Inhibitory effects of cordycepin on cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinases

The in vitro effect of cordydepin was tested using various protein kinase preparations. These included cyclic AMP-dependent protein kinase (A-PK) from bovine heart, cyclic GMP-dependent protein kinase (G-PK) from fetal guinea pig lung, and two cyclic nucleotide-independent nuclear protein kinases (PK-I and PK-II) prepared from rat hepatoma 3924A and rat liver. The 50 per cent inhibitory concentrations (id₅₀) of cordycepin for A-PK and G-PK ranged from 1.5–5.0 × 10^?4M and 2.5–8.0 × 10^?4 M, respectively, depending on the presence or absence of cyclic AMP and cyclic GMP in the assay. The id₅₀ of cordycepin with either hepatoma 3924A or rat liver PK-I and PK-II was 4.5 × 10^?5 M and 1.0 × 10^?3 M. respectively. The inhibitory effect of cordycepin was competitive with respect to ATP in all cases. The K_{m} for ATP was increased 3-fold and 5-fold by 5 × 10^?4 M cordycepin for G-PK and A-PK, respectively, while the K_m for ATP was increased 10-fold and 4-fold by 1 × 10^?3 M cordycepin for PK-I and PK-II, respectively.     

Influence of sex and sexual hormones in the bradykinin-receptor interaction in the guinea pig ileum

Strips of the ileal longitudinal muscle layer of guinea pigs have the following affinities for bradykinin: in males, K_n = 1.55 × 10^?8 M with S. D. = 0.23 × 10^?8 M; in females. K_n = 0.39 × 10^?8 M with S. D. = 0.04 × 10^?8 M. No difference between males and females was found in the K_n values for acetylcholine or for histamine. Castration of males and hormonal treatment after castration modify K_n values for bradykinin: in castrated males, K_n = 0.84 × 10^?8 M with S. D. = 0.01 × 10^?8 M; in castrated males treated with testosterone, K_n = 1.33 × 10^?8 M with S. D. = 0.34 × 10^?8 M; and in castrated males treated with β-estradiol. K_n = 0.45 × 10^?8 M with S. D. = 0.02 × 10^?8 M. No difference was observed in the maximum contractility to bradykinin among the five groups of animals. The state of the bradykinin receptors in the castrated animals seems to be influenced by the sexual hormones in the following way: administration of testosterone to the animal changes the receptors to a state of lower affinity for bradykinin: administration of β-estradiol changes the receptors to a state of higher affinity for bradykinin. The results of the hormonal effects were discussed in terms of an allosterically controlled receptor or the synthesis of modified receptors.     

Phosphorylation of selected substrates by semipurified cytoplasmic kinases of three P815 murine leukemias exhibiting collateral sensitivity (P815/TG), sensitivity (P815) or resistance (P815/ARA-C) to 1-beta-D-arabinofuranosylcytosine

Partial separation and purification of the kinases phosphorylating 2'-deoxycytidine (EC 2.7.1.74) (D-K), pyrimidine nucleoside-5'-monophosphate (EC 2.7.4.14) (PM-K) and nucleoside-5'-diphosphate (EC 2.7.4.6) (NDP-K) were achieved by high performance liquid chromatography on Micro Pak TSK-gel 3000 SW columns. Using the standard conditions for all three kinases investigated, the following observations were made: a comparison of the D-K activities using deoxycytidine (dCyd) or l-β-d-arabinofuranosylcytosine (ara-C) as substrate in peak fractions derived from homogenates of murine neoplasms P815, either sensitive (P815) or resistant to ara-C (P815/ara-C) or resistant to 6-thioguanine (P815/TG), revealed comparable specific activities for dCyd and somewhat lower specific activities for ara-C in fractions derived from P815 and P815/TG cells, while substantially reduced specific activities were observed for both substrates in fractions derived from P815/ara-C cells. The 5'-monophosphate of ara-C (ara-CMP) exhibited a higher specific activity than 2'-deoxycytidine-5'-monophosphate (dCMP) in peak fractions with PM-K activity derived from all three cell lines. The 5'-diphosphate of dCyd (dCDP) was phosphorylated to comparable extents by peak fractions with NDP-K activity derived from all three cell lines. The 5'-diphosphate of ara-C (ara-CDP) is a substrate with specific activities comparable to dCDP in peak fractions with NDP-K activity derived from P815/ara-C and P815/TG cell lines, but with somewhat lower specific activities than the dCDP substrate in P815-derived fractions. The ratios of total enzyme activities recovered after injection of a crude P815 cell homogenate were: 1:260:20,000 for D-K (dCyd as substrate), PM-K (dCMP as substrate) and NDP-K (CDP as substrate); their yield was 100% for D-K and NDP-K, and 40% for PM-K activities. The purification achieved ranged from 5 to 33 times, with substantial reductions in the number of bands observed in disc electrophoresis when compared with those in the crude extracts. Experiments evaluating the inhibitory activity of ara-C and its 5′-mono-, di- and triphosphates upon the phosphorylation by these semipurified kinases of dCR and its 5′-phosphates revealed the possibility that ara-CDP and, less so, ara-CTP effectively inhibit the phosphorylation of dCDP to dCTP in the de novo biosynthesis of the latter and, thus, provide insufficient amounts of dCTP for DNA synthesis.     

