Antitumor effects of bis-thiosemicarbazones— inhibition of deoxyribonucleic acid synthesis in vitro

A group of bis-thiosemicarbazones was evaluated for potential antitumor activity, using the L1210 murine leukemia in cell culture. Drug levels required to inhibit DNA synthesis by 50 per cent, under standard conditions, were determined. The most potent of the agents examined had the structure X[CH₂CR₁=NNHCSNHR₂]₂ where X = C or S and R₁ = R₂ = CH₃. Optimal activity was also obtained with R₁ = H and R₂ = CH₃ only when X = S. The most potent derivatives inhibited DNA synthesis by 50 per cent within 10 min at 10^?6 M levels (id₅₀). Metal chelates of several compounds tested were extremely potent inhibitors of DNA synthesis ($\text{id}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{M or less}$). Insolubility in water and short duration of action in vivo may limit effectiveness of the bis-thiosemicarbazones.     

Effect of diphenylhydantoin on the uptake and catabolism of l-[3H]norepinephrine in vitro in rat cerebral cortex tissue

The effect of diphenylhydantoin on the accumulation of [³H]norepinephrine in vitro was examined in brain slices prepared from rat cerebral cortex. High concentrations of diphenylhydantoin (10^?3 M) caused a significant reduction in the 5-min accumulation of [³H]norepinephrine. On the other hand, 10^?5–10^?4 M diphenylhydantoin facilitated the 20-min accumulation of [³H]norepinephrine. This facilitative action of diphenylhydantoin was (1) associated with a reduction in oxidative catabolism of [³H]norepinephrine and (2) abolished by the 2-hr pretreatment of rats with 100 mg/kg of nialamide (i.p.). The inhibitory action of diphenylhydantoin on the oxidative catabolism of [³H]norepinephrine was observed in both whole and lyzed crude synaptosomal preparations. When diphenylhydantoin and pargyline were compared, it was found that pargyline $\text{(}\text{id}{}_{50}\text{= 1.5 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ was 37 times more effective than diphenylhydantoin $\text{(}\text{id}{}_{50}\text{= 5.5 × 10}{}^{\mathrm{?5}}\text{M}\text{)}$ in inhibiting the oxidative deamination of [³H]norepinephrine. These results suggest that diphenylhydantoin alters norepinephrine metabolism in cerebral cortex slices by an inhibitory action on (1) monoamine oxidase activity and (2) the neuronal uptake system.     

Determination of extraction constant,true partition coefficient and formation constant of ion-pair complexes of quaternary ammonium salts,tetrabutylammonium bromide and isopropamide iodide.with some organic anions by a solvent extraction technique

A new method of determining the extraction constant (K_e, the true partition coefficient (TPC) and the formation constant (K_f) of ion-pairs, was developed by the solvent extraction technique. K_e and TPC were estimated from the reciprocals of the intercept and the slope of the regression line obtained by plotting

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{vs}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}$

in the following equation.

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{=}\text{1}\text{K}{}_{\mathrm{e}}\text{+}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{x}\text{1}\text{TPC}$

where [A^T_W] and [B^T_W] are the total concentrations of the cationic compound A and that of the anionic compound B in the aqueous phase respectively, APC is the apparent partition coefficient of A, dA is the partition coefficient of cation A⁺. K_f, which is expressed by K_e/TPC, was then calculated. These constants were determined for the ion-pair extraction of tetrabutylammonium bromide and isopropamide iodide with 4 organic anions, i.e. benzoic acid, p-toluenesulfonic acid, salicylic acid and taurodeoxycholic acid. This new method might be applicable to other ion-pairs without further assumptions except that the molar ratio of the ion-pair formation be 1 : 1.     

