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W Sadée

Publications and source records attributed to W Sadée.

At least 109 records · Page 6Linked to original sources
PubMed1985

In vivo receptor binding of opioid drugs at the mu site.

The in vivo receptor binding of a series of opioid drugs was investigated in intact rats after s.c. administration of [3H]etorphine tracer, which selectively binds to mu sites in vivo. Receptor binding was determined by a membrane filtration assay immediately after sacrifice of the animals and brain homogenization. Coadministration of unlabeled opioid drugs together with tracer led to a dose-dependent decrease of in vivo tracer binding. Estimates of the doses required to occupy 50% of the mu sites in vivo established the following potency rank order: diprenorphine, naloxone, buprenorphine, etorphine, levallorphan, cyclazocine, sufentanil, nalorphine, ethylketocyclazocine, ketocyclazocine, pentazocine, morphine. In vivo-in vitro differences among the relative receptor binding potencies were only partially accounted for by differences in their access to the brain and the regulatory effects of Na+ and GTP, which are expected to reduce agonist affinities in vivo. The relationship among mu receptor occupancy in vivo and pharmacological effects of the opioid drugs is described.

Animals↗
PubMed1984

Opiate receptor binding-effect relationship: sufentanil and etorphine produce analgesia at the mu-site with low fractional receptor occupancy.

The analgesic activity of the opiate agonists etorphine and sufentanil and the antagonistic effects of diprenorphine and naloxone have been related to the occupancy of 3 classes of opiate binding sites previously defined in vivo in order to establish their pharmacological significance. Sufentanil binds specifically in vivo to the first type of site (site 1), exhibiting approximately 1100-fold selectivity over site 2, whereas etorphine displays approximately 20-fold selectivity for site 1 over site 2. Neither agonist has a measurable affinity to the third type of binding site. The binding data suggest that site 1 is analogous to the mu site previously identified in vitro. Both agonists produce analgesia in the rat tail flick test at the same low fractional occupancy of site 1 (approximately 2% at the ED50) while they display much lower and quite different occupancies at site 2. Both of the opiate antagonists naloxone and diprenorphine reduce the potency of sufentanil and etorphine by a factor of 2 at 50% occupancy of site 1 alone. These results provide strong evidence that these 4 drugs exert their effects by interaction with site 1 (mu sites) which therefore may be regarded as the receptor responsible for analgesic action in this test. The assumption of a direct relationship between antagonistic effect and fractional occupancy appears to be valid for naloxone and diprenorphine at site 1, while the agonists exert their action at a very low fractional occupancy implying a non-linear binding-effect process.

Animals↗
PubMed1984

Discrimination of three types of opioid binding sites in rat brain in vivo.

Opiate receptor sites in the rat brain were defined in vivo by measuring the binding of etorphine, sufentanil, diprenorphine, and naloxone in saturation and cross-competition experiments. The binding data were analyzed simultaneously, using a computerized curve-fitting technique with an extended least-squares nonlinear regression program. Three types of binding sites could be distinguished: site 1 (18 pmoles/g of brain), site 2 (15 pmoles/g of brain), and site 3 (20 pmoles/g of brain). Site 1 is bound selectively by sufentanil (the ratio of the apparent equilibrium dissociation constants K2/K1 approximately equal to 1200), etorphine (K2/K1 approximately equal to 20), and naloxone (K2/K1 approximately equal to 15), and it resembles the mu binding site previously demonstrated in vitro. Diprenorphine binds to both site 1 and site 2 with high affinity and a slight (approximately 3.7-fold) selectivity for site 1 over site 2. The latter site may represent a mixture of the delta and kappa binding sites. The third site displays relatively high affinity for naloxone, but it is clearly different from sites 1 and 2, as it exhibits a lack of affinity for sufentanil, etorphine, and diprenorphine. This binding site population does not resemble any of the known opiate binding sites. Recent in vitro binding studies revealed that site 3 (now termed lambda site) is highly labile in vitro and was, therefore, not previously detected. These results suggest significant differences between in vitro and in vivo opioid receptor binding characteristics.

Animals↗
PubMed1984

Interaction among the distinct effects of adenine and guanine depletion in mouse lymphoma cells.

Toxic guanine depletion was shown previously to result in a dramatic reduction of DNA synthesis, while toxic adenine depletion failed to affect DNA synthesis (M. B. Cohen and W. Sadée, Cancer Res., 43: 1587-1591, 1983). In this study, relative DNA synthesis rates were measured in mouse lymphoma S49 cells over 24 hr after drug exposure and were compared to cell growth curves. DNA synthesis inhibition by mycophenolic acid (guanine starvation) was achieved at lower drug concentrations than was the inhibition of cell growth. This result further supports the hypothesis (reference above) that guanine starvation specifically affects cells in S phase while it allows cells with full DNA complement to divide. In contrast, L-alanosine (adenine starvation) failed to affect DNA synthesis for at least 24 hr at a concentration that inhibits cell growth by 80%. The dramatically different effects of guanine and adenine starvation on DNA synthesis can thus be used to assess the magnitude of each when blocking early de novo purine biosynthesis by 6-methyl-mercaptopurine ribonucleoside (6- MMPR ). The results suggest that, although 6- MMPR effects primarily resemble those of guanine depletion, adenine starvation measurably contributes to the overall toxicity of 6- MMPR . Drug combination experiments with L-alanosine, mycophenolic acid, and 6- MMPR suggest that the basic mechanisms underlying the toxic effects of guanine and adenine starvation act synergistically.