Characterization of adenosine deaminase from the malarial parasite, Plasmodium lophurae, and its host cell,the duckling erythrocyte

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) was purified and characterized from the malarial parasite, Plasmodium lophurae, and its host cell, the duck (Anas domesticus) erythrocyte, using chromatofocusing (Pharmacia) and adenosine affinity columns. Gel filtration of the enzymes gave molecular weights of 33,800 (P. lophurae) and 36,500 (duck erythrocyte); both enzymes had broad pH optima (pH 6.8 to 8.0), similar stabilities when stored as crude lysates, and like K_m values with adenosine: 2.74 ± 0.88 × 10^?5 M (parasite) and 1.74 ± 0.27 × 10^?5 M (erythrocyte). The P. lophurae adenosine deaminase had a pI of 5.37 ± 0.09, and the duck erythrocyte enzyme had a pI of 4.72 ± 0.09, as determined by chromatofocusing. The parasite enzyme exhibited a specific activity in the crude lysate that was an average 60-fold higher than that of the erythrocyte enzyme. The pattern of elution from the adenosine affinity column, as well as kinetic studies with three adenosine analogs, revealed distinct differences in the binding characteristics of the two enzymes. The P. lophurae adenosine deaminase was weakly retarded by the affinity column, whereas the duck erythrocyte enzyme was strongly retarded. With 9-β-d-arabinosyladenine as substrate, the K_m values were similar (2.29 ± 0.98 × 10^?4 M for P. lophurae and 1.10 ± 0.21 × 10^?4 M for the duck erythrocyte). Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was a potent inhibitor of the duck erythrocyte enzyme with 100% inhibition at 1.3 μM, whereas the parasite adenosine deaminase was not inhibited at 422 μM even when incubated for 24 hr. Inhibitor studies with coformycin, a tight-binding inhibitor, resulted in K_i values of 7.14 × 10^?11 M for P. lophurae and 1.86 × 10^?10 M for the duck erythrocyte. The molar equivalencies, E_t, and catalytic numbers, k₃, were slightly different for both enzymes. The E_t values were 2.80 × 10^?10 M (P. lophurae) and 3.13 × 10^?10 M (duck erythrocyte); the k₃ values were 5.18 × 10³ min^?1 and 4.36 × 10³ min^?1 respectively.     

Relevance of PepT1 in the Intestinal Permeability and Oral Absorption of Cefadroxil

Purpose

To determine the contribution of intestinal PepT1 on the permeability and oral absorption of the β-lactam antibiotic drug cefadroxil.

Methods

The effective permeability (P _eff ) of cefadroxil was evaluated in wild-type and PepT1 knockout mice following in situ single-pass intestinal perfusions. The plasma concentration-time profiles of cefadroxil were also examined after oral gavage.

