RNA labeled with [¹⁴C]uridine and l-[methyl-³H]methionine showed preferential inhibition of base methylation in mononucleotides, but not of 2′-O-methylation in dinucleotides, for all three drugs. This differential effect persisted to varying degrees in ?18S and 4S nuclear RNA separated by electrophoresis. The reduction in base methylation in 4S RNA was associated with seven of the eight methylated nucleosides in 4S RNA separated by two-dimensional thin-layer chromatography. These results indicate that tubercidin, 8-azaadenosine and formycin can preferentially inhibit the base methylation of nuclear RNA relative to its synthesis.     

Adenosine deaminase from human erythrocytes: Purification and effects of adenosine analogs

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified about 3000-fold from human erythrocytes. The molecular weight of the enzyme was estimated to be 33,000. With the partially purified erythrocytic adenosine deaminase, K_m and V_max values relative to adenosine were: adenosine, 25 μM, 100 per cent; formycin A, 1000 μM, 753–850 per cent; 8-aza-adenosine, 130 μM, 310 per cent; 6-chloropurine ribonuclcoside, 1000 μM, 91 per cent; 2,6-diaminopurine ribonucleoside, 74 μM, 91 per cent; 2'-deoxyadenosine. 7 μm, 60 per cent; xylosyladenine, 33 μm, 62 per cent; arabinosyi adenine, 100 μM, 47 per cent; 3'-deoxyadenosine (cordycepin), 41 μM, 100 per cent; 3'-amino3'-deoxyadenosine. 133 μM, 89 per cent: 4'-thioadenosine, 13 μM, 43 per cent; and 6-methylselenopurine ribonucleoside, 27 μM, 88 per cent. Apparent K_ti values of reaction products and some adenosine analogs using adenosine as a substrate were as follows; inosine. 116 μM; 2'-deoxyinosine, 60 μM; guanosine, 140 μM; 2-fluoroadenosine, 60 μM; 2-fluorodeoxyadenosine. 19 μM; N⁶-methyladenosine, 17 μM; N₁-methyladenosine, 275 μM; 6-thioguanosine, 92 μM; 6-thioinosine, 330 μM; 6-methylthioinosine, 270 μM; arabinosyl 6-thiopurine, 360 μM; and coformycin, 0.01 μM. Tubercidin (7-deaza-adenosine) and toyocamycin were devoid both of substrate and inhibitor activity. Also. N⁷-methylinosine, N⁷-methylguanosine and dipyridamole (Persantin®) did not inhibit the enzymic activity.     

Effects of 8-azaadenosine and formycin on cell lethality and the synthesis and methylation of nucleic acids in human colon carcinoma cells in culture

The cytocidal and biochemical effects of formycin and 8-azaadenosine in the presence and absence of the adenosine deaminase inhibitor, 2′-deoxycoformycin, were studied in human colon carcinoma (HT-29) cells in culture. Logarithmically growing cells were unaffected by 24-hr exposure to either 10^?6M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10^?5M of each analog. In the presence of 10^?6M 2′-deoxycoformycin, a 3- and 30-fold potentiation of the cytocidal activity of 8-azaadenosine and formycin, respectively, was produced. Inhibition of DNA synthesis but not RNA synthesis by 8-azaadenosine paralleled its cytocidal activity; however, neither variable correlated closely with the cytotoxic effects of formycin. In addition, the methylation of nuclear RNA was unaffected by both drugs while the methylation of 5-methyl-deoxycytidine in DNA was inhibited to a lesser extent than DNA synthesis. Measurements of the incorporation of [³H]formycin and [³H]8-azaadenosine into nuclear RNA and DNA in the presence and absence of 2′-deoxycorformycin indicated that formycin substitution in RNA and DNA was enhanced 10- and 20-fold, respectively, while [³H]8-azaadenosine incorporation into both nucleic acids was increased 6- to 7-fold. These results suggest that the incorporation of formycin into nucleic acids, particularly DNA, correlates closely with its lethal effect on cell viability. On the other hand, the cytocidal activity of 8-azaadenosine more clearly parallels its inhibitory effect on DNA synthesis rather than its substitution into nucleic acids.     

Formycin B Elimination from the Cerebrospinal Fluid of the Rat

The goal of this study was to determine whether specific transport systems are involved in nucleoside elimination from the cerebrospinal fluid (CSF). First, in vitro studies were carried out in isolated choroid plexus tissue slices from rat to ascertain the mechanisms of transport of formycin B, a model nucleoside analogue. ³H-Formycin B accumulated against a concentration gradient in the presence of an Na⁺ gradient in the isolated ATP-depleted choroid plexus tissue slices. This accumulation was reduced by high concentrations of unlabeled formycin B. Nitrobenzylthioinosine (NBMPR), an equilibrative nucleoside transport inhibitor, inhibited the uptake of formycin B in the absence of an Na⁺ gradient. These data suggest that both equilibrative and secondary active Na⁺-nucleoside transport systems are present in rat choroid plexus. In vivo, formycin B, together with inulin as a bulk flow marker, was injected into the lateral ventricle of the anesthetized rat with the aid of a stereotaxic device, and CSF was sampled from the cisterna magna at various times after injection. Twelve rats were randomized and divided into a low- and a high-dose group. The CSF clearance (CL_CSF) of formycin B was significantly higher than the CL_CSF of inulin in both animal groups (P < 0.01), indicating that formycin B is cleared from CSF by a pathway(s) in addition to bulk flow. Formycin B CL_CSF was significantly lower in the high-dose group than in the low-dose group (P < 0.05), suggesting a saturable CSF elimination. The CL_CSF of formycin B was also significantly reduced in animals treated with NBMPR (P < 0.05). These data are consistent with the in vitro studies and collectively suggest that formycin B is eliminated from the CSF by a pathway(s) in addition to bulk flow. At least one pathway is saturable and may represent an equilibrative nucleoside transport system which can be inhibited by NBMPR.