Drug effects on response duration differentiation I: differential effects of drugs of abuse

Rats were trained to respond under a response duration differentiation schedule in which responses on a lever were reinforced if lever press durations were greater than or equal to 1.00 s but were also less than 1.30 s. Dose-effect curves were generated for cocaine, methamphetamine, pentobarbital, phencyclidine, delta-9-tetrahydrocanninabol (⁹-THC), and morphine. All drugs produced dose-dependent decreases in accuracy (the percentage of total response durations that were reinforced); however, the degree to which changes in accuracy were accompanied by changes in response rates varied among drugs. Pentobarbital and morphine affected primarily longer (> 1.3 s) response durations, phencyclidine and ⁹-THC affected primarily shorter response durations, whereas cocaine and methamphetamine affected both shorter and longer response durations. High doses of methamphetamine and cocaine increased the dispersion of response duration distributions with increasing dose, whereas higher doses of pentobarbital, ⁹-THC and morphine did not increase dispersion of response duration distributions as much. These data show that behavior under this novel schedule is differentially sensitive to a number of pharmacologic manipulations, and that the schedule can provide a useful addition to the analysis of drug effects upon behavior.     

A Simple and Rapid Fluorometric Determination Method of α1-Acid Glycoprotein in Serum Using Quinaldine Red

We examined different fluorescent probes suitable for fluorometric determination of ₁-acid glycoprotein (AGP) in serum. Quinaldine red (QR) was shown to bind strongly and selectively to AGP. Taking advantage of the enhanced fluorescence of QR in the presence of AGP, we developed a direct method for the determination of serum AGP without removal of other serum proteins such as albumin. AGP concentrations in serum of healthy volunteers and patients correlated well with results from the conventional single radial immunodiffusion (SRID) method (r = 0.93, slope = 1). The newly developed method is faster and has a larger analytical concentration range than the SRID method. This method can also be used to determine AGP in serum of experimental animals, and it can serve to monitor AGP serum concentrations for pharmacokinetic evaluation of basic drugs.     

If current mediatesβ-adrenergic enhancement of heart rate but not contractility in vivo

Background: The hyperpolarization-activated I_f current in the sinoatrial (SA) node participates in the spontaneous diastolic depolarization responsible for pacemaking function. Both sympathetic and parasympathetic control of heart rate is thought to involve modulation of I_f. This study tested whether -adrenoceptor activation of heart rate, but not contractile state, could be reduced by blockade of I_f channels in the intact, anesthetized pig.Methods: Both isopterenol (ISO, 0.1 g/kg/min i.v. for 5 min) and norepinephrine (NE, 0.3 g/kg/min i.v. for 5 min) were used sequentially to activate -adrenoceptors in five metomidathydrochloride-anesthetized pigs. Left ventricular pressure and dP/dt, aortic blood pressure and cardiac output were measured. I_f channels were then blocked selectively with 0.3 mg/kg i.v. zatebradine (ULFS49) and the test doses of ISO and NE were repeated. Following a further high dose (10 mg/kg, i.v.) of zatebradine, the test doses of ISO and NE were repeated once again.Results: Before I_f blockade, ISO and NE elicited reproducible increases in both heart rate and left ventricular dP/dt. Whereas NE caused an increase in both systolic (56%) and diastolic (53%) aortic pressure and a modest heart rate increase (22%), ISO caused a decrease in diastolic aortic pressure (–22%) and a marked increase in heart rate (81%). Low dose zatebradine reduced basal heart rate from 98±6 to 66±3 bpm, p<0.05; cardiac output fell by 20%, stroke volume increased by 18% and total peripheral resistance was unchanged. ISO after low-dose zatebradine still elicited marked increases in heart rate (66±3 to 105±5 bpm, p<0.05) and left ventricular dP/dt (774±94 to 3364±206 mmHg/s, p<0.05) and reduced aortic diastolic pressure (37 ±2 to 33±1 mmHg, p<0.05). NE after low-dose zatebradine increased heart rate (73±4 to 89±5 bpm, p<0.05), left ventricular dP/dt (810 ±95 to 3372±196 mmHg/s, p<0.05) and both systolic and diastolic aortic pressures. High dose zatebradine caused no further reduction in heart rate (77±4 vs 82±6 bpm, NS) but left ventricular dP/dt decreased (798 ±92 to 418±50 mmHg/s, p<0.05) as did both systolic and diastolic aortic pressures. Subsequent administration of ISO had no effect on heart rate but increased left ventricular dP/dt from 418±50 to 3468±256 mmHg/s (p<0.05) and systolic aortic pressure increased from 58±7 to 90 ±3 mmHg (p<0.05). NE administered after high dose zatebradine also increased left ventricular dP/dt (580±54 to 2608±182 mmHg/s, p<0.05) while heart rate fell (86±4 to 74±6 bpm, p<0.05). Both systolic and diastolic aortic pressures mereased substantially during the NE infusion after high dose zatebradine.Conclusion: Zatebradine dosedependently inhibits -adrenoceptormediated heart rate increases while leaving -adrenoceptor-mediated increases in myocardial contractile state intact. This observation can be explained by a selective blockade of the hyperpolarization-activated current I_f by low concentrations of the drug.     

