Enantioselective pharmacodynamics of the nonsteroidal antiinflammatory drug ketoprofen: in vitro inhibition of human platelet cyclooxygenase activity.

The pharmacological activity of ketoprofen enantiomers was investigated in humans by an in vitro method. The antiplatelet effect of ketoprofen was assessed by measuring the inhibition of platelet thromboxane B2 (TXB2) generation during the controlled clotting of whole blood obtained from each of four healthy volunteers. Ketoprofen was added separately to whole blood as a range of concentrations of (1) predominantly (S)-ketoprofen, (2) racemic ketoprofen, and (3) predominantly (R)-ketoprofen. (S)-Ketoprofen was found to be solely active at inhibiting human platelet TXB2 production; (R)-ketoprofen was devoid of such activity and did not modify the potency of its optical antipode. A relationship between the percentage inhibition of TXB2 generation and the unbound concentration of (S)-ketoprofen in serum was modelled according to a sigmoidal Emax equation. The mean (+/- SD) serum unbound concentration of (S)-ketoprofen required to inhibit platelet TXB2 generation by 50% (EC50) was 0.320 (+/- 0.062) ng/ml. This value for ketoprofen is considerably lower than previously reported values for (S)-ibuprofen and (S)-naproxen.     

Role of capsule in Klebsiella pneumoniae virulence: lack of correlation between in vitro and in vivo studies

In vitro and in vivo models were used to investigate the role of capsule on the virulence of Klebsiella pneumoniae. We showed that capsule expression reduces dramatically the ability of the K. pneumoniae to bind to epithelial cells when compared to its non-capsulated variant. The presence/absence of capsule had no effect on the colonization of the gastrointestinal tract, while in the urinary tract we established that capsule is an important virulence factor. Our study demonstrates the caution needed when extrapolating from results of in vitro studies and emphasizes the necessity of in vivo models in studies of bacterial virulence.     

Reversible interaction between androgen binding protein and testicular macromolecules causing inhibition of androgen binding activity.

Sertoli cells in culture isolated from immature rat testes secrete androgen binding protein (ABP) in the culture medium. Binding activity of ABP in concentrated medium was estimated with equilibrium dialysis against 1 nM dihydrotestosterone at 4 degrees C. The ABP protein activity was inhibited approximately 50% through addition of cytosol preparations from testis or liver, but not from brain tissue, to the concentrated culture medium; this inhibition remained constant for at least two days. The inhibitor is probably a macromolecule, because the activity could not be removed by charcoal treatment and dialysis. The percent inhibition of ABP binding activity was increased when increasing amounts of cytosol were added, it decreased in the presence of increased concentrations of androgens, but it was not influenced by variations of the concentration of ABP. Inhibition of androgen binding to ABP by cytosols in the presence of 1 nM testosterone could be reversed after dialysis in the presence of 10 nM testosterone. These results suggest a reversible competition between testosterone and the testicular macromolecule for ABP. The occurrence of this interaction between ABP and a testicular macromolecule can explain the variable results of estimated ABP binding activity in testis cytosol preparations.     

In vitro embryotoxicity of a series of para-substituted phenols: structure, activity, and correlation with in vivo data.

The embryotoxicity of phenol and twelve para-substituted congeners on mid-gestation rat embryos was evaluated in vitro. Through application of correlative procedures and stepwise regression, equations describing the relationship between physical-chemical properties and various measures of activity were developed. Embryotoxicity was quantified by the log of the reciprocal of the potency estimates for reduction in selected growth parameters and induction of four morphological defects. In general, co-cultured hepatocytes ameliorated embryotoxicity; only phenol-induced embryotoxicity was enhanced by the presence of hepatocytes. In the absence of hepatocytes, measures of growth retardation were positively correlated with molar refractivity of the phenols. With hepatocytes, lipophilicity became important for describing the potential to induce growth deficits. The structural defects had varying correlation patterns in both culture systems. Potencies of these congeners in vitro were also compared to maternal and developmental potencies observed in vivo (Kavlock, Teratology, 41:43-59, '90). Two of the congeners were very toxic in both systems. For the remaining congeners, one maternal toxicity measure was found to be positively correlated with embryotoxicity for growth and development in vitro without hepatocytes. With hepatocytes, a broad spectrum of correlations, both positive and negative, were observed between in vivo developmental toxicity endpoints and in vitro embryotoxicity. Data from preliminary dosimetry studies suggest that phenol congeners may accumulate in embryos exposed in vitro more readily than with in vivo exposure. Potency calculations based on dosimetry information may demonstrate better correlations between data and allow additional relationships between chemical structure and activity to be developed.     

