Liver growth and mixed-function oxidase activity: Dose-dependent stimulatory and inhibitory effects of α-hexachlorocyclohexane

Summary -hexachlorocyclohexane (-HCH) elicits liver growth and stimulation of hepatic microsomal mixed-function oxidase. In the present study the extent of these changes was determined in rats 2, 4 and 6 days after treatment with doses of -HCH ranging from 1 to 600 mg/kg. Above the respective threshold doses liver mass, liver DNA, and the rate of aminopyrine demethylation increased in proportion to the log dose. Threshold doses were calculated to be 30 mg/kg for the increase of liver weight and DNA, and 5 mg/kg for the stimulation of aminopyrine demethylation.Liver mass and liver DNA continued to increase up to the highest dose used; in contrast, the rate of aminopyrine demethylation declined at doses exceeding 200 mg/kg. This decline seems to be due to inhibition by -HCH retained in the microsomal fraction: -HCH is a potent, apparently competitive inhibitor of aminopyrine demethylation, and gaschromatographic determinations revealed that the amount of -HCH retained in the microsomes is sufficient to produce considerable inhibition of the enzymatic reaction.Abbreviations -HCH -1,2,3,4,5,6-hexachlorocyclohexane, -benzene hexachloride - EL 241 [,-bis(p-chlorophenyl)-3-pyridinemethanol] - RLW relative liver weight = liver weight in percent of body weight     

Differential inhibition of biphenyl hydroxylation in perfused rat liver

Summary A differential inhibition of biphenyl hydroxylation by -naphthoflavone and metyrapone was observed in isolated pefused rat liver. -Naphthoflavone inhibited 2- and 4-hydroxylation in livers from -naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in -naphthoflavone-stimulated livers.Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl preferentially formed sulphates in livers from untreated animals but after induction glucuronidation was as effective as sulphation or even exceeded sulphation. Only glucuronic acid conjugates of 2-hydroxybiphenyl were detected.     

Effect of ethanol on the metabolism of lithocholic acid in rat liver

Summary Lithocholic acid 24-C¹⁴ is converted by 20000x g-supernatant of rat liver homogenate into several products of higher polarity. 3-6-dihydroxy-5-cholanic acid is the main metabolite, whereas 7-hydroxylation occurs only to a small extent. Besides of the hydroxylations conjugation with taurine and the formation of a 3-sulfate ester of lithocholic acid can be demonstrated. Addition of ethanol to the enzymatic system results in an inhibition of the formation of 3,6-dihydroxy-5-cholanic acid, whereas no effect can be observed with respect to the formation of the other products. This inhibition is present also in 20000x g-supernatant obtained from rats pretreated with ethanol 100 min before being preparation from ethanol-pretreated rats amounts to 68% of the controls. The chrome P-450 mediated oxidative mechanism, which has been shown to be involved in microsomal 6-hydroxylation of bile acids.This work was supported by the Deutsche Forschungsgemeinschaft.     

Influence of the decarboxylase inhibitor benserazide on the antihypertensive effect and metabolism of alpha-methyldopa in patients with essential hypertension

Summary In a single-blind study, the dopa-decarboxylase inhibitor benserazide (375 mg/day for 3 days and 750 mg/day for further 3 days) and a placebo were given orally in combination with individually effective doses of alpha-methyldopa (mean 1.5 g/day) to 3 hospitalized patients with essential hypertension. Alpha-methyldopa (-MD) alone lowered blood pressure from 165/107 to 136/93 mm Hg (P<0.05). Benserazide did not alter the hypotensive effect of -MD, although the decarboxylation of -MD was markedly reduced, as shown by the urinary excretion of alpha-methyldopamine (-MDA). During administration of -MD alone, the ratio -MD/-MDA in urine of the 3 patients was 8:1, 7:1 and 22:1, respectively. When benserazide 375 mg/day was added the ratio rose to 31:1, 31:1 and 35:1; the ratio was 37:1, 18:1 and 46:1 at the higher dose of inhibitor. In a double-blind crossover study the effect on blood pressure of 3 weeks of treatment with -MD (mean 1.75 mg/day), benserazide (375 mg/day), placebo and their combinations were compared in 5 hypertensive subjects. Again, benserazide did not influence the antihypertensive action of -MD. To study whether benserazide entered the CNS, a single oral dose of¹⁴C-benserazide of 125 mg was given to 2 patients who were to undergo diagnostic lumbar puncture. Two hours after intake of the labelled drug, when radioactivity in blood had reached a maximum, the concentration of radioactivity in spinal fluid was less than 1% of the plasma level. Thus, the antihypertensive action of -MD was not influenced by oral doses of the decarboxylase inhibitor benserazide. The results suggest that benserazide in doses up to 750 mg/day does not affect central decarboxylation of -MD and that this antihypertensive agent lowers blood pressure by a central action.The study was supported by the Deutsche Forschungsgemeinschaft. Part of the results has been presented at the Spring Meeting 1976 of the Deutsche Pharmakologische Gesellschaft (Planz et al. 1976)     