Regioselectivity and Enantioselectivity of Metoprolol Oxidation by Two Variants of cDNA-Expressed P4502D6

Purpose. The oxidative metabolism of metoprolol was investigated in two human lymphoblastoma cell-lines transfected with variants of cDNA for cytochrome P4502D6. Methods. The regioselective and enantioselective features of the oxidations of deuterium-labeled pseudoracemic metoprolol were characterized by GC/MS analysis of the substrate and products. Results. There were significant differences between the two P4502D6 variants in the formation kinetics of O-demethylmetoprolol and -hydroxymetoprolol. The h2D6-Val microsomes highly favored the formation of the O-demethylmetoprolol regioisomer 6.3:1 and 2.8:1, respectively from (R)-metoprolol-d₀ and (S)-metoprolol-d₂, while the corresponding ratios for h2D6v2 microsomes were much lower. For both variants, O-demethylmetoprolol formation favored the (R)-substrate 1.5 to 2-fold, while -hydroxymetoprolol formation was non-enantioselective. Similar Km values of metoprolol oxidation, 10-20 µM, were observed for the two microsomal preparations. Conclusions. The regioselectivity, enantioselectivity, and Km values for the h2D6-Val microsomes resemble those observed for the native P4502D6 in human liver microsomes, whereas the h2D6v2 microsomes deviated remarkably in regioselectivity.     

Use of Drug Kinetics in Dermis to Predict in VivoBlood Concentration After Topical Application

Purpose. To use the drug kinetics in dermis to predict the in vivo blood concentration after topical administration. Methods. A two-step pharmacokinetic model was established. The first step was to calculate the drug input rate or flux from the skin to the systemic circulation using the drug kinetic parameters in dermis. These parameters include (a) distance over which the drug concentration declines by 50%, (b) drug concentration at the epidermal-dermal junction, and (c) minimal plateauing drug concentration in the muscle layer. These parameters were experimentally determined from the drug concentration-tissue depth profiles in the dermis, after the application of a topical dose of ddI (200 mg/kg) to rats. The second step was to use the drug input rate together with the systemic disposition pharmacokinetics of ddI in rats to predict the plasma concentration-time profiles. The model-predicted plasma concentration-time profiles were compared with the observed profiles, to determine the validity of the proposed pharmacokinetic model. Results. The observed steady state concentration (C_ss) in individual animals (n = 6) deviated from the predicted values by 3 to 55% with 3 of 6 rats showing a <15% deviation. The mean observed C_ss of all animals deviated from the mean predicted values by less than 15%. Conclusions. The close agreement between the observed and the model-predicted drug concentrations indicates that the systemic drug input can be calculated from the drug kinetics in the dermis.     