Mechanisms by which quipazine,a putative serotonin receptor agonist,alters brain 5-hydroxyindole metabolism

Quipazine (2-[1-piperazinyl] quinoline maleate) was shown to increase serotonin and decrease 5-hydroxyindoleacetic acid concentrations in whole brain, several brain regions, and the spinal cord of rats 1 hr after its administration (10 mg/kg, i.p.). In animals with transected spinal cords, quipazine induced stronger activation of extensor reflexes than 5-hydroxytryptophan, chlorimipramine, or Lilly 110140. This response could be blocked by methiothepin. In slices of rat cerebral cortex, quipazine inhibited the uptakes of [³H]-serotonin (EC₅₀ = 10^?6 M) and [³H]-norepinephrine ($\text{EC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?6}}\text{m}$); it was equipotent with Lilly 110140 in inhibiting serotonin uptake, but less potent than chlorimipramine ($\text{EC}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{m}$). Quipazine administration to rats did not inhibit monoamine oxidase activity, and actually elevated brain tryptophan levels. These observations suggest that the effects of quipazine on brain serotonin and 5-hydroxyindoleacetic acid concentrations could have been caused by direct activation of central serotonin receptors (which would secondarily decrease impulse flow along serotonergic neurones), or by the inhibition of serotonin reuptake, or by both mechanisms.     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.     

Correlation of biologic data with physico-chemical properties among the vinca alkaloids and their congeners.

Displacement of [³H]vinblastine binding to tubulin by other Vinca alkaloid derivatives has been demonstrated to be a competitive process, allowing for determination of the association constant of each drug. Correlation of LD₅₀ data and anti-P-388 activity was found with log P and log K_a, according to the equations: $\text{log}\text{LD}{}_{50}\text{= 0.129 (}\text{log}\text{P)}{}^2\text{? 0.522}\text{log}\text{P ? 0.479}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 4.652}\text{log}\text{P ? 388 = 0.222 (}\text{log}\text{P)}{}^2\text{? 1.059}\text{log}\text{P ? 0.520}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 5.366}$. Vincristine and desacetylvinblastine were the two most active agents in this series. That the latter drug had significant biologic activity was of considerable interest, since it is known to be a human metabolite.     

The effects of cadmium, manganese and aluminium on sodium-potassium-activated and magnesium-activated adenosine triphosphatase activity and choline uptake in rat brain synaptosomes

The effects of Cd²⁺, Mn²⁺ and Al³⁺ on rat brain synaptosomal sodium-potassium-activated and magnesium-activated adenosine triphosphatase (Na-K-ATPase and Mg-ATPase) activity and choline uptake were studied. All three types of metal ions inhibited Na-K-ATPase activity more markedly than Mg-ATPase activity. The rank order of inhibition of Na-K-ATPase was: $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.4 μ}\text{M}\text{) >}\text{Mn}{}_{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 955 μ}\text{m}\text{) >}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 8.3}\text{mM}$). The rank order of inhibition of Mg- was:$\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 316 μ}\text{M}\text{>}\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.5}\text{mM}\text{>}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 21.9}\text{mM}\text{).}\text{Al}{}^{\mathrm{3+}}$ was most potent in inhibiting synaptosomal choline uptake ($\text{ic}{}_{50}\text{= 24μ}\text{M}$ in the absence of Ca²⁺ and 123 μ.M in the presence of 1 mM Ca²⁺). $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 363 μ}\text{M}\text{)}$ was a more effective inhibitor of choline uptake than $\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 1.2?1.5}\text{mM}\text{)}$ . The presence of 1 mM Ca²⁺ did not alter choline uptake, nor did it antagonize the inhibitory actions of the three metals. Our observations that Cd²⁺ and Al³⁺ inhibited synaptosomal choline uptake, but did not show parallel inhibitory effects on Na-K-ATPase activity directly contradicts the ionic gradient hypothesis. These results are also discussed in relation to the in vivo neurotoxicity of cadmium, manganese and aluminium.     

Kinetic studies on the deethylation of ethoxybenzamide: A comparative study with isolated hepatocytes and liver microsomes of rat

Kinetic parameters (K_m and V_max) of ethoxybenzamide deethylation in isolated rat hepatocytes and liver microsomes were compared. Adjustment of cofactors in microsomal deethylation, such as NADPH and Mg²⁺, to give optimum conditions, and appropriate correction of the apparent kinetic parameters for nonspecific binding and microsomal yield resulted in good agreement among the kinetic parameters of isolated hepatocytes [$\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 0.0863 μmole · min}{}^{\mathrm{?1}}\text{· (g liver)}{}^{\mathrm{?1}}$ and K_m = 0.459 mM] and microsomes [V_max = 0.124 μmoles · min^?1 · (gliver)^?1 and K_m = 0.378 mM].     