Results

The P _eff (cm/s) of cefadroxil in wild-type mice was 0.49?×?10^?4 in duodenum, 0.80?×?10^?4 in jejunum, 0.88?×?10^?4 in ileum and 0.064?×?10^?4 in colon. The P _eff (cm/s) in PepT1 knockout mice was significantly reduced in small intestine, but not in colon, as shown by values of 0.003?×?10^?4, 0.090?×?10^?4, 0.042?×?10^?4 and 0.032?×?10^?4, respectively. Jejunal uptake of cefadroxil was saturable (Km?=?2–4 mM) and significantly attenuated by the sodium-proton exchange inhibitor 5-(N,N-dimethyl)amiloride. Jejunal permeability of cefadroxil was not affected by L-histidine, glycine, cephalothin, p-aminohippurate or N-methylnicotinamide. In contrast, cefadroxil permeability was significantly reduced by glycylproline, glycylsarcosine, or cephalexin. Finally, PepT1 ablation resulted in 23-fold reductions in peak plasma concentrations and 14-fold reductions in systemic exposure of cefadroxil after oral dosing.

Conclusions

The findings are definitive in demonstrating that PepT1 is the major transporter responsible for the small intestinal permeability of cefadroxil as well as its enhanced oral drug performance.     

Choline and diethylcholine transport into a cholinergic clone of neuroblastoma cells.

The transport of choline and diethylcholine has been investigated in a cholinergic clone (S20F₃) of mouse neuroblastoma cells. Choline transport was linear for the first 20 min of incubation and was temperature dependent at low concentrations (< 1 × 10^?5 M). Diethylcholine transport was linear for the first 10 min of incubation and was also temperature dependent at low concentrations. High affinity (K_m < 1 × 10^?6 M) and low affinity (K_m > 1 × 10^?5 M) components of transport were found for both compounds. The transport system had a greater apparent affinity (lower K_m) for choline than for diethylcholine (3-fold), but maximal transport velocities were about equal. Each compound competitively inhibited the other's high affinity transport. Hemicholinium (1 × 10^?5 M) slightly inhibited high affinity choline transport but triethylcholine (1 × 10^?6?1 × 10^?4 M) did not. Choline transport was also found to be dependent on the pH and pCO₂ of the medium.     

Inhibition of rat brain monoamine oxidase by formamidines and related compounds

The efficacy of the pesticide chlordimeform or N′-(4-chloro-o-tolyl)-N,N′-dimethylform-amidine, six chlordimeform metabolites, and eleven related compounds as inhibitors of the oxidative deamination of radiocarbon-labelled biogenic amines by rat brain monoamine oxidase was examined. The I₅₀ value for chlordimeform with tyramine as substrate was 6.0 × 10^?5M. Inhibition following prolonged pre-incubation of chlordimeform with monoamine oxidase increased with time. and this was attributed, at least in part, to the formation of the more potent monoamine oxidase inhibitor 4′-chloro-o-formotoluidide, a known chlordimeform metabolite and degradation product. 4′-Chloro-o-formotoluidide was the most potent monoamine oxidase inhibitor examined yielding I₅₀ values of 2.6 × 10^?6m. 1.5 × 10^?6M and 3.2 × 10^?6M, with tyramine, dopamine, and serotonin, respectively, as substrates. The N-demethyl (demethylchlordimeform) and N-didemethyl chlordimeform metabolites gave I₅₀ values with tyramine of 3.3 × 10^?5M and 7.5 × 10^?5M, respectively. Three additional metabolites, 4-chloro-o-toluidine, 5-chloroanthranilic acid, and N-formyl-5-chloroanthranilic acid, were weak inhibitors with I₅₀ values of 1 × 10^?4m or higher. The other formamidine compounds also inhibited the oxidative deamination of tyramine; I₅₀ values ranged from 9.3 × 10^?5m to 7.5 × 10^?6m. Lineweaver-Burk plots revealed that chlordimeform, demethylchlordimeform, and 4′-chloro-o-formotoluidide were competitive inhibitors of the oxidative deamination of β-phenylethylamine, tyramine, dopamine, tryptamine, and serotonin. Inhibition was reversible since activity was restored by washing.     