Differential modulation by muscimol and baclofen on antinociception induced by morphine, β-endorphin,d-Pen2,5-enkephalin and U50,488H administered intracerebroventricularly in the mouse

The present study was designed to investigate the modulatory effects of stimulation of GABA_A and GABA_B receptors at supraspinal sites on antinociception induced by supraspinally administered -, -, -, and -opioid receptor agonists. The effects of the GABA_A and GABA_B receptor agonists, muscimol and baclofen respectively, on the antinociception induced by morphine (a -receptor agonist), -endorphin (an -receptor agonist), D-Pen^2,5-enkephalin (DPDPE, a -receptor agonist) and U50,488H ({trans-3,4-di-chloroN-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeocetamide}; a -receptor agonist) injected intracerebroventricularly (i.c.v.) were studied. The anti-nociception was assayed using the tail-flick and hot-plate tests. Muscimol at doses of 25–200 ng, administered i.c.v. alone did not affect the latencies of tail-flick and hot-plate thresholds, but attenuated dose-dependently the inhibition of the tail-flick and hot-plate responses induced by i.c.v. administered morphine (2 g), -endorphin (1 g), DPDPE (10 g), and U50,488H (60 g). Baclofen (1.25–10 ng) administered i.c.v. alone did not affect the latencies of the tail-flick and hot-plate responses, but attenuated dose-dependently the inhibition of the tail-flick and hot-plate responses induced by -endorphin and U50,488H, without affecting morphine-or DPDPE-induced responses. Our results indicate that activation of GABA_A receptors at the supraspinal sites by i.c.v. injection of muscimol antagonizes antinociception induced by supraspinally administered -, -, -, and -opioid receptor agonists. On the other hand, activation of GABA_B receptors at supraspinal sites by i.c.v. baclofen antagonizes antinociception induced by i.c.v. administered - and -opioid agonists, but not - or -opioid agonists.     

Pharmaceutical care in Denmark: perceived importance of medicine‐related problems and participation in postgraduate training

Objective: To investigate the readiness of Danish community pharmacists to provide pharmaceutical care. Special focus was on information provided to patients on medicinerelated problems and participation in postgraduate training.Method: National crosssectional survey distributed to all Danish community pharmacies (N = 288). Response rate 75.7%; nonresponders were interviewed by telephone to establish their reasons for not participating.Main outcome measures: Prioritisation of information to patients regarding medicinerelated problems. Postgraduate courses selected by pharmacists during the past three years. Results: In accordance with the profile of Danish community pharmacists, respondents were primarily female and half were below 45 years of age. The main reason given for not responding was lack of time. The prioritisation of information to patients regarding medicinerelated problems resulted in three clusters of answers. The overall topics of the clusters were: information related to the technicalpractical use of medicines; information related to pharmacotherapeutic aspects of medicine use; and information related to the function of the medicine. The clusters accounted for 61.9% of the total variance. The most frequently attended postgraduate courses were on quality assurance and specific diseases. Conclusion: The patient information prioritised by Danish community pharmacists is primarily of a technicalpractical nature. The postgraduate training pursued is primarily technical without much focus on the philosophy of pharmaceutical care. These factors contribute to the lack of proper readiness of pharmacists to practice pharmaceutical care.     

