Feasibility of analysing [13C]urea breath tests for Helicobacter pylori by gas chromatography-mass spectrometry in the selected ion monitoring mode

Background : The [¹³C]urea breath test for Helicobacter pylori is nonradioactive, as well as noninvasive, but few clinical laboratories have the expensive isotope ratio mass spectrometer used for analysis.
Methods : To demonstrate the feasibility of analysing [¹³C]urea breath tests with a gas chromatograph-mass spectrometer routinely used for drug testing, ¹³CO₂ standards for breath tests and breath samples from patients in a multiple-blind study were analysed. The breath samples were also analysed by isotope ratio mass spectrometry, and the diagnoses were compared with biopsy results.
Results : The precision of the enrichment measurements by gas chromatography–mass spectrometry was 1.1 parts per thousand, and the calculated differences in enrichment between standard gases equaled the certified values. The sensitivity (94%), specificity (94%), and percentage agreement (94%) for diagnosis of Helicobacter pylori ( n =34) were as high or higher than for analysis of replicate breath samples by isotope ratio mass spectrometry and comparable to the values reported for diagnosis of the bacterium by other currently accepted tests.
Conclusions : The study demonstrates that a gas chromatograph–mass spectrometer can be used to analyse [¹³C]urea breath tests, thus potentially lowering the cost of the test and increasing the number of laboratories that can perform the test.     

Urea breath tests for detecting Helicobacter pylori

The urea breath test (UBT) is the most sensitive and specific non-invasive test for the detection of Helicobacter pylori infection both before and after treatment. Labelling of the urea with either ¹³C or ¹⁴C has relative advantages and disadvantages. ¹³C-UBTs are both safe and well-validated, and have the additional advantage that they can be used in children. However, the initial capital costs of ¹³CO₂ analysis are large compared to those for ¹⁴CO₂. The protocol details for use of the ¹³C-UBT are variable: a test meal is important if urea solution is to be used and a single sample time point at 30 min is adequate. The recent development of novel formulations of ¹³C urea and new analytical techniques for the measurement of ¹³CO₂ should allow reduction in the length of the test and its cost; they may herald a more widespread clinical application of this useful test.     

Caffeine based measures of CYP1A2 activity correlate with oral clearance of tacrine in patients with Alzheimer''s disease

Aims To study the potential utility of caffeine based probes of CYP1A2 enzyme activity in predicting the pharmokinetics of tacrine in patients with Alzheimer's disease.
Methods The pharmokinetics of a single 40  mg oral dose of tacrine were measured in 19 patients with Alzheimer's disease. Each patient also received 2  mg  kg⁻¹ [¹³C-3-methyl] caffeine orally and had breath and urine samples collected.
Results Tacrine oral clearance (CL   F ⁻¹  kg⁻¹ ), which varied 15-fold among the patients, correlated significantly with the 2  h total production of ¹³CO₂ in breath ( r =0.56, P =0.01), and with each of two commonly used urinary caffeine metabolite ratios: the raxanthine/caffeine ratio' (1,7X+1, 7U)/1,3,7X) ( r =0.76, P =0.0002) and the 'caffeine metabolic ratio' (AFMU+1X+1U)/1, 7U)( r =0.76, P =0.0001).
Conclusions These observations support a central role for CYP1A2 in the in vivo disposition of tacrine and the potential for drug interactions when tacrine treated patients receive known inducers or inhibitors of this enzyme. The magnitude of the correlations we observed, however, are probably not suffcient to be clinically useful in individualizing tacrine therapy.     