Characterization of uronic-acid-rich inhibitor of calcium oxalate crystallization isolated from rat urine

Human urine contains several macromolecules which inhibit calcium oxalate crystallization. Uronic-acid-rich protein (UAP), a glycoprotein with a molecular weight of approximately 35 kDa, is one such inhibitor. Here we report the characterization of UAP extracted from rat urine using three chromatographic steps including diethylaminoethanol (DEAE)-Sephacel, Sephacryl S-300 and Mono Q column and compare it with human UAP. The molecular weight of rat UAP (UAP_r) is similar to that of human UAP (UAP_h), being approximately 35 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid compositions are identical, they contain a high percentage of aspartic and glutamic acids and they react positively in the carbazole reaction, suggesting that they contain uronic acid. The inhibitory activities of UAP_h and UAP_r were assayed on a calcium oxalate crystallization system in vitro using [⁴⁵Ca]calcium chloride. Both exert a strong inhibition, suggesting that UAP_r, like UAP_h, plays an important role in preventing and reducing calcium oxalate crystallization in the urine. On Western blot analysis, both UAP_h and UAP_r immunoreact with inter--trypsin inhibitor (ITI) antibody. Nevertheless, using the Ouchterlony immunodiffusion technique, there was no precipitation line between ITI antibody and UAP. Therefore, we hypothesize that UAP is related to ITI and that they may have the same epitope but are not completely identical. We conclude that UAP belongs to the ITI superfamily of macromolecules which contribute to the regulation of the calcium oxalate crystallization process.     

Interleukin-1Β, interleukin-6, and growth hormone levels in human follicular fluid

Purpose To investigate possible relationships of interleukin-1 (IL-1, interleukin-6 (IL-6), and growth hormone (GH) with biochemical variables in human follicular fluid (FF) and selected in vitro fertilization (IVF) parameters.Methods A total of 67 FF samples (n=67 patients undergoing oocyte retrieval for IVF) was evaluated. IL-1, IL-6, GH, hLH, FSH, PRL, hCG, testosterone, total protein, fibrinogen, sialic acid, ₁-antitrypsin, plasminogen levels, and spectrophotometric absorbance at 458 nm were analyzed for selected FF. IL-6 and GH levels of serum and FF samples were also compared (n=23).Results Immunoreactive levels of IL-1, IL-6, and GH were detected in all FF samples. A positive correlation existed for IL-6 (r=0.5069, P=0.0161 when serum-to-FF levels were compared (concentration ratio, 11.857). Smaller-volume follicles (<4 ml) were associated with high IL-1 levels (P=0.0229, and an additional tendency of IL-1 to decrease with increasing embryo cleavage and scoring was observed. With the exception of a weak positive correlation between follicular IL-1 and testosterone levels (r=0.3128, P=0.025, no other relationship with biochemical variables or IVF parameters (etiology, e.g., endometriosis) could be implicated.Conclusions Substantially higher IL-6 levels occurred in FF compared to serum, thus supporting intrafollicular production. Interleukin- 1,IL-6, and GH levels in FF are, however, unsuitable markers for in vitro fertilization outcome.     

Interleukin-1α (IL-1α) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) as potential inducers of supravital chemotaxis

The phenomenon of artificially induced local leucocyte reactions during the supravital period could be of practical importance, but has not yet been comprehensively investigated. For a more detailed evaluation, experiments with the chemotactic agents interleukin-1 (IL-1) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were performed by subcutaneous injection into various anatomical regions (back, abdomen, limbs) of NMRI-mice (National Medical Research Institute) and pigs 0–5 min after circulatory arrest. Phosphate buffered saline (PBS) without effective components was administered to equivalent areas of the animals as a control. Tissue specimens were collected at 6 h postmortem (mice) and 12–14 h postmortem (pigs), cut into serial sections, stained with H & E and examined under the microscope. A leucocyte reaction did not develop in pigs (n = 10, 30 tissue samples) following injection of FMLP, however, dermal, subcutaneous and perivascular infiltration of leucocytes (in particular mononuclear cells and a few granulocytes) was found in 3 out of 30 tissue specimens in murine experiments. In addition intravascular cell accumulations were detected in 2 out of 30 samples. The injection of IL-l to mice gave similar results, i.e. aggregations of leucocytes and intravascular cell accumulations in 4 out of 30 and 3 out of 30 tissue samples, respectively. In negative controls no leucocyte reaction was detectable. This shows that potent chemotactic factors such as IL-1 and FMLP administered in the early supravital period can induce moderate local leucocyte reactions in animal models in at least some cases. A clear morphological differentiation between vital and supravital chemotaxis does not seem to be possible. The supravitally stimulated accumulations of leucocytes are interpreted as an aggregation of resident macrophages in combination with a slight migration of blood leucocytes. Presumably, these alterations are restricted to the very early supravital period as long as sufficient energy reserves are available. It must be stated that the observed changes are reactions, not spontaneous actions, so that the general validity of the phenomenon of leucocyte infiltration as a vital parameter is not affected.     