(−)δ9 THC as an hypnotic

(–) ⁹ THC was found to significantly decrease the time it takes to fall asleep in physically healthy insomniacs. Once asleep, interruptions of sleep were not significantly altered over the whole night. The (–) ⁹ THC tended to be associated with some decrease in awakenings in the first half of the night.The primary side effect experienced by the subjects at all dose levels in the Pre-Sleep phase was temporal disorganization and mood alterations. There was an increase in intensity of side effects and number of subjects affected with increasing dosage.The most significant side effect, however, was a hangover phenomenon, or continued high the next day, with some residual of temporal disorganization. It increased in intensity and duration with increase in dosage. This hangover seems severe enough to eliminate the consideration of the 30 mg dose range of (–) ⁹ THC for clinical use as an hypnotic.Dr. Cousens is a 3rd Year Resident in Psychiatry at the Napa State Hospital, Napa, California     

Inhibition by opiate narcotics of rat flexor α-motoneurones

Summary Specific effects of opiate narcotics on rat flexor -motoneurones were studied in ventral roots of laminectomized rats under halothane anaesthesia. The -motoneurones were activated by tetanic stimulation of the cut ipsilateral common peroneal nerve, exciting up to group II- but not group III- and C-afferents. Morphine (0.5–3.0 mg/kg i.v.) reduced or completely suppressed the discharge rate of flexor -motoneurones in a dose-dependent manner. This effect was antagonized by naloxone (0.5 mg/kg i.v.) and mimicked by levorphanol (1.0 mg/kg i.v.), but not by an equal dose of its steroisomer dextrorphan, suggesting that the effect described is a specific one. After spinalization, the inhibitory effect of morphine was abolished. Previous studies had shown that opiates (e.g. morphine, given in a dose of 2 or 4 mg/kg i.v.) excite rat extensor -motoneurones, an effect opposite to the opiate narcotic action on flexor -motoneurones. The action of opiates leading to an inhibition of flexor -motoneurones may contribute to akinesia and catalepsy, and opioid-induced muscular rigidity. From the results presented it appears that morphine produces a reciprocal change in the activity evoked in extensor and flexor reflex pathways.Some of these results were presented at the 18th Spring Meeting of the German Pharmacological Society, Mainz, March 16–18, 1977These studies were supported by a grant (B-3) from the Sonder-forschungsbereich 33 Nervensystem und biologische Information of the Deutsche Forschungsgemeinschaft     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

Dehydro-digitoxosides of digitoxigenin: Formation and importance for the digitoxin metabolism in the rat

Summary Only in the presence of NADPH and oxygen digitoxosides of digitoxigenin can be metabolized by rat liver microsomes. The metabolism includes 12-hydroxylation, formation of digitoxosides less the terminal digitoxose and of lipophilic metabolites. The lipophilic metabolites of digitoxin (dt-3), digitoxigeninbis-digitoxoside (dt-2), and of — mono-digitoxoside (dt-1) are formed by oxidation of the axial OH-group of the terminal digitoxosyl yielding the corresponding dehydro-digitoxosides. The structure could be confirmed by comparison with the synthetic compounds 15-dehydro-dt-3, 9-dehydro-dt-2, and 3-dehydro-dt-1, respectively.The terminal dehydro-digitoxosyl can be split off by liver microsomes even in the absence of NADPH. However, no further cleavage of the resulting digitoxosides could be detected. A cleavage rate of 2.8 nmoles/mg microsomal protein × 30 min was observed for both 15-dehydro-dt-3 and 9-dehydro-dt-2 (substrate concentration 50 M). In contrast to that, only 0.4 nmole 3-dehydro-dt-1 was cleaved under equal conditions. For the cleavage of 15-dehydro-dt-3 an apparent K _m of 200 M and a V _max of 440 pmoles dt-2 formed per mg microsomal protein x min was measured.Our results indicate that not the native but only the dehydro-digitoxosides can be cleaved. Therefore, two successive monoxygenase catalysed oxidations are necessary for the cleavage of the sugar chain of dt-3 before dt-1, the main substrate of conjugation enzymes, can be formed. Moreover, digitoxigenin will not be formed because of the high stability of 3-dehydro-dt-1.     