1-methyl substituent and stereochemical effects of 2-phenylcyclopropylamines on the inhibition of rat brain mitochondrial monoamine oxidase A and B

(E)-2-Phenylcyclopropylamine ((E)-TCP), (Z)-2-phenylcyclopropylamine ((Z)-TCP), (E)-1-methyl-2-phenylcyclopropylamine ((E)-MTCP), and (Z)-1-methyl-2-phenylcyclopropylamine ((Z)-MTCP) were synthesized and used to determine to what extent 1-methyl substitution and stereochemistry of 2-phenylcyclopropylamines affect inhibition of monoamine oxidase (MAO). Inhibition of rat brain mitochondrial MAO-A and B by the compounds were measured using serotonin and benzylamine as the substrate, respectively and IC₅₀ values obtained with 95% confidence limits by the method of computation. For the inhibition of MAO-A, (E)-MTCP (IC₅₀=6.2×10^?8M) was found to be 37 times more potent than (Z)-MTCP (IC₅₀=2.3×10^?6 M), whereas the activity of (E)-TCP (IC₅₀=2.9×10^?7 M) was slightly less than that of (Z)-TCP (IC₅₀=2.3×10^?7 M). Similarly, for the inhibition of MAO-B, (E)-MTCP (IC₅₀=6.3×10^?8 M) was 7 times more potent than (Z)-MTCP (IC₅₀=4.7×10^?7 M) and (E)-TCP (IC₅₀=7.8×10^?8 M), 0.6 times as potent as (Z)-TCP (IC₅₀=4.4×10^?8 M). The results suggested that while without 1-methyl group, potency of a (Z)-isomer was comparable to that of (E)-isomer, the methyl group in its (Z)-position was very unfavorable to the inhibition of MAO and that in its (E)-position, the methyl group contributed positively to the potency as found by the fact that (E)-MTCP was 1–5 times more potent than (E)-TCP. In view of the selective inhibition of MAO-A or B, all compounds elicited 4–10 times higher preference for the inhibition of MAO-B over MAO-A and 1-methyl substitution as well as the stereochemical factors did not significantly influence the selectivity.     

Angiotensin II binding to zona glomerulosa cells from rabbit adrenal glands.

Tritiated angiotensin II binds in a highly specific manner to zona glomerulosa cells prepared from the adrenal cortex of male rabbits. The reaction is a time-dependent process which obeys second-order kinetics (k₁ = 2.4 × 10⁵ M^?1 sec^?1) and reaches saturation in 5–7 min. Dissociation of the angiotensin II-cell complex is rapid (t_{1 2} = 100 sec) and obeys first-order kinetics for the first 3 min (k_?1 = 6.9 × 10^?3 sec^?1).Increased binding was observed with NaCl concentrations from 0 to 40 × 10^?3 M; however, concentrations from 40 to 140 × 10^?3 M decreased binding. Neither MgCl₂ nor Cll₂ at concentrations of 0 to 4 × 10^?3 M alter the binding of angiotensin II to zona glomerulosa cells. A significant decrease in binding was observed with increasing concentrations of KCl (0 to 140 × 10^?3 M).Temperature studies indicate that initially binding is more rapid at 37° (37° > 25° > 0°). However, binding of angiotensin II decreases after 3 min at 37° and after 7 min at 25°. Binding at 0° did not reach a plateau in the 15-min period studied.     

Kinetic studies on dose-triphosphoinositide responses and dose-contraction responses in rabbit iris.

Correlative studies on the dose-triphosphoinositide (TPI) breakdown and -phosphatidic acid (PA) labeling and dose-tension relationship to acetycholine (ACh). carbachol and acetyl-β-methacholine were investigated in the rabbit iris smooth muscle. Concentrations of these agonists between 1 × 10^?8 M and 1 × 10^?4 M caused a concentration-dependent TPI breakdown and PA labeling, and concentrations between 1 × 10^?7 M and 1 × 10^?3 M caused a concentration-dependent contraction of the iris. The ed₅₀ values for the various cholinergic muscarinic agonists were determined from the dose-response curves. Good correlation between the ed₅₀ values determined by the biochemical and pharmacological methods, being of the order of 1–8 × 10^?6 M. were observed. The apparent dissociation constants of ACh and atropine were estimated by measuring the effects of these agents on TPI metabolism and iris muscle contraction. Atropine, at concentrations between 1 × 10^?10 M and 1 × 10^?9 M, produced a parallel shift to the right of the ACh dose-response curve in both the biochemical and pharmacological methods. Both biochemical and pharmacological responses were inhibited in a competitive manner by atropine. For the TPI, PA and contraction responses, the K_B values (determined with ACh as agonist) were 1.7 × 10^?10, 2 × 10^?9 M and 1.14 × 10^?10 M, respectively, and the corresponding pA₂ values were 10.30, 10.35, and 9.90 respectively. It was concluded that the findings of similar K_B values for the atropine-muscarinic receptor, along with similar pA₂ values from the Schild plots, with the TPI, PA and contraction responses in the rabbit iris could suggest a close relationship between the biochemical and pharmacological responses.     

