Multiplex profiling identifies clinically relevant signalling proteins in an isogenic prostate cancer model of radioresistance.

The exact biological mechanism governing the radioresistant phenotype of prostate tumours at a high risk of recurrence despite the delivery of advanced radiotherapy protocols remains unclear. This study analysed the protein expression profiles of a previously generated isogenic 22Rv1 prostate cancer model of radioresistance using DigiWest multiplex protein profiling for a selection of 90 signalling proteins. Comparative analysis of the profiles identified a substantial change in the expression of 43 proteins. Differential PARP-1, AR, p53, Notch-3 and YB-1 protein levels were independently validated using Western Blotting. Pharmacological targeting of these proteins was associated with a mild but significant radiosensitisation effect at 4Gy. This study supports the clinical relevance of isogenic in vitro models of radioresistance and clarifies the molecular radiation response of prostate cancer cells.

n-Nonanoyl-CCL14 (NNY-CCL14), a novel inhibitor of allergic airway inflammation is a partial agonist of human CCR2.

BACKGROUND: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. AIM OF THE STUDY: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. METHODS: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. RESULTS: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca(2+)]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. CONCLUSIONS: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.

Genetic dissection of glucocorticoid receptor function in the mouse brain.

In the brain, glucocorticoids exert functions in neurogenesis, synaptic plasticity and behavioural responses, as well as in the control of hypothalamic-pituitary-adrenal axis activity. The generation of mice harbouring germline mutations that result either in loss or in gain of glucocorticoid receptor function provided a useful tool for understanding the role of glucocorticoids in the brain in vivo. The improvement of genomic technologies additionally allowed the establishment of mouse models with function-selective point mutations of the receptor as well as the generation of mice harbouring spatially and/or temporally restricted loss of glucocorticoid receptor, specifically within the brain. These models will provide the opportunity to better understand the mechanisms involved in glucocorticoid signalling within the nervous system.

A comparison of red blood cell thiopurine metabolites in children with acute lymphoblastic leukemia who received oral mercaptopurine twice daily or once daily: a Pediatric Oncology Group study (now The Children's Oncology Group).

INTRODUCTION: Mercaptopurine is an important antimetabolite for treatment of childhood acute lymphoblastic leukemia (ALL). It has been prescribed to be given daily without therapeutic monitoring of drug levels. After first-pass metabolism by hepatic xanthine oxidase (XO), mercaptopurine is converted into two major intracellular metabolites, thioguanine nucleotide (TGN) and methylated mercaptopurine metabolites (including methylated thioinosine nucleotides), which are cytotoxic in vitro. Its short plasma half-life and S-phase-dependent pharmacokinetics suggest that biologically active concentration and exposure duration may be critical to cell kill. METHODS: Pediatric Oncology Group (POG) 9605, a randomized, open label phase III study of standard-risk ALL, was designed to compare daily with twice-daily mercaptopurine during continuation therapy. Red blood cell (RBC) TGN and methylated mercaptopurine metabolite levels were measured as surrogates of leukemic cell levels in a randomly selected subset of patients. TGN and methylated mercaptopurine metabolites were analyzed quantitatively by high-performance liquid chromatography (HPLC) and reported in ng/8 x 10.8 RBC. Statistical inferences utilized multiple linear regression. RESULTS: One hundred eighteen patients received mercaptopurine 75 mg/m(2) daily and 108 received 37.5 mg/m(2)/dose twice daily. Descriptive statistics for the daily group showed the median TGN was 42 ng (mean and standard deviation [SD] = 48 +/- 35, quartiles 29-64). For the twice daily group, it was 40 ng (mean and SD = 40 +/- 27, quartiles 26-53). For methylated mercaptopurine metabolites, the daily group median was 2,020 ng (mean and SD = 2,278 +/- 1,559, quartiles 1,247-3,162); the twice daily group median was 1,275 ng (mean and SD = 1,580 +/- 1,240, quartiles 599-2,369). When adjusted for the covariables: actual dosage, days on study, age at diagnosis, white blood cell count, gender, Black race compared with not, and Hispanic compared with not, daily dosing resulted in significantly higher average methylated mercaptopurine metabolites by 668 (standard error [SE] = 179, P = 0.001) and a trend toward higher average TGNs by 6.2 (SE = 4.2, P = 0.14). CONCLUSIONS: Daily dosing of mercaptopurine resulted in higher mean red cell methylated mercaptopurine metabolites when compared to split (twice a day dosing). The data were inconclusive with respect to TGNs. The relationships of methylated mercaptopurine metabolites and TGNs to clinical outcomes will be elucidated as part of the maturing 9605 data.