Inhibition of hepatic microsomal lipid peroxidation by drug substrates without drug metabolism

Experiments were performed to study the mechanism of action of drug substrates on lipid peroxidation in rat hepatic microsomes. Addition of the drug substrates, aniline, β-diethylaminoethyl diphenylpropylacetate (SKF-525A), aminopyrine, benzo[a]pyrene or ethylmorphine, to hepatic microsomes causes almost complete inhibition of NADPH-induced (enzymatic) lipid peroxidation. These substrates also produce similar inhibition of ascorbate-induced (non-enzymatic) lipid peroxidation in microsomes in which drug-metabolizing enzymes were inactivated by heat treatment. The substrate concentrations producing half-maximal inhibition ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ are also similar for NADPH- and ascorbate-induced lipid peroxidation. Addition of metyrapone, an inhibitor of drug metabolism, has no effect on either the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values or on the maximal substrate inhibition of NADPH-induced lipid peroxidation. All five drug substrates also inhibit Fe²⁺-stimulated oxidation of linoleic acid. These results demonstrate that inhibition of lipid peroxidation in hepatic microsomes by drug substrates is independent of drug metabolism and is probably due to the antioxidant properties of the substrates.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.     

Adenosine receptor interactions and anxiolytics

$\text{[}{}^3\text{H}\text{]-N}{}^6\text{-}\text{cyclohexyladenosine}$ and [³H]-1,3-diethyl-8-phenylxanthine label the A₁ subtype of adenosine receptor in brain membranes. The affinities of methylxanthines in competing for A₁ adenosine receptors parallel their potencies as locomotor stimulants. The adenosine agonist N⁶-(-phenylisopropyl) adenosine is a potent locomotor depressant. Both diazepam and $\text{N}{}^6\text{-(}\text{l}\text{-}\text{phenylisopropyl}\text{)}\text{adenosine}$ cause locomotor stimulation in a narrow range of subdepressant doses. Combined stimulant doses of the two agents depress motor activity, as do larger doses of either one, given separately.Evidence supporting and against the hypothesis that some of the actions of benzodiazepines are mediated via the adenosine system is reviewed. A number of compounds interact with both systems, probably because of physico-chemical similarities between adenosine and diazepam. It is concluded that of the four classic actions of benzodiazepines, the sedative and muscle relaxant (but not anxiolytic or anticonvulsant) actions could possibly be mediated by adenosine.     

Studies of the metabolism of N-methyl containing anti-tumour agents: 14CO2 breath analysis after administration of 14C-labelled N-methyl drugs, formaldehyde and formate in mice

The ¹⁴CO₂; content of the breath was analysed after administration of the following N-¹⁴ CH₃ labelled drugs to mice: aminopyrine, hexamethylmelamine (HMM), pentamethylmelamine (PMM), procarbazine and caffeine. Except for aminopyrine, the ¹⁴CO₂; exhalation rate plots declined monophasically with half lives of 91 min ([¹⁴C]-HMM), 97 min ([¹⁴C]-PMM), 68 min ([¹⁴C]procarbazine) and 92 min ([¹⁴C]caffeine). The ¹⁴CO₂ exhalation rate peaked rapidly after aminopyrine administration and declined bi-phasically with an initial $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 15 min and a terminal $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 126 min. The ¹⁴CO₂; plots after both [¹⁴C]-HMM and [¹⁴C]aminopyrine were influenced by pre-treatment of mice with proadifen. Pretreatment with phenobarbitone shortened the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of the ¹⁴CO₂ appearance rate after [¹⁴C]HMM by 24% but did not change the ¹⁴CO₂ curve after administration of [¹⁴C] aminopyrine. The ¹⁴CO₂ exhalation rate plots after administration of H¹⁴CHO and H¹⁴COOH were virtually identical with that obtained after [¹⁴C]aminopyrine and not influenced by either proadifen or phenobarbitone pretreatment. The ¹⁴CO₂ exhalation rate profile obtained on metabolism of [¹⁴C]aminopyrine in mice thus appears to be determined by the rate of the oxidation of formaldehyde or formate to CO₂. Only 24% of the label injected with the N-methyl moieties of [¹⁴C]HMM and 21% of the label in [¹⁴C]procarbazine were exhaled as ¹⁴CO₂, whereas 49% of the N-¹⁴CH₃ in [¹⁴C]aminopyrine were metabolized to ¹⁴CO₂. It remains to be determined whether this difference and the difference in the shapes of the ¹⁴CO₂ exhalation profiles obtained with the cytotoxic N-¹⁴CH₃ drugs as compared to [¹⁴C]aminopyrine, are related to the biochemical processes mediating their antineoplastic activity.     