Inhibition of superoxide anion production in macrophages by anti-inflammatory drugs.

The inhibition by anti-inflammatory drugs of the production of Superoxide anions (O₂^?) by isolated guinea pig macrophages was studied spectrophotometrically using NADH and lactate dehydrogenase. id₅₀ values were: 4 × 10^?7M (diclophenac sodium), 1 × 10^?6M (oxyphenbutazone), 1 × 10^?5M (indomethacin), 4 × 10^?5M (phenylbutazone), 7 × 10^?5M (mefenamic acid), 8 × 10^?5 M (flufenamic acid), 8 × 10^?5M (colchicine), 3 × 10^?4M (aspirin), 3 × 10^?4M (benzydamine), 10^?3M < (dexamethasone) and 10^?3M < (gold sodium thiomalate). They seemed to block the cell membrane-associated mechanism to produce Superoxide anions, since most of them did not abolish the generation of superoxide anions from the xanthine oxidase plus hypoxanthine system. Cytochalasin B, pyrogallol, ascorbate, NEM, l-epinephrine and chlorpromazine also inhibited, the production of Superoxide anion, but many non anti-inflammatory drugs were ineffective. This technique was evaluated as a screening method in vitro for nonsteroidal anti-inflammatory drugs.     

Differential toxicity of carrier-bound methotrexate toward human lymphocytes,marrow and tumor cells

Methotrexate that was covalently linked to poly-l-lysine (mol. wt 3,000 and 60,000) (MTX-PLL 3K and 60K) was more inhibitory to the growth of five cell lines from human solid tumors (IC₅₀ 5?10 × 10^?8 M and 1?2.6 × 10^?8 M respectively) than to the growth of five lines of human lymphocytes (IC₅₀ 5?8 × 10^?7 M and 2?5 × 10^?7 M). In contrast, both methotrexate that was covalently linked to human serum albumin (MTX-HSA), and the free drug, were equally toxic to the two classes of cells, with IC₅₀ of 3?15 × 10^?7 M and 2?7 × 10^?8 M, respectively, for the cell types. Uptake studies showed that, whereas MTX and MTX-HSA were transported equally well into WI-L2 lymphocytes, human bone marrow cells, and an astrocytoma tumor line, uptake of MTX-PLL by the astrocytoma cells at 37° was three to four times greater than uptake by WI-L2 lymphocytes or marrow cells. [³H]Deoxyuridine ([³H]-Urd) incorporation studies indicated that low concentrations of MTX-PLL 60K (5 × 10^?7 M) resulted in inhibition of the target enzyme dihydrofolate reductase (DHFR) in the astrocytoma cells, but no iinhibition of DHFR occurred in WI-L2 lymphocytes or marrow cells until concentrations were reached where the carrier itself became toxic (5 × 10^?6 M). Two inhibitors of the lysosomal enzymes, chloroquine and lupuptin, were able to reverse the toxicity of MTX-PLL 60K against the astrocytoma cell line, increasing its IC₅₀ from 2 × 10^?8 to 2 × 10^?7 M. Both lysosomal inhibitors had no effect on the toxicity of MTX-PLL 60K against the WI-L2 lymphocytes or of MTX or MTX-HSA against either cell type, indicating that the increased toxicity of MTX-PLL 60K against the tumor cells was due, in part, to the ability of the lysosomes of these cells to convert MTX-PLL 60K either to the free drug or to a derivative that was effective in inactivating DHFR. These results suggest that comparable differential toxicity between marrow and tumor cells might also be achieved in vivo if MTX-PLL is infused over long periods at a rate that would maintain a constant serum concentration sufficient to kill tumor cells without affecting bone marrow cells.     