Quantitative tests of liver function measure hepatic improvement after sustained virological response: results from the HALT-C trial

Backgroud  The impact of virologic response on hepatic function has not been previously defined.
Aim  To determine the relationships of quantitative liver function tests (QLFTs) with virological responses to peginterferon (PEG) ± ribavirin (RBV) in patients with chronic hepatitis C and to use serial QLFTs to define the spectrum of hepatic improvement after sustained virological response (SVR).
Methods  Participants ( n  = 232) were enrolled in the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial, had failed prior therapy, had bridging fibrosis or cirrhosis and were retreated with PEG/RBV. All 232 patients had baseline QLFTs; 24 patients with SVR and 68 nonresponders had serial QLFTs. Lidocaine, [24-¹³C]cholate, galactose and ^99mTc-sulfur colloid were administered intravenously; [2,2,4,2-²H]cholate, [1-¹³C]methionine, caffeine and antipyrine were administered orally. Clearances ( Cl ), breath ¹³CO₂, monoethylglycylxylidide (MEGX), perfused hepatic mass (PHM) and liver volume were measured.
Results  Rates of SVR were 18–26% in patients with good function by QLFTs, but ≤6% in patients with poor function. Hepatic metabolism, measured by caffeine k _elim ( P  = 0.02), antipyrine k _elim ( P  = 0.05) and antipyrine Cl ( P  = 0.02) and the portal circulation, measured by cholate Cl _oral ( P  = 0.0002) and cholate shunt ( P  = 0.0003) and PHM ( P  = 0.03) improved after SVR.
Conclusion  Hepatic dysfunction impairs the virological response to PEG/RBV. SVR improves hepatic metabolism, the portal circulation and PHM.     

Influence of different proton pump inhibitors on activity of cytochrome P450 assessed by [(13)C]-aminopyrine breath test

Aminopyrine is metabolized by cytochrome P450 (CYP) in the liver. The investigators evaluated influences of different PPIs on CYP activity as assessed by the [(13)C]-aminopyrine breath test ([(13)C]-ABT). Subjects were 15 healthy volunteers with different CYP2C19 status (5 rapid metabolizers [RMs], 5 intermediate metabolizers [IMs], and 5 poor metabolizers [PMs]). Breath samples were collected before and every 15 to 30 minutes for 3 hours after oral ingestion of [(13)C]-aminopyrine 100 mg on day 8 of each of the following regimens: control; omeprazole 20 mg and 80 mg, lansoprazole 30 mg, and rabeprazole 20 mg. Changes in carbon isotope ratios in carbon dioxide ((13)CO(2)/(12)CO(2)) in breath samples were measured by infrared spectrometry and expressed as delta-over-baseline (DOB) ratios (‰). Mean areas under the curve of DOB from 0 to 3 h (AUC(0-3h) of DOB) were significantly decreased by omeprazole 20 mg and lansoprazole 30 mg but not by rabeprazole 20 mg. Conversely, higher PPI dose (ie, omeprazole 80 mg) seemed to further decrease AUC(0-3h) of DOB in RMs but increased it in PMs. Omeprazole and lansoprazole at the standard doses inhibit CYP activity but rabeprazole does not, whereas high-dose omeprazole seems to induce CYPs.     

Histamine H2-receptor antagonists have no clinically significant effect on the steady-state pharmacokinetics of voriconazole

Aims  Voriconazole, a new triazole antifungal agent, is metabolized mainly by cytochrome P450s CYP2C19 and CYP2C9, and also by CYP3A4. The aim of this open-label, placebo-controlled, randomized, three-way crossover study was to determine the effects of cimetidine and ranitidine on the steady-state pharmacokinetics of voriconazole.
Methods  Twelve healthy male subjects received oral voriconazole 200 mg twice daily plus cimetidine 400 mg twice daily, voriconazole 200 mg twice daily plus ranitidine 150 mg twice daily, and voriconazole 200 mg twice daily plus placebo twice daily. Treatment periods were separated by at least 7 days.
Results  When cimetidine was administered with voriconazole, the maximum plasma voriconazole concentration ( C _max) and the area under the plasma concentration–time curve of voriconazole (AUC_τ) was increased by 18.3%[90% confidence interval (CI) 6.0, 32.0] and 22.5% (90% CI 13.3, 32.5), respectively. Concomitant ranitidine had no significant effect on voriconazole C _max or AUC_τ. Time of C _max ( t _max) elimination half-life ( t _1/2) or terminal phase rate constant ( k _el) for voriconazole were similar in all three treatment groups. Most adverse events were mild and transitory; two subjects were withdrawn due to adverse events.
Conclusions  Coadministration of the histamine H₂-receptor antagonists cimetidine or ranitidine does not affect the steady-state pharmacokinetics of voriconazole in a clinically relevant manner.     