Plasma pharmacokinetics and cerebrospinal fluid penetration of thioguanine in children with acute lymphoblastic leukemia: a collaborative Pediatric Oncology Branch, NCI, and Children's Cancer Group study.

PURPOSE: In preclinical studies, thioguanine (TG) has been shown to be more potent than the standard acute lymphoblastic leukemia (ALL) maintenance agent, mercaptopurine (MP), suggesting that TG may be more efficacious than MP in the treatment of childhood ALL. As part of a pilot trial in which TG was used in place of MP, we studied the plasma pharmacokinetics of oral TG and measured steady-state plasma and CSF TG concentrations during a continuous intravenous infusion (CIVI) in children with newly diagnosed standard-risk ALL. METHODS: Nine plasma samples were collected after each patient's first 60 mg/m2 oral TG dose during maintenance. CIVI TG (20 mg/m2/h over 24 h) was administered during the consolidation phase of therapy, and simultaneous plasma and CSF samples were collected near the end of the infusion. TG was measured by reverse-phase HPLC with ultraviolet detection. Erythrocyte TG nucleotide (TGN) concentrations were measured 7 days after a course of CIVI TG and prior to the start of each maintenance cycle. RESULTS: After oral TG (n = 35), the mean (+/- SD) peak plasma concentration was 0.46 +/- 0.68 microM and the AUC ranged from 0.18 to 9.5 microM.h (mean 1.5 microM.h). Mean steady-state plasma and CSF TG concentrations during CIVI (n = 33) were 2.7 and 0.5 microM, respectively. The mean (+/- SD) TG clearance was 935 +/- 463 ml/min per m2. Plasma TG concentrations did not correlate with erythrocyte TGN concentrations after oral or CIVI TG. The 8-OH-TG metabolite was detected in plasma and CSF. CONCLUSIONS: TG concentrations that are cytotoxic to human leukemia cell lines can be achieved in plasma after a 60 mg/m2 oral dose of TG and in plasma and CSF during CIVI of TG.

Bioequivalence evaluation of lansoprazole 30-mg capsules (Lanfast and Lanzor) in healthy volunteers.

The bioequivalence of two lansoprazole 30-mg capsules was determined in healthy human, adult volunteers after a single dose in a randomized cross-over study. The study was conducted at Pharmaconsult, Flemington Pharmaceutical Corp., New Jersey, USA. Reference (Lanzor, Laboratoires Houde, Paris, France) and test (Lanfast, Julphar, UAE) were administered to volunteers with 240 ml water after overnight fasting. Blood samples were collected at specified time intervals, plasma was separated and analyzed for lansoprazole using a validated HPLC method. The pharmacokinetic parameters AUC(0-t), AUC(0-~), C(max), T(max), T(1/2) and elimination rate constant were determined from plasma concentration-time profile of both formulations and found to be in good agreement with previously reported values. The calculated pharmacokinetic parameters were compared statistically to evaluate bioequivalence between the two brands, using the statistical modules recommended by the Food and Drug Administration. The analysis of variance (ANOVA) did not show any significant difference between the two formulations and 90% confidence intervals fell within the acceptable range (80-120%) for bioequivalence. Based on these statistical inferences it was concluded that the two formulations exhibited comparable pharmacokinetic profiles and that Julphar's Lanfast is bioequivalent to Lanzor of Lab. Houde.

Translocation of acylated pardaxin into cells.

Acylated pardaxin is translocated through the cytoplasmic membrane and is accumulated in the nucleoli of NG108-15 and chromaffin cells. The uptake is time- and dose-dependent and temperature-sensitive. However, the binding of acylated 125I-pardaxin cannot be reduced by competition with pardaxin acylated with Rudinger's reagent. In this respect, acylated pardaxin resembles the Tat protein 37-71 fragment. Metabolic inhibitors do not significantly reduce the uptake of acylated 125I-pardaxin. Acylated pardaxin might be useful as a vector to translocate other molecules.