Inhibition of superoxide anion production in non-stimulated guinea pig peritoneal exudate cells by anti-inflammatory drugs.

Inhibitory effects of anti-inflammatory drugs on the production of superoxide anion (·O₂^? by isolated non-treated guinea pig peritoneal exudate cells (PEC) was studied spectro-photometrically using NADH and lactate dehydrogenase (LDH). Values of ID₅₀ were; diclofenac sodium (2 × 10^?5M), indomethacin (3 × 10^?5M), oxyphenbutazone (8 × 10^?5M), fenamic acid (1 × 10^?4M), ibuprofen (1 × 10^?4M), benzydamine (3 × 10^?4M), aspirin (10^?3M<) and dexamethasone (10^?3M<). The mechanism of inhibition seemed to block plasma membrane associated NAD(P)H oxidase(s) activity which produces ·O₂^? ID₅₀ values of other drugs; superoxide dismutase (SOD, 2 × 10^?8M), cytochalasin B(1 × 10^?7M) and NEM (6 × 10^?6M). d-Mannitol radical scavenger), 1,3-diphenyl-isobenzofuran (singlet oxygen scavenger) and sodium azide (mitochondrial electron transport inhibitor and singlet oxygen scavenger) were negative.Superoxide radical itself or oxygen-centered radical(s) derived from ·O₂^? is supposed recently as a rate-limiting factor for prostaglandin (PG) synthetase. Whether the inhibition of non-steroidal anti-inflammatory drug (NSAID) on ·O₂^? production is linked directly to PG biosynthesis or not, ·O₂^? was already demonstrated in our laboratory to make a role for the development of rat carageenan foot oedema. It may serve as a new in vitro sceening method of NSAID, to check the inhibitory potency of a compound on ·O₂^? production by guinea pig PEC.     

Tight binding inhibitors--VI. Interactions of deoxycoformycin and adenosine deaminase in intact human erythrocytes and sarcoma 180 cells

The inactivation and reactivation of adenosine deaminase (ADA) by deoxycoformycin was studied in intact human erythrocytes and murine Sarcoma 180 cells in vitro. The second-order rate constant (k₁) for the association reaction between deoxycoformycin and intraerythrocytic ADA was calculated to be 5.1 × 10³M^?1 sec^?1. This is about 300 to 500-fold lower than the k₁ values determined either with hemolyzed human erythrocytes (k₁ = 1.4 × 10⁶ M^?1 sec^?1) or with partially purified human erythrocytic ADA (k₁ = 2.6 × 10⁶ M^?1 sec^?1). In intact erythrocytes only slight reactivation (<10 per cent) of the inhibited ADA (EI complex) was detectable over 24 hr, whereas with hemolysates about 50 per cent reactivation of the inhibited ADA was observed in about 25 hr (k₂ = 7.7 × 10^?6sec^?1). The k¹ values with intact and supernatant fractions from homogenized Sarcoma 180 cells were determined to be 1.1 × 10⁴M^?1 sec^?1 and 4.2 × 10⁶ M^?1 sec^?1 respectively. With intact Sarcoma 180 cells, negligible reactivation of ADA was seen during a 48-hr period. Preliminary studies indicate an important role for the erythrocytic nucleoside transport system on the apparent k₁ values and the rate of inactivation of ADA by deoxycoformycin in intact cells.     