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-l-lysine analogue of methotrexate, $\text{N}{}^{\mathrm{\alpha }}\text{-(}\text{4-amino-4-deoxy-10-methylpteroyl}\text{)-N}{}^{\mathrm{\epsilon }}\text{-(5-[N,N-}\text{dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)-}\text{l}\text{-lysine}$, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [³H]methotrexate transport with a K_i of 7.02 μM, a value virtually identical to the K_t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.     

Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide: A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?7}}\text{M}\text{)}$ but not of acetylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 4 × 10}{}^{\mathrm{?4}}\text{M}\text{)}$. For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K_i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K_d of 4.5 × 10^?7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.     

Effects of GABA receptor agonists on [3H] flunitrazepam binding to rat cerebellar and hippocampal membranes

The effects of GABA agonists on [³H] flunitrazepam binding were examined in membranes from CNS areas proposed to contain different populations of benzodiazepine binding site subtypes ($\text{BZ}{}_1\text{BZ}{}_2$). Since these effects were broadly similar in both cerebellar and hippocampal membranes, it seems unlikely that GABA receptors interacting with the proposed $\text{BZ}{}_1\text{BZ}{}_2$ binding sites haue markedly different properties. Sodium chloride clearly facilitated the potentiating effect of muscimol on [³H] flunitrazepam binding but produced a complex interaction uith the effects of GABA itself and the partial agonist imidazoleacetic acid in the phosphate buffer system used.     

Effects of exposure to ozone on susceptibility to experimental tuberculosis

Exposure of mice to 1.96 $\text{mg}\text{m}{}^3$ ozone (O₃) 3 $\text{h}\text{day}$, 5 days/week, for up to 8 weeks beginning at 1 or 2 weeks after challenge with Mycobacterium tuberculosis R 1Rv resulted in significant enhancement of bacterial titers in the lungs at 5 through 8 weeks after challenge when compared to mice exposed to filtered air. Exposure to lower concentrations of O₃ did not produce any significant changes compared to controls.Exposure of guinea pigs to 2.9 $\text{mg}\text{m}{}^3$ O₃ for 3 h immediately after challenge with M. tuberculosis resulted in a suppression of the cutaneous delayed hypersensitivity response, without affecting the serum hemagglutination antibody titers. However, exposure of guinea pigs to 0.98 $\text{mg}\text{m}{}^3$ O³ 3 $\text{h}\text{day}$ for 5 days, initiated within 3 h after the infectious challenge, enhanced hemagglutination antibody titers initially, but the delayed hypersensitivity reaction did not differ from controls.     

Dopaminergic effects of buspirone,a novel anxiolytic agent

The novel anxiolytic drug buspirone raised striatal levels of the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) 1 hr after oral administration. This effect was dose-dependent with a peak at 60 min. No changes were observed in the levels of 3-methyxytyramine (3MT), the extraneuronal metabolite of dopamine. Noradrenaline, serotonin and its metabolite 5-hydroxyindoleacetic acid (5HIAA) were not affected. Buspirone displaced [³H]spiroperidol from striatal binding sites, with an ic₅₀ (1.8 × 10^?7 M), comparable to that of clozapine ($\text{ic}{}_{50}\text{= 1.4 × 10}{}^{\mathrm{?7}}\text{M}$) but considerably lower than that of haloperidol (4.7 × 10^?9 M). Buspirone was only a weak inhibitor of dopamine-stimulated adenyl cyclase. Buspirone was not active on the binding of trifluoperazine to calmodulin and did not modify calmodulin-induced activation of phosphodiesterase (PDE). Repeated administration of buspirone did not increase the number of DA receptors. These data show that, although buspirone has antidopaminergic activity, it can hardly be classified as a classic neuroleptic agent.     