The role of the coagulation cascade in brain edema formation after intracerebral hemorrhage

Summary The coagulation cascade has a potential role in brain edema formation due to intracerebral hemorrhage. In this study blood and other solutions were injected stereotactically into the right basal ganglia in rats. Twenty-four hours following injection, brain water and ion contents were measured to determine the amount of brain edema. Intracerebral blood resulted in an increase in brain water content. The amount of brain edema surrounding the intracerebral hematoma was reduced by a thrombin inhibitor Na-(2-Naphthalenesulfonylglycyl)-4-amidino-DL-phenylalaninepiperidide, (-NAPAP) infused into the hematoma after the clot had been allowed to solidify. The inhibitor did not alter the actual size of the clot mass. An artificial clot composed of fibrinogen, thrombin, and styrene microspheres also produced brain edema. A fibrin clot led to edema formation even in the absence of mass effect provided by the microspheres. The single component responsible for production of brain edema in all these models was thrombin. The edema was formed in response to a fibrinogen-independent pathway. These results indicate that the coagulation cascade is involved in brain edema that develops adjacent to an intracerebral hematoma.     

The stereoisomers of 17α-[123I]iodovinyloestradiol and its 11α-methoxy derivative evaluated for their oestrogen receptor binding in human MCF-7 cells and rat uterus,and their distribution in immature rats

We studied the potential of both stereoisomers of 17-[¹²³I]iodovinyloestradiol (E- andZ-[¹²³I]IVE) and of 11-methoxy-17-[¹²³I]iodovinyloestradiol (E-andZ-[¹²³I]MIVE) as suitable radioligands for the imaging of oestrogen receptor(ER)-positive human breast tumours. The 17-[¹²³I]iodovinyloestradiols were prepared stereospecifically by oxidative radio-iododestannylation of the corresponding 17-tri-n-butylstannylvi-nyloestradiol precursors. Competitive binding studies were performed in order to determine the relative binding affinity (RBA) of the unlabelled 17-iodovinyloes-tradiols for the ER in both human MCF-7 breast tumour cells and rat uterine tissue, compared with that of diethylstilboestrol (DES). Target tissue uptake, retention and uptake selectivity of their¹²³I-labelled analogues were studied in immature female rats. All four 17-iodovi-nyloestradiols showed high affinity for the ER in human MCF-7 cells, as well as rat uterus. Their RBA for the ER showed the following order of decreasing potency: RBA of DES >Z-IVE >Z-MIVE >E-MIVE E-IVE. Neither of these 17-iodovinyloestradiols showed any significant binding to the sex hormone binding globulin in human plasma. The biodistribution studies showed ER-mediated uptake in the uterus, ovaries and pituitary, that ofE- andZ-[¹²³I]MIVE being higher than that ofE- andZ-[¹²³I]IVE. High target-to-non-target tissue uptake ratios, especially at longer periods after injection (up to 24 h), were exhibited by both isomers of [¹²³I]MIVE. The uterus-to-blood uptake ratio was higher for^E-[¹²³I]MIVE. However, the uterus-to-fat uptake ratio appeared to be higher for theZ-isomer of [¹²³I]MIVE, especially at 24 h after injection. Metabolic properties and temperature effects, which play a more important role in vivo, probably cause the discrepancies seen between in vitro and in vivo binding results. On the basis of their in vitro binding properties and in vivo distribution characteristics we conclude thatE- andZ-[¹²³I]MIVE could be suitable radioligands for the diagnostic imaging of ER in human breast cancer. Therefore, further studies with these radioligands in mature normal and tumour-bearing rats are warranted.