Biochemical studies of a new antitumor agent, O2,2'-cyclocytidine

Cyclocytidine, O²,2′-cyclocytidine (cyclo-C), structurally related to 1-β-d-arabinofuranosyl cytosine (ara-C), inhibits the incorporation of ³H-thymidine into DNA of L1210 leukemia cells both in vivo and in vitro, and human normal marrow cells and leukemic cells in vitro; but it has no effect on uridine or l-valine incorporation. The inhibition is proportional to the dose, and on an equimolar basis in vivo, cyclo-C shows a lesser but longer lasting effect than ara-C. Cyclo-C has no effect on thymidine incorporation into DNA of L1210 leukemic cells resistant to ara-C. Cyclo-C is stable in 0.1 M Tris buffer, pH 7.0, at 37° for the incubated 4-hr period, but at pH 9.0 for 1hr, > 90 per cent is hydrolyzed to ara-C. When cyclo-C was incubated at 37° for 60 min with plasma from various species, the supernatant from boiled human plasma, or Eagle's minimum essential medium, the only product found was ara-C. Ara-C was found in dog's plasma and urine 2 hr after the i.v. injection of ¹⁴C-cyclo-C and in mouse urine 1 hr after the injection (i.p.). The above results suggest that cyclo-C is hydrolyzed to ara-C and may thus serve as a reservoir of ara-C. Intermittent treatment with cyclo-C may, therefore, replace the current clinical practice of 5-day continous intravenous infusion of ara-C.     

Sorafenib Decreases Tumor Exposure to an Anti-carcinoembryonic Antigen Monoclonal Antibody in a Mouse Model of Colorectal Cancer

In this investigation, we test the hypothesis that treatment with sorafenib, an anti-angiogenic agent, decreases tumor vascularization and, consequently, hinders the delivery of monoclonal antibodies (mAb) to xenograft tumors. Severe combined immunodeficiency mice bearing carcinoembryonic antigen (CEA) expressing tumor xenografts were divided into control and sorafenib-treated groups. Sorafenib was administered to the latter group at 50 mg/kg IP every 48 h, starting 4 days post-tumor implantation. When tumors attained a size of 200–300 mm³, mice were evaluated for (a) tumor microvessel density (using immunohistochemical analysis), (b) tumor macromolecular extravasation (using Evans Blue Dye (EBD)), (c) pharmacokinetics of an anti-CEA mAb, T84.66, following an intravenous dose of 10 mg/kg, and (d) intra-tumoral spatial distribution of T84.66 (using autoradiography). Sorafenib treatment resulted in a substantial reduction in tumor growth rate, a visible reduction in tumor microvessel density, and in a 46.4% decrease in EBD extravasation in tumor tissue (p?<?0.0455). For control and treated mice, no significant difference was found for the area under the mAb plasma concentration-time curve (AUC_(0–7d): 1.67?×?10³?±?1.28?×?10² vs. 1.76?×?10³?±?1.75?×?10² nM?×?day, p?=?0.51). However, tumor AUC_(0–7d) was reduced by 40.8% in sorafenib-treated mice relative to that observed in control mice (5.61?×?10²?±?4.27?×?10¹ vs. 9.48?×?10²?±?5.61?×?10¹ nM?×?day, p?<?0.001). Sorafenib therapy was also found to markedly alter mAb tumor spatial distribution. The results collectively suggest that sorafenib treatment causes a significant reduction in mAb delivery to, and distribution within, solid tumors.     

Inhibition by (−)-Deprenyl of Agonist-Evoked Contractions in Rabbit Aorta

Abstract: The effect of (?)-deprenyl, a relatively selective MAO-B inhibitor, was examined for its ability to inhibit the contractions of rabbit isolated aorta evoked by various agonists and potassium. (?)-Deprenyl (10^?5?3 × 10^?4 M) antagonized the contractions evoked by noradrenaline (10^?8?3 × 10^?4 M); pA₂: 5.10. The antagonism was reversible. It was attenuated by cocaine (3x M); pA₂: 4.38, unchanged by corticosterone (4 × 10^?5 M); pA₂ 4.79 and enhanced by cocaine (3 × 10^?5 M) plus corticosterone (4 × 10^?5 M); pA₂: 5.48. (+)-Deprenyl (10^?6?10^?4 M) did not alter the contractions evoked by noradrenaline (3 × 10^?9?10^?4 M). Clorgyline (10^?5 and 10^?4 M) antagonized the noradrenaline-evoked contractions. Pargyline (10^?4 and 3 × 10^?4 M) had no effect. (?)-Deprenyl (10^?5?3 × 10^?4 M) antagonized the contractions evoked by phenylephrine (10^?8?10^?4 M); pA₂: 5.10. Removal of the endothelium did not alter the antagonism; pA₂: 5.35. (?)-Deprenyl (10^?5?3 × 10^?4 M) antagonized the contractions evoked by either 5-hydroxytryptamine (3 × 10^?8?3 × 10^?4 M); pA₂: 4.61 or by histamine (10^?6?3 × 10^?2 M); pA₂: 4.84. (?)-Deprenyl (3 × 10^?4 M) caused a non-competitive antagonism of the contractions evoked by potassium (1.5-5.5 × 10^?2 M). It is concluded that (?)-deprenyl is a weak inhibitor of postjunctional α-adrenoceptors, 5-hydroxytryptamine (5-HT₂) receptors, and histamine (H₁) receptors.     