Event-related potential (ERP) asymmetries to emotional stimuli in a visual half-field paradigm.

To investigate the hypothesis of a right hemispheric superiority in negative emotional processing, event-related potentials (ERPs) were recorded from 17 sites (Fz, Cz, Pz, F3/4, F7/8, C3/4, T7/8, P3/4, P7/8, O1/2) in a visual half-field paradigm. While maintaining fixation, right-handed women viewed pictures of patients with dermatological diseases before (negative) and after (neutral) cosmetic surgery. A principal components analysis with Varimax rotation performed on ERPs revealed factors identified as N1, N2, early P3, late P3, and slow wave. Repeated measures analyses of variance performed on factor scores revealed a significant effect of emotional content for all factors except for N1. However, asymmetries in emotional processing were restricted to N2 and early P3, with maximal effects over the right parietal region. N2-P3 amplitude was augmented for negative and reduced for neutral stimuli over right hemisphere regions. Visual field presentation interacted with these asymmetries in enhancing amplitudes contralaterally for early but ipsilaterally for late ERP components. Overall, findings for N2 and P3 support theories of an asymmetry in emotional processing.

[Anxiety and anxiolysis: on the need for differentiated characterization of anxiety from the experimental and biopsychological viewpoint]. / Angst und Anxiolyse: Zur Notwendigkeit einer differenzierten Kennzeichnung von Angst aus experimental- und biopsychologischer Sicht.

Characterization and operationalization of anxiety point to the problem of the homogenity of the construct "anxiety". Results of experimental studies show that one and the same increase in subjective anxiety can be related to systematically different autonomic-physiological processes. Accordingly, (1) an unspecific increase in ANS-arousal is not, as usually assumed, a basic characteristic of anxiety and (2) indicators of experienced anxiety, which often have a key-role in inferring a state of anxiety, do not allow a sufficient differentiation of this state. The integration of the results in experimentally based biopsychological theories of emotion and the application to the preoperative situation underline the necessity of a more differentiated characterization of anxiety, also and especially with regard to possibilities of a pharmacological anxiolysis.

[Are psychophysiologic changes in the "public speaking" paradigm an expression of emotional stress?]. / Sind psychophysiologische Veränderungen im Paradigma "Offentliches Sprechen" Ausdruck emotionaler Belastung?

Public Speaking is often used to induce anxiety. The emotional stress elicited by the anticipation of delivering a public speech, however, is usually confounded with the mental stress of speech preparation. In order to separate the effects of the emotional from those of the mental stress factor, only half of the subjects were informed about the topic of the speech at the beginning of the anticipation period, whereas the other half were told about the topic at a later time. In a pilot study, the effects of this variation were tested under a condition in which the subjects were told that the speech would be videotaped as an anxiety-provoking and under an emotionally neutral control condition. The main experiment involved an additional condition with a simulated audience designed to intensify the impact of the emotional stress component ("strong anxiety"). Self-reports on present state and cardio-vascular and electrodermal responses were measured in n = 12 subjects in both studies. Knowledge of the speech topic did not affect the subjective anxiety-inducing effects of public speaking. Physiologically arousing effects, however, could be shown without knowledge of the topic only in some variables and only under the "strong anxiety"-provoking condition. In studying public speaking anxiety, confounding with the mental stress of speech preparation should therefore be avoided and a more differentiated interpretation of the physiological effects should be made.

Heat-stable antigen is expressed by murine keratinocytes and delivers costimulatory signals in T-cell activation.

Heat-stable antigen (HSA), expressed by various antigen-presenting cells (APC), has been described as a costimulatory molecule for CD4+ T cells. Recently, we observed that HSA also serves as an important costimulatory molecule on epidermal Langerhans cells (LC). During these studies, low levels of HSA staining were also detected on normal murine keratinocytes (KC). To investigate whether HSA also is involved in T-cell activation by KC, normal murine KC or the spontaneously transformed KC cell-line PAM 212 were treated with PDB or PMA to induce HSA-expression. FACS analyses showed induction of HSA expression on normal murine KC, as well as PAM 212 cells. In functional assays PDB or PMA-treated normal or transformed KC were far more potent inducers of primary allogeneic T-cell responses than untreated KC. Addition of anti-HSA-specific mAb 20C9 specifically inhibited the costimulatory activity of KC, an effect that was even more pronounced when CTLA-4Ig was added to the cultures. Cleavage of HSA on KC surfaces by a phosphoinositol-specific phospholipase C (PI-PLC) also significantly inhibited the costimulatory capacity of KC for naive CD4+ T cells. In aggregate, our data indicate that expression of HSA on activated KC contributes to the capacity of these cells to induce proliferation of allogeneic T cells.