Relative dispersion of intravascular transit times during isolated human limb perfusions for recurrent melanoma

Aims We have characterized the relative dispersion of vascular and extravascular markers in the limbs of three patients undergoing isolated limb perfusions with the cytotoxic melphalan for recurrent malignant melanoma both before and after melphalan dosing.
Methods A bolus of injectate containing [ ⁵¹Cr] labelled red blood cells, [¹⁴C]-sucrose and [³H]-water was injected into an iliac or femoral artery and outflow samples collected at 1  s intervals by a fraction collector. The radioactivity due to each isotype was analysed by either gamma [ ⁵¹Cr] or beta [ ¹⁴C and ³ H] counting. The moments of the outflow fraction-time profiles were estimated by a nonparametric (numerical integration) method and a parametric model (sum of two inverse Gaussian functions).
Results The availability, mean transit time and normalised variance (CV² ) obtained for labelled red blood cells, sucrose and water were similar before and after melphalan dosing and with the two methods of calculation but varied between the patients.
Conclusions The vascular space is not well-stirred but characterized by a CV² similar that reported previously for in situ rat hind limb and rat liver perfusions. A flow-limited blood-tissue exchange was observed for the permeating indicators. Administration of melphalan did not influence the distribution characteristics of the indicators.     

Inhibition of CYP2C9 by selective serotonin reuptake inhibitors in vitro: studies of phenytoin p-hydroxylation

Aims Inhibition of cytochrome P450 (CYP) activity by selective serotonin reuptake inhibitors (SSRIs) has frequently been reported with regard to pathways mediated by CYP2D6, CYP3A4/5, and CYP1A2. Little data exist on the capability of SSRIs to inhibit CYP2C9.
Methods We investigated the effect of SSRIs on p -hydroxylation of phenytoin (PPH), an established index reaction reflecting CYP2C9 activity, in an in vitro assay using liver tissue from six different human donors.
Results In control incubations (without inhibitor), 5-( p -hydroxy-phenyl)-5phenylhydantoin (HPPH) formation rates were: V _max 0.023  nmol min⁻¹  mg⁻¹; K _m 14.3  &mgr;m. Average inhibition constants ( K _i ) differed significantly among the SSRIs, with fluvoxamine having the lowest K _i (6  &mgr;m ) followed by R-fluoxetine (13  &mgr;m ), norfluoxetine (17  &mgr;m ), RS-fluoxetine (19  &mgr;m ), sertraline (33  &mgr;m ), paroxetine (35  &mgr;m ), S-fluoxetine (62  &mgr;m ), and desmethylsertraline (66  &mgr;m ). Thus, assuming comparable molar concentrations at the site of inhibition, fluvoxamine can be expected to have the highest probability of interfering with the metabolism of CYP2C9 substrates. S-fluoxetine is on average a 5 fold weaker CYP2C9 inhibitor than either R-fluoxetine or the racemic mixture.
Conclusions These findings are consistent with published case reports describing SSRI-related increments in plasma phenytoin levels. Because phenytoin has a narrow therapeutic index, plasma levels should be closely monitored when SSRIs are coadministered.     

Inhibitory effects of antiarrhythmic drugs on phenacetin O-deethylation catalysed by human CYP1A2

Aims The aim of the study was to clarify whether the pharmacokinetic interaction between theophylline and mexiletine is mediated by inhibition of CYP1A2 and to assess the possible interaction potential of other antiarrhythmic drugs with drugs metabolized by CYP1A2.
Methods The inhibitory effects of mexiletine and 10 antiarrhythmic drugs on phenacetin O -deethylation, a marker reaction of CYP1A2, were studied using human liver microsomes and cDNA-expressed CYP1A2.
Results Propafenone and mexiletine inhibited phenacetin O -deethylation with I C ₅₀ values of 29 and 37  μm, respectively. Disopyramide, procainamide and pilsicainide produced negligible inhibition of phenacetin O -deethylation (I C ₅₀>1  mm ). Amiodarone, bepridil, aprindine, lignocaine, flecainide and quinidine inhibited phenacetin O -deethylation in a concentration-dependent manner, although the inhibitory effects were relatively weak with I C ₅₀ values ranging from 86 to 704  μm. Propafenone and mexiletine selectively abolished the high-affinity component of phenacetin O -deethylation in human liver microsomes. In addition, propafenone and mexiletine inhibited phenacetin O -deethylation catalysed by cDNA-expressed CYP1A2.
Conclusions These data suggest that, among the antiarrhythmic drugs studied, propafenone and mexiletine are relatively potent inhibitors of CYP1A2, which may cause a drug-drug interaction with drugs metabolized by CYP1A2.     