Tight-binding inhibitors-II. Non-steady state nature of inhibition of milk xanthine oxidase by allopurinol and alloxanthine and of human erythrocytic adenosine deaminase by coformycin.

The non-steady state nature of the inhibition of milk xanthine oxidase by allopurinol and alloxanthine was demonstrated, and the kinetic data presented are consistent with the known mechanisms of inhibitions by these inhibitors. With the use of human erythrocytic adenosine deaminase and its tight-binding inhibitor, coformycin, it was demonstrated that the classical methods of enzyme kinetics based on the steady state assumptions are grossly inadequate for determining the inhibition mechanisms or inhibition constants for tight-binding inhibitors. The application of the Ackermann-Potter plot, I₅₀. the Easson-Stedman plot (or Henderson plot), and the rates of association and dissociation of enzyme-inhibititor complex were presented and their usefulness was evaluated. The molar equivalency and the catalytic number of human erythrocytic adenosine deaminase were estimated at about 1.0 × 10^?10 mole/unit and 1.0 × 10⁴ min^?1 respectively. It was also demonstrated that the K_i, value of coformycin for this enzyme does not exceed 1.2 × 10^?10 M, and that the second-order rate constant for the association of the enzyme with coformycin is approximately 2 × 10⁶ M^?1 sec^?1. The biphasic nature of the dissociation of the deaminase-coformycin complex (EI complex) indicates that the EI complex undergoes a slow conformational change. The implications of these new kinetic approaches for the study of tight-binding inhibitors, including transition-state analogs, were discussed.     

The binding of quinidine to protein fractions of normal human sera.

In contrast to previous assumptions, albumin is not the only protein in normal human serum responsible for binding quinidine. Human serum proteins were fractionated by gel filtration and floatation. Quinidine binding was determined by equilibrium dialysis. The binding to low (LDL) and high (HDL) density lipoproteins exhibited two binding sites on each protein, and the dissociation constants K and number of binding sites n were calculated. LDL: K₁ = 2 × 10^?5, n₁ = 1 and K₂ = 5·2 × 10^?4, n₂ = 97. HDL: K₁ = 1·9 × 10^?5, n₁ = 0·1 and K₂ = 1·1 × 10^?3, n₂ = 14·7.     

In vitro hydroxylation of diphenylhydantoin and its inhibition by other commonly used anticonvulsant drugs

Using a crude 9,000 g rat liver musomal preparation, in vitro studies were carried out on (a) the metabolism (hydroxylation) of diphenylhydantoin (DPH), (b) the effect of other commonly used anticonvulsants on this hydroxylation. DPH hydroxylation exhibited saturation kinetics at a DPH substrate concentration of approximately 10^?4M. Mean K_m and V_m values for the reaction were 9.3 × 10^?5 M and 23.3 μg/m1 respectively. The linear Hill plot with an interaction coefficient of 1.0 suggests that there is no cooperativity between different DPH molecules during DPH receptor binding process. The anticonvulsants ethosuximide, sodium phenobarbitone, sodium valproate and sulthiame all exhibited inhibition of DPH hydroxylation to varying degrees. K_i, inhibition constants for the four anticonvulsants were respectively 1.1 × 10^?2, 9 × 10^?4, 1.8 × 10^?2 and 8.8 × 10^?4M. Inhibition of DPH hydroxylation by sodium phenobarbitone and sulthiame was strong and competitive in nature. Ethosuximide showed a weak competitive type of inhibition and sodium valproate a weak uncompetitive type of inhibition.