In vitro and in vivo antimycobacterial activity of antiinflammatory drug, diclofenac sodium

The non-steroidal antiinflammatory drug diclofenac sodium exhibited remarkable inhibitory action against both drug sensitive and drug resistant clinical isolates of Mycobacterium tuberculosis, as well as other mycobacteria. This agent was tested in vitro against 45 different strains of mycobacteria, most of which were inhibited by the drug at 10-25 microg/ml concentration. When tested in vivo, diclofenac, injected at 10 mg/kg body weight of a Swiss strain of white mice, could significantly protect them when challenged with a 50 median lethal dose of M. tuberculosis H37 Rv102. According to Chi-square test, the in vivo data were highly significant (P<0.01).     

Beta-Endorphin: lack of correlation between opiate activity and immunoreactivity by radioimmunoassay.

A sensitive radioimmunoassay for human β-endorphin has been developed. When natural human β-lipotropin and various synthetic analogs of β-endorphin were assayed for their immunoreactivity, lack of correlation was found between opiate activity and immunoreactivity. These data suggest that residues 6–15 of β-endorphin are the antigenic determinant.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

In vitro and in vivo inhibition by zopiclone of benzodiazepine binding to rodent brain receptors.

Up to now the only drugs known to be able to inhibit the binding of benzodiazepines to rodent brain receptors are members of this chemical family.Zopiclone (RP 27 267), a new drug with a pharmacological profile similar to that of chlordiazepoxide and nitrazepam but entirely different chemically from benzodiazepines, has been tested for its ability to inhibit benzodiazepine binding. $\text{In vitro}$ and $\text{in vivo}$ studies have shown that zopiclone is able to inhibit the binding of [³H] diazepam and [³H] flunitrazepam to brain receptors. The potency of zopiclone is quite comparable to that of diazepam and nitrazepam $\text{in vitro}$ and to that of chlordiazepoxide $\text{in vivo}$.These results confirm the pharmacological similarities existing between zopiclone and the benzodiazepines.     

In search of a correlation between the electron affinity and the radiosensitizing activity of some glyoxylic compounds.

The electron affinity of some glyoxylic compounds with radiosensitizing properties was evaluated using different approaches. The half-wave reduction potentials were measured by polarography, and the molecular structures in solution were determined by NMR spectroscopy. The electron affinity of the unhydrated and hydrated molecules was theoretically evaluated by means of a semi-empirical quantum mechanical procedure, the MNDO. The possible correlations between these values and the radiosensitizing efficiency of these glyoxylic compounds is discussed.     

Absence of correlation between antiestrogenic activity and binding affinity for the estrogen receptor.

We have compared the binding to the estrogen receptor (R) of different androgens and antiestrogens with their antiestrogenic activities on uterine growth. We found that estradiol (E₂)¹ and hydroxytamoxifen, a potent antiestrogen, displayed the same affinity for R. Conversely, androgens which have a much lower affinity for R and a much higher dissociation rate than E₂, behave at high doses as full estrogens, with no significant antiestrogenic activity. We conclude that there is no correlation between the dissociation rate from R and the antiestrogenic activity of R ligands and that one cannot discriminate between estrogen and antiestrogen ligands by simply evaluating their $\text{in vitro}$ binding to the cytosol R.     

Irreversible protein binding of metabolites of ethynylestradiol in vivo and in vitro