Longitudinal effects of hepatitis C virus treatment on hepatic mitochondrial dysfunction assessed by C-methionine breath test

Background  Hepatitis C virus (HCV) infection is characterized by remarkable levels of oxidative stress induced by virus interactions with hepatic mitochondria.
Aim  To examine hepatic mitochondrial function in HCV-infected patients assessed by a non-invasive ¹³C-methionine breath test (MeBT) and to explore longitudinal effects of antiviral treatment.
Methods  Twenty-one patients with chronic hepatitis C undergoing antiviral treatment with pegIFNα and ribavirin and 20 healthy controls were studied. MeBT was performed at baseline, week 12, end-of-treatment and after 24 weeks of follow-up in all patients with early virological response ( n  = 15).
Results  Twelve patients achieved sustained virological response (SVR); three patients relapsed for HCV-RNA replication. Cumulative percentage ¹³C-exhalation (cPDR_1.5h) was significantly decreased in HCV-infected individuals compared to controls irrespective of genotype and fibrosis stage ( P  < 0.001). Antiviral treatment induced a further decay in cPDR_1.5h ( P  < 0.01). After treatment cessation, ¹³C-exhalation returned at least to baseline values in all patients. SVR was even associated with a mean cPDR_1.5h increase of 70% compared to baseline.
Conclusions  Hepatitis C virus infection and antiviral treatment synergistically impair hepatic mitochondrial function, which may return to normal after sustained virus elimination. MeBT may be a valuable diagnostic instrument for monitoring hepatic mitochondrial function in particular in patients with mitochondrial comorbidities.     

The induction effect of rifampicin on activity of mephenytoin 4''-hydroxylase related to M1 mutation of CYP2C19 and gene dose

Aims To determine the induction effect of rifampicin on the activity of 4'-hydroxylase in poor metabolizers (PMs) with m₁ mutation of S-mephenytoin 4'-hydroxylation and the relationship of the effect with gene dose.
Methods Seven extensive metabolizers (EMs) of S-mephenytoin 4'-hydroxylation and five PMs with m₁ mutation were chosen to take rifampicin 300  mg day⁻¹ orally for 22 days. Prior to and after rifampicin treatment, each subject was given racemic mephenytoin 100  mg. The 4'-hydroxymephenytoin (4'-OH-MP) excreted in the 0–24  h urine and mephenytoin S/R ratio in the 0–8  h urine were determined by h.p.l.c. and GC, respectively.
Results In all EMs, the excretion of 4'-OH-MP in the 0–24  h urine was increased by 146.4±17.9%, 0–8  h urinary mephenytoin S/R ratio was decreased by 77.3±8.8%, the percentage increase in the 0–24  h excretion of 4'-OH-MP in those CYP2C19 homozygous (wt/wt) was greater than that in those heterozygous (wt/m₁ and wt/m₂ ) (203.9±42.5% vs 69.6±4.1%). 0–8  h urinary mephenytoin S/R ratio of those PMs with m₁ mutation was decreased by 9.6%, the amount of 4'-OH-MP excreted in the 0–24  h urine was increased by 80.1±48.0%.
Conclusions The activity of 4'-hydroxylase of PMs with m₁ mutation of S-mephenytoin 4'-hydroxylation can be induced by rifampicin and the inducing effect of rifampicin on 4'-hydroxylase is gene dependent.     