Experimental models for aggression and inventories for the assessment of aggressive and autoaggressive behavior.

This paper reviews principles realized in questionnaires for the assessment of aggression as well as in experimental models suitable for inducing aggression for the validation of questionnaire scales and for providing experimental models for testing aggression-reducing drug effects. Existing self-rating scales based on factor analysis were shown to measure certain parameters of reactions concerning modes of expression of aggression and its objects. In observer rating scales situations are usually also specified. A final scale containing 9 situations and 17 reactions grouped into seven factors is presented. It could be shown to differentiate between certain types of aggression provoking situations. Furthermore, models suitable for eliciting aggression were developed in three different departments of psychology. They are based on frustration by blockade of goals and critique or subtraction of positive reinforcers ("Tower of Hanoi" and "Superball Game" in Würzburg, "Unsolvable Maze Computer Task" in Berlin) and by a competitive condition combined with application of aversive stimuli by a coplayer ("Modified Buss Machine" in Giessen). All experimental conditions were suitable for inducing anger and emotional arousal, negative ratings of confederates or experimenters, and partly also physiological changes. The results seem promising enough to test the relationship between artificially induced aggression and pathologiocal aggression in further research.

Processing of tetanus and botulinum A neurotoxins in isolated chromaffin cells.

Tetanus and botulinum A neurotoxins were introduced into the cytosol of chromaffin cells by means of an electric field in which the plasma membrane is forced to form pores of approximately 1 micron at the sites facing the electrodes. As demonstrated by electron microscopy, both [125I] and gold-labelled tetanus toxin (TeTx) diffuse through these transient openings. Dichain-TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in which the heavy and light chains are interconnected by an additional peptide bond, was far less effective than dichain-TeTx at blocking exocytosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [125I] dichain-TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different methods, was less than 10. The long duration of tetanus poisoning may result from the persistence of intracellular toxin due to scarcity of free cytosolic proteases. This may also hold for the slow recovery from botulism.

Effect of Streptococcus pneumoniae and influenza A virus on middle ear antimicrobial pharmacokinetics in experimental otitis media.

Antimicrobial treatment failures in children with acute otitis media and concomitant viral respiratory tract infection prompted us to study the effects of influenza A virus infection on middle ear antimicrobial drug penetration. Using a chinchilla model of Streptococcus pneumoniae we compared middle ear elimination rates in 4 groups of chinchillas: (1) control, (2) influenza A virus inoculation alone intranasally, (3) both influenza A and S. pneumoniae inoculation directly into the middle ear, and (4) S. pneumoniae inoculation alone into the middle ear. After infection was established, a solution containing amoxicillin, sulfamethoxazole, and trimethoprim was instilled into the middle ear and removed 4 hours later. The rate constant of elimination and half-life were calculated from measured drug concentrations initially and at 4 hours. S. pneumoniae infection alone significantly shortened the middle ear elimination half-life compared with the control group: amoxicillin, 2.65 +/- 0.73 vs. 6.63 +/- 2.55 hr; sulfamethoxazole, 1.75 +/- 0.28 vs. 2.74 +/- 0.6 hr; and trimethoprim, 1.06 +/- 0.14 vs. 1.56 +/- 0.34 hr (n = 16 ears, p values all < 0.01). The combined influenza virus and S. pneumoniae infection significantly lengthened the half-life compared with the S. pneumoniae infection alone: amoxicillin, 5.65 +/- 6.44 vs. 2.65 +/- 0.73 hr; sulfamethoxazole, 2.5 +/- 0.85 vs. 1.75 +/- 0.28 hr; and trimethoprim, 1.26 +/- 0.42 vs. 1.06 +/- 0.14 hr (n = 16 ears, p values all < 0.01). Influenza virus produced the longest half-lives for all 3 antimicrobials: amoxicillin 25.52 +/- 14.96 hr; sulfamethoxazole, 5.46 +/- 0.87 hr; and trimethoprim, 2.57 +/- 0.75 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of two otitis media models for the study of middle ear antimicrobial pharmacokinetics.