Intrathecal substance P depresses spinal motor and sensory responses to stimulation of nociceptive afferents — Antagonism by naloxone

Summary The effect of an injection of substance P into the subarachnoid space was studied on a motor and a sensory response elicited by supramaximal stimulation of the sural nerve in spinal rats. Substance P 10 g depressed the reflex activation in the electromyogram recorded from the ipsilateral tibialis anterior muscle; the depression was significant 5 and 10 min after the injection. Substance P 10 g reduced the activity in ascending axons of the spinal cord evoked by stimulation of afferent C fibres; the effect developed slowly, lasted longer than 60 min and was abolished by an i.v. injection of naloxone 0.2 mg/kg. Only half the number of ascending axons tested showed a depression by substance P, and the administration of a higher dose (50 g) did not produce an effect in a greater number of axons. Substance P did not influence the activity evoked in ascending axons by stimulation of afferent A and A fibres. The depression by substance P of ascending nociceptive activity was antagonized by an i.v. injection of naloxone 0.2 mg/kg. When naloxone 0.2 mg/ng i.v. was administered alone, it increased the activity in ascending axons activated by afferent C fibre stimulation. It is concluded that (i) substance P depresses spinal nociceptive activity without the intermediation of endorphinergic neurons, and (ii) naloxone antagonizes tonic inhibition of the spinal nociceptive system mediated by endogenous opioid peptides and, by facilitating excitatory transmission through disinhibition, neutralizes the depression produced by substance P.Supported by the Sonderforschungsbereich 38 Membranforschung     

Effects of intracerebroventricular administration of prostaglandin D2 on behaviour,blood pressure and body temperature as compared to prostaglandins E2 and F2α

The present work examined some central nervous actions of prostaglandin D₂ (PGD₂), which is the most prevalent prostaglandin in rodentorain. The effects of PGD₂ were compared with those of PGE₂ and PGF₂. The prostaglandins were administered intracerebroventricularly (ICV) to conscious rats using the method of Herman (1970). All three prostaglandins studied produced depressive behavioral effects, causing obvious sedation at doses of 2.0 g and 20.0 g ICV. PGD₂ and PGE₂ significantly reduced spontaneous motor activity at doses of 2.0 g and 20.0 g ICV. PGF₂ was less effective; only 20.0 g significantly inhibited motor activity. At a dose of 20.0 g ICV all three compounds were shown to block convulsions induced by pentylenetetrazol. PGD₂, the most effective prostaglandin in this respect, was still slightly anticonvulsive at a dose of 2.0 g ICV. PGF₂ hat the weakest anticonvulsive potency. PGE₂ and PGF₂ (2.0 g and 20.0 g ICV) caused a marked hypertensive effect, whereas PGD₂ at the same dose levels only produced a small increase in blood pressure. PGE₂ and PGF₂ (2.0 g and 20.0 g) also exerted marked pyrogenic actions. The effects of PGD₂ on body temperature were variable. When given at a dose of 20.0 g ICV, it caused slight hyperthermia whereas a lower dose (2.0 g ICV) induced a moderate fall in body temperature. These findings suggest a relationship between the actions of the different prostaglandins on blood pressure and body temperature.A preliminary report was given at the Spring Meeting of the Deutsche Pharmakologische Gesellschaft, March 1983 (Förstermann and Heldt, 1983)     

Centralα-2 adrenoceptors are responsible for a clonidine-induced cue in a rat drug discrimination paradigm

Clonidine produces an interoceptive discriminative stimulus or cue in rat drug discrimination studies. This cue may be mediated by its-2 adrenoceptor agonist properties and/or its affinity for the non-adrenoceptor imidazoline preferring receptor. Six rats were trained to respond differentially after receiving clonidine (0.02 mg kg^–1, IP) or a saline vehicle. The-2 adrenoceptor agonists clonidine, UK14, 304 and rilmenidine, which bind to the imidazoline preferring receptor, and guanabenz which does not, dose-dependently substituted for (i.e. > 80% total responding was clonidine associated) the clonidine-induced cue in doses up to 0.02, 0.16, 1.25 and 0.32 mg kg^–1, respectively. Furthermore, the cue was blocked when clonidine was given in combination with 30-min pretreatments of the highly selective-2 adrenoceptor antagonists RX811059 (2.5 mg kg^–1) and fluparoxan (3 mg kg^–1). Since the clonidine-induced cue was substituted for by guanabenz, which does not act at the imidazoline-preferring receptor, and antagonised by RX811059 and fluparoxan it appears to be mediated by-2 adrenoceptors. Moreover, abolition of the clonidine-induced cue did not occur with the peripherally acting-2 adrenoceptor antagonist L659, 066 suggesting it involves central as opposed to peripheral sites.     