Evaluation of a [13C]-dextromethorphan breath test to assess CYP2D6 phenotype

A [(13)C]-dextromethorphan ([(13)C]-DM) breath test was evaluated to assess its feasibility as a rapid, phenotyping assay for CYP2D6 activity. [(13)C]-DM (0.5 mg/kg) was administered orally with water or potassium bicarbonate-sodium bicarbonate to 30 adult Caucasian volunteers (n=1 each): CYP2D6 poor metabolizers (2 null alleles; PM-0) and extensive metabolizers with 1 (EM-1) or 2 functional alleles (EM-2). CYP2D6 phenotype was determined by (13)CO(2) enrichment measured by infrared spectrometry (delta-over-baseline [DOB] value) in expired breath samples collected before and up to 240 minutes after [(13)C]-DM ingestion and by 4-hour urinary metabolite ratio. The PM-0 group was readily distinguishable from either EM group by both the breath test and urinary metabolite ratio. Using a single point determination of phenotype at 40 minutes and defining PMs as subjects with a DOB 相似文献   

Baseline microbiota activity and initial bifidobacteria counts influence responses to prebiotic dosing in healthy subjects

Background  Dietary intervention with prebiotics can cause changes in the colonic microbiota and their metabolic activities.
Aim  To investigate whether the response to prebiotic dosing is influenced by the baseline metabolic activity of the colonic flora and bifidobacteria counts.
Methods  The 4-week effect of lactulose (10 g bid.; n  = 29) and oligofructose-enriched inulin (10 g bid.; n  = 19) was evaluated in healthy human volunteers. Lactose-[¹⁵N, ¹⁵N]-ureide was used to study the colonic NH₃-metabolism. Urine (48 h) and faeces (72 h) were collected and analysed for p-cresol and ¹⁵N-content by gas chromatography–mass spectrometry and isotope ratio mass spectrometer, respectively. Faecal bifidobacteria were quantified by real-time polymerase chain reaction.
Results  After the 4-week prebiotic administration period, the urinary excretion of p-cresol and ¹⁵N was significantly decreased in both groups ( P  < 0.05) corresponding to a significantly higher faecal excretion of ¹⁵N ( P  < 0.05). The decrease in urinary ¹⁵N and p-cresol excretion significantly correlated with baseline ¹⁵N and p-cresol levels ( P  < 0.05), indicating that subjects with higher baseline levels showed a higher response to prebiotic dosing. Furthermore, a significant correlation was seen between baseline bifidobacteria counts and the effect of prebiotic intake ( P  < 0.05).
Conclusion  The response to prebiotic dosing, as indicated by the fate of NH₃, p-cresol and bifidobacteria, is determined by the initial colonic conditions.     

Mucosal interleukin-1β production and acid secretion in enlarged fold gastritis

Background : We have previously shown that eradication of Helicobacter pylori increases acid secretion in H. pylori -associated enlarged fold gastritis.
Aim : To investigate whether locally produced interleukin-1β is possibly involved in the inhibition of acid secretion in H. pylori gastritis.
Methods : IL-1β release from the gastric body mucosa was determined by short-term culture of biopsy specimens in 13 patients with enlarged fold gastritis (all H. pylori -positive), five H. pylori -positive and 10 H. pylori -negative patients without enlarged folds. The acid-inhibitory effect of locally produced IL-1β was examined by [¹⁴C]-aminopyrine uptake assay using isolated rabbit gastric glands.
Results : IL-1β release was significantly greater in patients with enlarged fold gastritis, significantly correlated with both basal and tetragastrin-stimulated acid outputs in the H. pylori -positive patients ( r  = −0.591 and r  = −0.641, respectively; P  < 0.01), and significantly decreased with concomitant increases in acid secretions after eradication of H. pylori . [¹⁴C]-aminopyrine uptake was inhibited by IL-1β in a dose-dependent manner.
Conclusions : Increased production of IL-1β caused by H. pylori infection is possibly involved in the inhibition of acid secretion in enlarged fold gastritis.     