We compared two models of acute otitis media that estimate middle ear antimicrobial pharmacokinetics. Using a crossover study design, we compared a systemic drug administration model with a diffusion model we devised that measures the disappearance of antimicrobials from the middle ear. We induced acute otitis media in 14 chinchillas by inoculating S. pneumoniae into the middle ear, then administered 3 antimicrobials: amoxicillin, trimethoprim, and sulfamethoxazole. Next we collected middle ear fluid samples to analyze drug concentrations and compare rate constants for the systemic and diffusion models by analysis of variance. We found that amoxicillin K values were not affected by model testing sequence (p = 0.827) or model type (systemic versus diffusion, p = 0.310), nor were sulfamethoxazole K values: model testing sequence (p = 0.917), model type (p = 0.963). Trimethoprim K values were also not affected by model testing sequence (p = 0/760), but were by model type (p = 0.0001). Trimethoprim elimination from the diffusion model was faster (K = 0.33 +/- 0.17 versus 0.57 +/- 0.09 hr-1) than from the systemic model, although it appears this was caused by sampling before drug distribution into the middle ear was complete. In conclusion, it appears K values derived from either systemic antimicrobial administration or direct middle ear instillation are similar for assessing middle ear antimicrobial pharmacokinetics, and these models can be used interchangeably to study factors affecting otitis media treatment response.

Improved method for autoradiographic localization of beta-adrenoceptors using photoaffinity labelling.

Contact autoradiography of tissue sections, using emulsion coated coverslips or X-ray films, is widely used to provide information about the regional distribution of receptors. This easy to perform, standard technique has the disadvantage of an image spread due to the gap between the radioactive source and the film. The present study describes a new technique which combines photoaffinity labeling of beta-adrenoceptors with "dipping" autoradiography and a modified trichrome stain. Incubation of 16 microns cryosections of rat lung tissue with the iodinated, photoaffinity labeling, non-selective, beta-adrenergic agonist [125I]-cyanopindololazide II ([125I]-CYPA II) (100 pmol/l) in the absence or presence of 1 mumol (+/-)-propranolol revealed strong, specific beta-adrenoceptor binding to alveolar parenchyma and bronchial epithelium of large and small bronchioles, lesser binding to smooth muscle bundles of large airways and only sparse binding to the smooth muscle of small bronchioles or peripheral branches of pulmonary artery. With standard autoradiographic techniques, a similar distribution of the label was obtained, although resolution and sensitivity were inferior. Staining of tissue sections through the photoemulsion by means of a modified Mallory's trichrome dye facilitated the discrimination between alveolar and bronchial epithelium, muscular and collagenous tissues. In conclusion, the photoaffinity labeling of beta-adrenoceptors with [125I]-CYPA II allows the use of "dipping" autoradiography. This technique, in combination with trichrome staining through the photoemulsion, results in an improved autoradiographic image together with a better association of the label with distinct histological structures and the higher sensitivity of the method.

Cellular pharmacology of 6-mercaptopurine in acute lymphoblastic leukemia.

PURPOSE: The cellular pharmacology of 6-Mercaptopurine (6MP) in acute lymphoblastic leukemia (ALL) is reviewed. DESIGN: Relevant studies on the clinical pharmacology of 6MP were reviewed. RESULTS: 6MP is one of the major drugs used in maintenance therapy of acute lymphoblastic leukemia (ALL). It is also used to treat steroid unresponsive inflammatory bowel disease. 6MP is an inactive prodrug that requires absorption, cellular uptake, and intracellular anabolism to nucleotides for cytotoxic activity. These nucleotides are ultimately incorporated into DNA and RNA, resulting in cell death. Two analogs of 6MP, azathioprine and 6-thioguanine, are also anabolized to the same intracellular metabolites, suggesting they should be therapeutically equivalent to 6MP. 6MP may be anabolized to nonmethylated nucleotides or may undergo methylation by the enzyme thiopurine methyltransferase to S-methylated nucleotides, which are also cytotoxic. CONCLUSION: Recent studies of 6MP pharmacokinetics in children with ALL have suggested that a higher systemic exposure, as measured by a greater area under the plasma concentration time curve or a higher concentration of 6MP metabolites in red blood cells, is associated with a decreased risk of relapse.