Adenine↗
PubMed1983

An opiate binding site in the rat brain is highly selective for 4,5-epoxymorphinans.

In vitro binding studies have demonstrated the existence of multiple opiate receptor types. An additional site in the rat brain (termed the lambda site) is distinct from the established types by its selectivity for 4,5-epoxymorphinans (such as naloxone and morphine). While the lambda site displays a high affinity for naloxone in vivo and in vitro in fresh brain membrane homogenates, these sites rapidly convert in vitro to a state of low affinity. The regional distribution of the lambda site in the brain is strikingly different from that of the classic opiate receptor types.

Animals↗
PubMed1983

Opiate receptor binding affected by guanine nucleotide. Partial loss of sensitivity to guanosine-5'-(beta,gamma-imido)triphosphate evident in vitro.

The sensitivity of the receptor dissociation rate of the opiate agonist [3H]etorphine to the effects of guanosine-5'-(beta, gamma-imido)triphosphate and Na+ was measured in washed rat brain membrane homogenates after in vivo labeling. Comparison to the previously measured rapid in vivo dissociation curve (t1/2 approximately 50 s) (Perry, D. C., Rosenbaum, J. S., Kurowski, M., and Sadée, W. (1982) Mol. Pharmacol. 21, 272-279) revealed that brain homogenization and membrane washing procedures significantly prolonged the dissociation rate, even when measured in the presence of Na+ and guanyl nucleotide. The in vivo dissociation rate could only be reproduced in vitro when labeling occurred in vivo and brain homogenization occurred in the presence of these regulatory factors. The prolonged in vitro [3H]etorphine dissociation curve was predominately a result of a decreased sensitivity to guanine nucleotide rather than to Na+. These data suggest that partial functional uncoupling of the opiate receptor-effector system may occur in vitro.

Animals↗
PubMed1983

Guanine ribonucleotide depletion in mammalian cells. A target of purine antimetabolites.

In a previous report we demonstrated in mouse lymphoma (S-49) cells that DNA synthesis inhibition resulting from guanine starvation is associated with GTP rather than dGTP depletion. Since several effective anticancer drugs act via guanine depletion, it is important to test whether critical GTP depletion is unique to S-49 cells or also occurs in other cell lines. Mycophenolic acid-induced guanine starvation caused a drastic DNA synthesis inhibition in the human lymphoblastic T leukemia (CEM) and the mouse B leukemia (L1210) cell lines, which was again associated with GTP depletion rather than dGTP depletion. These results suggest that GTP depletion represents a common target of purine antimetabolites in mammalian cells.

Animals↗
PubMed1983

In vivo characterization of four types of opioid binding sites in rat brain.

Opioid receptor multiplicity was studied in the brain of intact rats by two experimental approaches. The first involved in vivo labeling by administration of [3H] tracers s.c. to intact rats. The second method was based on ex vivo labeling by incubating fresh brain homogenates immediately after sacrifice with suitable receptor type-specific tracers. The ex vivo labeling approach thus can measure the in vivo receptor occupancy of unlabeled ligands, provided that these ligands do not equilibrate between bound and free form over the assay period. The results suggest the presence of four separate types of opioid binding sites that resemble the mu, delta, kappa, and the newly identified lambda sites.

Analgesics, Opioid↗
PubMed1983

Metabolic activation of R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) to 5-fluorouracil by soluble enzymes.

There are two major R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) activation pathways to 5-fluorouracil, one that is mediated by microsomal cytochrome P-450 oxidation at C-5' of the tetrahydrofuran moiety and one that is mediated by soluble enzymes. This report demonstrates that the soluble enzyme pathway proceeds via enzymatic cleavage (possibly hydrolytic) of the N-1--C-2' bond to yield 5-fluorouracil and 4-hydroxybutanal, which is immediately further metabolized to gamma-butyrolactone or gamma-hydroxybutyric acid. The soluble activation pathway was present in liver, small intestine, and brain. Because of the limited distribution of cytochrome P-450 in body tissues and because of the lack of redistribution of 5-fluorouracil via the systemic circulation after ftorafur administration, we propose that the soluble enzyme pathway is at least in part responsible for organ toxicity and possibly antitumor effect. Distinction of the microsomal (C-5') and the soluble enzyme (C-2') activation pathways can be exploited in the design of more selective prodrug analogues.