Evidence for involvement of human CYP3A in the 3-hydroxylation of quinine

Aims Our previous studies using in vitro hepatic microsomal preparations suggested that the hepatic metabolism of quinine to form the major metabolite 3-hydroxyquinine is most likely catalysed by human P450 3A (CYP3A). The present study was carried out to investigate the kinetics and to identify and further characterise the human liver CYP isoforms involved in the metabolism of quinine.
Methods In vitro human microsomal techniques were employed.
Results The mean apparent K _m value for 3-hydroxyquinine formation was 83±19 (s.d.)  μm, ranging from 57  μm to 123  μm in microsomes from ten human livers. There was a 6.7-fold variation in V _max values (mean 547±416  pmol min⁻¹  mg⁻¹ ). Quinine 3-hydroxylation was inhibited by the specific CYP3A inhibitors, troleandomycin, midazolam and erythromycin. Inhibitors selective for CYP1A1/2, CYP2D6, CYP2E1, CYP2C9/10 or CYP2C19 had little or no effect on quinine 3-hydroxylation. Using microsomes from a panel of livers, significant correlations were found only between 3-hydroxyquinine activity and other CYP3A activities (caffeine 8-oxidation, omeprazole sulphoxidation, midazolam 1'-hydroxylation and midazolam 4-hydroxylation) and immunoreactive CYP3A content. There were no statistically significant correlations with activities selective for CYP1A2, CYP2C9 and CYP2E1. Competitive inhibition of quinine 3-hydroxylation was observed with a substrate known to be specifically metabolized by human CYP3A, i.e. midazolam, with an apparent K _i value of 11.0  μm.
Conclusions The present results strongly indicate that the conversion of quinine to 3-hydroxyquinine is the major metabolic pathway in human liver in vitro and that the reaction is catalysed by CYP3A isoforms.     

Differential inhibition of cytochrome P450 isoforms by the protease inhibitors, ritonavir, saquinavir and indinavir

Aims To compare the inhibitory potential of the HIV protease inhibitors saquinavir, ritonavir and indinavir against CYP1A2, CYP2C9, CYP2E1 and CYP3A4 catalysed metabolic reactions in human liver microsomes in vitro .
Methods Microsomes from six human livers were utilized in this study. The probe substrates were phenacetin (CYP1A2), tolbutamide (CYP2C9), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Metabolites were analysed by high performance liquid chromatography. I C ₅₀ (concentration of inhibitor giving 50% decrease in enzyme activity) and, where appropriate, K _i values were calculated.
Results Ritonavir was a very potent inhibitor of CYP3A4 mediated testosterone 6β-hydroxylation (mean K _i=0.019±0.004  μm, mean±s.d.; n =6) and also inhibited tolbutamide hydroxylation (I C ₅₀=4.2±1.3  μm, mean±s.d.; n =6). Inhibition of phenacetin O -deethylation and chlorzoxazone 6-hydroxylation was negligible. Indinavir was an order-of-magnitude less potent in inhibiting CYP3A4 ( K _i=0.17±0.01  μm ) and did not produce appreciable inhibition of the CYP1A2, CYP2C9 or CYP2E1 catalysed reactions. Saquinavir was the least potent CYP3A4 inhibitor ( K _i =2.99±0.87  μm ) and produced some inhibition of CYP2C9 (approximately 50% at 50  μm ).
Conclusions The HIV protease inhibitors have differential effects on CYP isozymes. There is obvious potential for clinically significant drug interactions particularly with ritonavir. Pharmacokinetic drug interaction studies are crucial to gain an overall understanding of the beneficial and potentially harmful effects of this important group of drugs.     