During incubation of 6,7-³H-ethynylestradiol with rat liver microsomes up to 20 % of the radioactivity was bound irreversibly to the microsomal proteins. Incubations in presence of albumin resulted in a further radioactive labelling of the albumin. The irreversible nature of the steroid-protein bond was established by solvent extraction and charcoal treatment. Further evidence was obtained after hydrolyzing the microsomal protein with trypsin and submitting the labelled tryptic peptides to ion exchange chromatography and electrophoresis. The labelled albumin was applied to sephadex gel filtration which showed the association of the ethynylestradiol radioactivity to the albumin peak.The binding reaction required supply of NADPH, could be stimulated by pretreatment of the animals with phenobarbital and was inhibited by CO and SKF 525 A. On these characteristics the concept was based that, in analogy to the well known binding of estradiol and estrone, 2hydroxylation is also an essential prerequisite for the binding of ethynylestradiol. The concept was confirmed by trapping off the 2-hydroxy-ethynylestradiol with glutathione, which led to a decrease of the ethynylestradiol-protein binding.Further evidence resulted from experiments $\text{in vivo}$, dosing rats with 6,7-3H-ethynylestradiol and 6,7-3H-estradiol 48 hrs prior to sacrifice and examining the amount of radioactivity irreversibly bound to the liver endoplasmic reticulum. 3H-ethynylestradiol caused a radioactive labelling of microsomes twice as much as that after ³H-estradiol.     

In vitro and in vivo studies demonstrating potent and selective estrogen inhibition with the nonsteroidal aromatase inhibitor CGS 16949A

CGS 16949A inhibited the conversion of [4-14C]androstenedione (A) to [4-14C]estrone by human placental microsomes in a competitive manner (Ki = 1.6 nM). Aminoglutethimide, also a competitive inhibitor, had a Ki = 0.7 microM in this assay system. The Km for the aromatization of A was 0.11 microM. Using ovarian microsomes from immature rats primed with pregnant mare's serum gonadotrophin and using [4-14C]testosterone conversion to [4-14C]estradiol as a measure of aromatase activity, the Km was 42 nM. At a substrate concentration 3-fold the Km, CGS 16949A was 180 times more potent as an inhibitor than aminoglutethimide, exhibiting half-maximal inhibition at 1.7 nM as compared to 0.3 microM. In vivo CGS 16949A lowered ovarian estrogen synthesis by gonadotropin-primed, androstenedione treated, immature rats by 90% at a dose of 260 micrograms/kg (PO). A dose of 100 mg/kg of aminoglutethimide was needed to produce this same effect. CGS 16949A at a dose of 4 mg/kg (PO) induced uterine atrophy (aromatase inhibition) without inducing adrenal hypertrophy - indicating a lack of inhibition of corticosterone secretion, while aminoglutethimide at 40 mg/kg (PO) induced adrenal hypertrophy without inducing uterine atrophy. CGS 16949A was neither androgenic nor estrogenic in rats using standard bioassays. The data suggest that CGS 16949A may serve as a potent and selective agent for modulating estrogen-dependent functions.     

In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein.

We constructed several deletion mutants of Escherichia coli single-stranded DNA binding protein (EcoSSB) lacking different parts of the C-terminal region. This region of EcoSSB is composed of two parts: a glycine and proline-rich sequence of approximately 60 amino acids followed by an acidic region of the last 10 amino acids which is highly conserved among the bacterial SSB proteins. The single-stranded DNA binding protein of human mitochondria (HsmtSSB) lacks a region homologous to the C-terminal third of EcoSSB. Therefore, we also investigated a chimeric protein consisting of the complete sequence of the human mitochondrial single-stranded DNA binding protein (HsmtSSB) and the C-terminal third of EcoSSB. Fluorescence titrations and DNA-melting curves showed that the C-terminal third of EcoSSB is not essential for DNA-binding in vitro. The affinity for single-stranded DNA and RNA is even increased by the removal of the last 10 amino acids. Consequently, the nucleic acid binding affinity of HsmtSSB is reduced by the addition of the C-terminus of EcoSSB. All mutant proteins lacking the last 10 amino acids are unable to substitute wild-type EcoSSB in vivo. Thus, while the nucleic acid binding properties do not depend on an intact C-terminus, this region is essential for in vivo function. Although the DNA binding properties of HsmtSSB and EcoSSB are quite similar, HsmtSSB does not function in E.coli. This failure cannot be overcome by fusing the C-terminal third of EcoSSB to HsmtSSB. Thus differences in the N-terminal parts of both proteins must be responsible for this incompatibility. None of the mutants was defective in tetramerization. However, mixed tetramers could only be formed by proteins containing the same N-terminal part. This reflects structural differences between the N-terminal parts of HsmtSSB and EcoSSB. These results indicate that the region of the last 10 amino acids, which is highly conserved among bacterial SSB proteins, is involved in essential protein-protein interactions in the E.coli cell.