Synthesis and pharmacological study of some alkylamini esters and amides of α,α-diarylaliphatic acids

Conclusions Sixteen alkylamino esters and amides of ,-diphenyl--ethoxyacetic acid and ,-diphenylpropionic acid have been synthesized and investigated pharmacologically. All the compounds prepared possess spasmolytic and cholinolytic properties. Derivatives of ,-diphenyl--ethoxyacetic acid are also characterized by the presence of analgesic and anti-cough action. Loading the alcoholic or acid part of the molecule, or replacement of the ester bond by an amide bond, leads to weakening of activity.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 1, pp. 9–14, January, 1970.     

Proteolytic maturation of transforming growth factor-α

Summary Transforming growth factor- (TGF-) is a mitogenic peptide hormone produced extracellularly, by tumor cells, and by virally and chemically transformed cells in culture. TGF- is almost certainly derived from its precursor protein (pro-TGF-) by limited endoproteolysis, but physiologically relevant processing enzyme(s) of the pro-TGF- protein and the cellular or subcellular compartment in which processing takes place are not known with certainty. We previously detailed [Cappelluti, E. and Harris, R.B., Biochemistry, 32 (1993) 551] the discovery, characterization and purification of novel, elastase-like enzymes (molecular weight 38 000) from oncogenically transformed rat liver epithelial cells or cultural Schwann cells transfected with SV40-large T antigen. The elastase-like enzyme appeared to be specifically induced in the transformed epithelial cells compared with the level of enzyme in the nontransformed parental cells. In the intervening time, other elastase-like serine proteinases have been implicated in processing pro-TGF- in other human carcinoma cell lines. We now report that the elastase-like enzymes, purified from transformed Schwann or liver epithelial cells, are inhibited in a time- and concentration-dependent fashion with three differently substituted monocyclic -lactam-based compounds originally developed as specific inhibitors of polymorphonuclear leukocyte elastase, thus further supporting the elastase-like character of the putative pro-TGF- processing enzymes. We also report the presence of the elastase-like enzyme in two different human malignant mammary cell lines, but even though MCF-7 cells receiving high doses of radiation in vitro show an increased level of expression of TGF-, the elastase-like enzyme does not appear to be induced in these cells following irradiation.     

Can two, four or eight‐hour urine collections after voluntary voiding be used instead of twenty ‐four hour collections for the estimation of creatinine clearance in healthy subjects

The accuracy of creatinine clearance estimations obtained from 4hour (16:0020:00, 20:0024:00, 08:0012:00, 12:0016:00) and 8hour (16:0024:00, 24:0008:00 and 08:0016:00) urine collections and the Cockcroft Gault formula compared with the standard 24hour collection, as well as the cyclical variation in creatinine excretion were studied in a group of 22 healthy subjects (Serum creatinine < 1.5mg/dl, Blood Urea Nitrogen < 50mg/dl) after voluntary voiding. The mean 4hour and 8hour creatinine clearances were not significantly different from the 24hour values. Clearance values from 8hour collections between 24:0008:00 and 16:0024:00 were found to be the most accurate and gave the best correlations. Furthermore only the mean absolute percentage deviations of the 8hour from the 24hour clearance values were significantly less than 20%. Significant cyclical variations in creatinine clearance over 24 hours were not observed. Time intervals between 23:0007:00 and 07:0009:00 were chosen for the comparisons between 8hour, 2hour, Cockcroft Gault creatinine clearance estimations and the 24hour values in 21 healthy subjects. The mean 2hour and 8hour creatinine clearances were not significantly different from the 24hour values. However, once again only the 8hour clearance values differed by less than 20% from the 24hour values and they were more accurate and better correlated than the 2hour values. As expected, in both groups of subjects, the percentage of clearance values that deviated by more than 20% from the 24hour values decreased as the length of the collection times increased. The Cockcroft Gault formula in both groups of volunteers gave less accurate clearance estimations, smaller correlation coefficients (not statistically significant in Group I subjects) and percentage deviations from the 24hour values greater than 20%. Undetected early stage renal insufficiency in three volunteers and the use of actual instead of normalized Scr values may have been the cause of these poor clearance estimations. In healthy subjects (Scr < 1.5mg/dl) 24hour creatinine clearance may be estimated from an 8hour urine collection with voluntary voiding if a 20% deviation from the 24hour value is considered clinically acceptable.     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

Enhancement of Solubility and Bioavailability of β-Lapachone Using Cyclodextrin Inclusion Complexes