Pharmacokinetics and effect on caffeine metabolism of the proton pump inhibitors, omeprazole, lansoprazole, and pantoprazole

Aims To study the pharmacokinetics of three proton pump inhibitors, omeprazole, lansoprazole, and pantoprazole, as well as any potential influence on CYP1A2 activity (measured by means of rate of caffeine metabolism) of these compounds at single dose and repeated dose administration.
Methods Fourteen healthy males, classified as 12 extensive metabolizers (EMs) and two poor metabolizers (PMs) according to the urinary S/R mephenytoin ratio, completed this open, randomized, three-way cross-over study. In each of the three 7-day treatment periods either omeprazole (20  mg), lansoprazole (30  mg) or pantoprazole (40  mg) in therapeutically recommended doses was administered once daily, and the pharmacokinetics of the proton pump inhibitors as well as the rate of caffeine metabolism was measured on days 1 and 7.
Results In the EMs there was an increase in AUC from day 1 to day 7 for omeprazole. In the PMs the AUC of both omeprazole and lansoprazole was unchanged during repeated dosing, while for pantoprazole there was a tendency to a slight decrease. The AUC at steady state was for all three proton pump inhibitors 5 fold higher in PMs compared with EMs, indicating that the same proportion of the dose, irrespective of compound, is metabolized by CYP2C19. No induction of CYP1A2 was evident for any of the compounds in either EMs or PMs.
Conclusions The ∼5 fold difference in AUC between EMs and PMs indicates that approximately 80% of the dose for all three proton pump inhibitors is metabolized by the polymorphically expressed CYP2C19. None of the three proton pump inhibitors, administered in therapeutically recommended doses, is an inducer of CYP1A2—neither in PMs nor in EMs.     

HIGH-LEVEL EXPRESSION OF RATD1A DOPAMINE RECEPTOR cDNA IN MOUSE FIBROBLASTLTK- CELLS BY n-BUTYRATE

1. In orefer to develop a simple, efficient system for the highlvel expression of dopamine recetors in eukaryotic cells, we have studied the effects of n-butyrate on the expression of rat D_1A dopamine receptor cDNA in mouse fibroblast LTK- cells as compared with those of n-butyrate on endogenous D₁ receptor levles in opossum kidney cells.
2. In the transfected LTK- cell mebranes with pRc/CMVD_1A receptor cDNA, a selective D₁ dopamine antagonist, [³H]-SCH 23390, exhibited a K_d of 0.9 ± 0.1 nmol/L and a B_max of 0.35 ± 0.05 pmol/mg protien (n = 5).
3. Addtion of n-butrate (2–10 mmol/L) to the culture medium for 48h dose-dependently increased the D_1A receptor level up to 1.5 ± 0.3 pmol/mg protien (n = 7), although the K_d values were not affected. The increase in receptor level was accompanined by an elevation of selective D₁ agoinist-induced adenyly cyclase activity.
4. In contrast, n-butyrate treatment (2–10 mmol/L) did not affect either endogenous D₁ receptor levels or fendoldopmainduced adenylyl cyclase activity in possum kidney cells.
5. These results sugges n-butyrate is a sueful tool for obtaining high-level expression of D_1A dopamine receptor cDNA in mouse fibroblast LTK- cells.     

Pharmacokinetics of recombinant human interleukin-2 in advanced renal cell carcinoma patients following subcutaneous application

Aims The aim of the study was to investigate the pharmacokinetics of recombinant human interleukin-2 (rhIL-2) in patients with metastatic renal cell carcinoma following different subcutaneous (s.c.) administration regimens.
Methods RhIL-2 was administered subcutaneously to 10 patients according to two different dosing regimens: group A received 20×10⁶  IU  m⁻² once daily and group B 10×10⁶ IU  m⁻² twice daily (every 12  h). Additionally, in all patients the influence of soluble interleukin-2 receptor (sIL-2R) on the pharmacokinetics of rhIL-2 was investigated.
Results The mean area under the serum concentration-time curve to 24  h (AUC(0,24  h)) was 627  IU  ml⁻¹  h in treatment group A and 1130  IU  ml⁻¹  h ( P =0.029) in treatment group B. In both study groups C _max and AUC(0,12  h) were not significantly different. Seventy-two  hours after the beginning of s.c. rhIL-2 therapy the sIL-2R increased significantly ( P =0.016), and sIL-2R levels over 1200  pmol  l⁻¹ seemed to reduce the AUC.
Conclusions In patients with metastatic renal cell cancer administration of 20×10⁶  IU  m⁻² of rhIL-2  s.c. in two daily doses (10×10⁶  IU  m⁻² every 12  h) provides better bioavailability and is preferable to the single dose administration.