Animals↗
PubMed1982

[3H]diprenorphine receptor binding in vivo and in vitro.

In order to investigate opiate receptor binding in vivo, [3H]diprenorphine was given s.c. to rats, and the tracer specifically bound to membraneous high affinity sites was determined with a rapid filtration technique after brain homogenization. Bound [3H]diprenorphine accounted for 70% of the total brain activity after tracer doses. The in vivo binding sites were saturable at 25-30 pmol/g brain. Fifty percent occupancy of the [3H]diprenorphine binding sites in vivo occurred at a dose (10-15 micrograms/kg) that is similar to the antagonistic ED50 of diprenorphine for reversing morphine analgesia. The in vitro binding capacity for [3H]diprenorphine was also approximately 30 pmol/g brain in fresh untreated Tris buffer brain homogenate; however, extensive homogenate dilution or standard membrane washing procedures resulted in a reduction of the [3H]diprenorphine binding site population to 13-22 pmol/g. These results indicate that the opiate receptor system is modified in vitro. Previous studies have shown that the [3H]diprenorphine tracer is retained at cerebral binding sites over several hours in vivo. A diffusion boundary model was proposed to account for the dose dependent tracer retention. In order to investigate the mechanism of the in vivo binding kinetics, [3H]diprenorphine dissociation was measured in brain homogenates after in vivo labeling, immediately following sacrifice of the animals to minimize in vitro artefacts. No differences were found in the dissociation curves at 'infinite' homogenate dilution in the presence or absence of saturating diprenorphine concentrations under various ionic incubation conditions. This result argues against cooperative binding. It is consistent with the hypothesis that the [3H]diprenorphine tracer is retained in vivo because of a diffusion boundary next to the binding sites (receptor micro-compartment) that is destroyed during brain homogenization.

Animals↗
PubMed1982

Differential regulation of the mu-, delta-, and kappa-opiate receptor subtypes by guanyl nucleotides and metal ions.

The effects of Na+, guanyl-5'-yl imidodiphosphate (Gpp(MH)p), and Mn2+ on the binding of dihydromorphine, ethylketocyclazocine, D-Ala2, D-Leu5-enkephalin, and diprenorphine to the opiate receptor were investigated. Three distinct binding sites, mu, delta, and kappa sites, were identified with the use of multiple tracer displacement curves. Moreover, this approach was used to determine the effects of Gpp (NH)p and metal ions on each individual binding site. At the mu and delta sites, Na+ and Gpp(NH)p each decreased and Mn2+ increased agonist binding affinities, with the exception of D-Ala2, D-Leu-enkephalin affinity which was not affected by Gpp(NH)p. None of these conditions markedly altered dihydromorphine and D-Ala2, Leu5-enkephalin binding to kappa sites, whereas the affinity of ethylke-tocyclazocine for kappa sites was decreased by Gpp(NH)p. Sodium ions lowered the capacity of mu sites and Gpp(NH)p reduced that of delta sites, while both agents increased the capacity of apparent kappa sites. These results demonstrate that each of the kinetically distinguishable binding sites is regulated differentially by metal ions and guanyl nucleotides. Simultaneous addition of Na+ and Gpp(NH)p greatly reduced the binding affinity of all three agonists at their respective high affinity sites (dihydromorphine at the mu site, D-Ala2, D-Leu5-enkephalin at the delta site, and ethylketocyclazocine at the kappa and mu sites). This result confirms previous observations that agonist binding is characterized by a large affinity reduction in the presence of both Na+ and guanyl nucleotides, and it extends this concept to each of the opiate receptor subtypes.

Animals↗
PubMed1982

Buprenorphine: differential interaction with opiate receptor subtypes in vivo.

The mixed agonist-antagonist buprenorphine previously was shown to display a bell-shaped dose-response curve with peak agonistic (antinociceptive) opiate effects at approximately 0.5 mg/kg s.c., 60 min after the dose and a gradual decline of the effects in the dosage range between 0.5 to 10 mg/kg (electrically induced vocalization test). In vitro, buprenorphine as well as the potent pure agonist etorphine possessed very high affinity for all of the opiate receptor subtypes that are labeled by the universal tracer [3H]diprenorphine. However, in vivo, [3H]buprenorphine and [3H]etorphine appeared to label preferentially a subset of the opiate receptor sites; the labeled subset may be identical to or include the mu receptor sites, since these sites were also labeled with high affinity by [3H]naloxone, a mu preferring antagonist ligand. Buprenorphine saturated this opiate receptor subpopulation in its agonistic dosage range (less than or equal to 0.5 mg/kg). Moreover, buprenorphine saturated the remaining receptor subpopulation, labeled in vivo by [3H]diprenorphine, over its antagonistic dosage range (0.5--10 mg/kg). Cooperative receptor binding behavior of buprenorphine was not detectable. These results are consistent with the hypothesis that noncompetitive autoinhibition occurs among the opiate receptor subtypes.

Allosteric Regulation↗