The Ah regulatory gene product. Survey of nineteen polycyclic aromatic compounds' and fifteen benzo[a]pyrene metabolites' capacity to bind to the cytosolic receptor

The capacity of 19 polycyclic aromatic compounds and 15 benzo[a]pyrene metabolites to displace $\text{[1,6-}{}^3\text{H}\text{]2,3,7,8-}\text{tetrachlorodibenzo}\text{-p-}\text{dioxine}\text{([}{}^3\text{H]Tcdd}\text{)}$ from the mouse liver cytosolic Ah receptor was examined. We compared our data with various parameters taken from previously published results: the capacity of seven polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase (AHH) activity in human cell cultures, the capacity of 10 polycyclic hydrocarbons to induce azo dye N-demethylase activity in rat liver, the capacity of 6 polycyclic hydrocarbons to shorten zoxazolamine paralysis times in the intact rat, and the capacity of 15 benzo[a]pyrene metabolites to induce AHH activity in rat hepatoma H-4-II-E cultures. An excellent correlation is seen between the capacity to displace the radioligand from the Ah receptor and the capacity to induce these monooxygenase activities. Differences in the rate of cellular uptake and formation of alkali-extractable metabolites of dibenzo[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene in Hepa-1 mouse hepatoma cell cultures do not account for differences in the capacity of these three polycyclic hydrocarbons to displace [³H]TCDD from the Ah receptor.     

Tight-binding inhibitors—IV. Inhibition of adenosine deaminases by various inhibitors

Three ADA (adenosine deaminase) inhibitors, DHMPR (1,6-dihydro-6-hydroxymethyl purine ribonucleoside); EHNA [erythro-9-(2-hydroxy-3 nonyl)adenine] ; and deoxycoformycin [(R)-3-(2-deoxy-β-d-erythro-pento-furanosyl)-3, 6,7,8-tetrahydroimidazo[4,5-d] [1,3-diazepin-8-ol] or Covidarabin, were compared with regard to their inhibitory behavior with ADAs from human erythrocytes and calf intestine. Marked differences in the times required for establishment of steady state between the enzyme and inhibitors were observed, e.g. DHMPR, virtually instantaneous; EHNA, 2–3 min; and deoxycoformycin, many hr. The parameters of the inhibition of human erythrocytic ADA by deoxycoformycin were as follows: the association rate constant (k₁) = 2.6 × 10⁶ M^?1 sec^?1 ; the dissociation rate constant of the enzyme-inhibitor complex (k₂) = 6.6 × 10^?6 sec^?1; K_i (from $\text{k}{}_2\text{k}{}_1\text{) = 2.5 × 10}{}^{\mathrm{?12}}\text{M}$ and K_i (from I₅₀) = 1.5 × 10^?11 M. The K_i values for EHNA and DHMPR, as determined by classical methods after attainment of steady state, were 1.6 × 10^?9 and 1.3 × 10^?6 M, respectively, for human erythrocytic ADA. The kinetic parameters for EHNA and calf intestinal ADA were as follows: K_i = 6.5 × 10^?9 M (by the method of I₅₀); k₁ = 0.7 × 10⁶ M^?1 sec^?1' and k₂ = 4.6 × 10^?3 sec^?1. On the basis of K_i values, the inhibitors. DHMPR, EHNA and deoxycoformycin (a transition state analog), were classified as readily reversible, semi-tight-binding and tight-binding inhibitors. The difficulties encountered in the kinetic analyses of different types of inhibitors and the methods for dealing with the problems of these inhibitors are discussed.