Purpose. To explore the use of cyclodextrins (CD) to form inclusion complexes with -lapachone (-lap) to overcome solubility and bioavailability problems previously noted with this drug. Methods. Inclusion complexes between -lap and four cyclodextrins (-, -, -, and HP-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and ¹H-NMR spectroscopy. Biologic activity and bioavailability of -lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). Results. Phase solubility studies showed that -lap solubility increased in a linear fashion as a function of -, -, or HP-CD concentrations but not -CD. Maximum solubility of -lap was achieved at 16.0 mg/ml or 66.0 mM with HP-CD. Fluorescence and ¹H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between -CD and HP-CD with -lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of -lap in -CD or HP-CD inclusion complexes (TD₅₀ = 2.1 M). Animal studies in mice showed that the LD₅₀ value of -lap in an HP-CD inclusion complex is between 50 and 60 mg/kg. Conclusions. Complexation of -lap with HP-CD offers a major improvement in drug solubility and bioavailability.     

Role of human liver microsomal CYP2C9 in the biotransformation of lornoxicam

Objective: The nature of the enzyme(s) catalysing the biotransformation of lornoxicam to one of its major metabolites, 5-hydroxy-lornoxicam, has been investigated in human liver microsomes. The reaction kinetics were characterised, the affinity of lornoxicam for three major human drug metabolising cytochrome P-450 isozymes (CYP2C9, CYP2D6 and CYP3A4) was determined, and inhibition of the reaction by known substrates (diclofenac, ibuprofen, mefenamic acid, phenytoin, tolbutamide and warfarin) and the prototype inhibitor (sulphaphenazole) of CYP2C9 was investigated. Results: Lornoxicam 5-hydroxylation displayed single enzyme Michaelis-Menten kinetics, with a K_M of 3.6 mol·l^-1 and a V_max of 2.6 nmol·h^-1·mg^-1 microsomal protein. The apparent affinity of lornoxicam was high for CYP2C9, but negligible for CYP3A4 and CYP2D6. Inhibition of lornoxicam 5-hydroxylation by CYP2C9 substrates and sulphaphenazole was comparable in all livers preparations, values predicted from their K_M or K_i for CYP2C9 determined in separate studies assuming competitive inhibition. Sulphaphenazole competitively and completely inhibited lornoxicam 5-hydroxylation (K_i=0.31 mol·l^-1) as well as lornoxicam clearance (K_i=0.33 mol·l^-1), partial metabolic clearance (f_m)=0.95). Conclusion: 5-Hydroxylation appears to be the only cytochrome P-450 catalysed metabolic reaction of lornoxicam by human liver microsomes and this major in vivo biotransformation pathway is catalysed virtually exclusively by CYP2C9.Supported in part by Hafslund Nycomed Pharma AG (Linz, Austria) and by a grant of the Forschungsförderungsfond der Gewerblichen Wirtschaft Österreichs     

Short-term effects of herbicides on primary productivity of periphyton in lotic environments

Freshwater algae are quite sensitive to herbicides that enter running water ecosystems through direct application, aerial drift, and/or watershed run-off. However, due to a lack of suitable methodologies, few studies examine the effects of such contamination on naturally occurring attached algal communities under field conditions (i. e., exposure regimes using pulsed doses or brief episodes of peak concentrations to simulate surface run-off during storm events). This paper describes a method for determining the acute short-term effects of four herbicides (hexazinone, atrazine, tebuthiuron and metolachlor) on the net primary productivity (NPP) of periphytic algae in the field using a portable bankside incubator; NPP was measured by monitoring changes in oxygen production (mg O₂ per m²) upper surface of rock substrate per h and mg O₂ h per mg chlorophyll using the light-dark technique. All herbicides with photosynthetic inhibition as a mode of action significantly reduced NPP. The lowest observed effect concentrations (LOECs) for the herbicides were 43 g hexazinone l^–1, 109 g atrazine l^–1 and 137 g tebuthiuron l^–1. The no observed effect concentrations (NOECs) for these chemicals were <43 g hexazinone l^–1, 93 g atrazine l^–1 and 52 g tebuthiuron l^–1. Metolachlor did not significantly reduce NPP at the concentrations that were tested (range 19.6–274 g l^–1). However, community respiration (which included respiration by invertebrates) was significantly reduced at the highest metolachlor concentration (274 g l^–1). Community respiration was not significantly affected by any concentration of the other three herbicides used.