Isotype-specific rabbit antibodies against chinchilla immunoglobulins G, M, and A.

Chinchillas have become a preferred animal model for studying otitis media, and are also useful in studying insulin release, gastrin physiology, intestinal infection, and hepatocellular pathophysiology. Immunopathologic studies in the model, however, have been limited by absence of specific antibody reagents against chinchilla immunoglobulins. We describe a method for preparing isotype-specific rabbit antibodies against the heavy-chain components of chinchilla immunoglobulins G, M, and A. Chromatographic techniques were used to isolate chinchilla immunoglobulins from serum and breast milk; heavy-chain fractions were isolated and used as antigens to produce isotype-specific antibodies in New Zealand White rabbits. Enzyme-linked immunosorbent assay of these antisera disclosed anti-light chain cross-reactivity, which was removed by affinity chromatography. The isolation and affinity purification techniques were highly reproducible. The availability of these reagents should greatly enhance the utility of the chinchilla in modeling human disease.

Mizoribine pharmacokinetics and pharmacodynamics in a canine renal allograft model of local immunosuppression.

We utilized a canine renal transplant model to estimate the first-pass extraction of mizoribine (MZB) during renal artery infusion and to compare the efficacy and toxicity of continuous intraarterial (i.a.) versus intravenous (i.v.) MZB delivery, with and without a background of oral cyclosporine. Five autotransplanted mongrel dogs with programmable, implantable pump/catheter systems were given MZB by both i.v. bolus (5 mg/kg) and i.a. infusion (5.0 mg/kg/day). Mean +/- SD elimination half-life was 3.02 +/- 0.81 hr, and the transplanted kidney removed as much as 47-59% (mean 56%) of locally infused MZB. With increasing local and systemic MZB delivery in a single autografted dog undergoing both i.a. and i.v. pump/catheter placement, renal extraction decreased from at least 47% (5.0 mg/kg/day) to 33% (7.5 mg/kg/day), finally to 18% (10.0 mg/kg/day). A dose of 3.0 mg/kg/day MZB did not significantly prolong survival of renal allograft recipients over that of untreated controls (median survival time [MST] = 8 days) when administered either locally (MST = 9 days) or systemically (MST = 12 days). All dogs receiving 4.0 mg/kg/day MZB i.a. died from rejection, and a survival advantage was still not realized (MST = 7 days). In contrast, 4.0 mg/kg/day i.v. prolonged survival over controls (MST = 14 days; P = 0.03) but not when directly compared with the i.a. group (P = 0.30), and produced death from severe debility in five of seven animals with significantly higher mean systemic MZB levels (P = 0.02). Four of six dogs receiving 5.0 mg/kg/day MZB i.a. (MST = 14 days) and two of four dogs receiving 5.0 mg/kg/day i.v. (MST = 14 days) died from severe debility, though survival in both groups was prolonged over control values (P = 0.01 and P = 0.05, respectively). Coadministration of a subtherapeutic dose of oral CsA (5 mg/kg/day) significantly prolonged the overall survival of dogs receiving MZB 4.0 mg/kg/day i.a. (MST = 23; P = 0.01) but not i.v. (MST = 11; P = 1.00), so that a significant difference in overall survival between the combined MZB i.a. + CSA and MZB i.v. + CSA groups was now realized in favor of the former (P = 0.04). We conclude that at local doses required to achieve immunosuppression, the transplanted kidney was not able to extract enough MZB to prevent death from systemic toxicity, presumably as a result of saturation of renal elimination mechanisms, so that an overall survival benefit was not realized.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversed-phase high-performance liquid chromatographic approach to determine total lymphocyte concentrations of 6-thioguanine, methylmercaptopurine and methylthioguanine in humans.

A reversed-phase high-performance liquid chromatographic (HPLC) procedure was developed to quantify intracellular lymphocyte 6-thioguanine, methylmercaptopurine and methylthioguanine. The free base of each metabolite was obtained by acid hydrolysis, which allowed for a total determination of thiopurine metabolites. 6-Thioguanine was analyzed on an octadecylsilane column using acetonitrile-10 mM sodium phosphate (11:89), pH 7, containing 0.06% tetrabutylammonium chloride. 6-Thioguanine was oxidized with potassium permanganate, and fluorescence was measured at 330 nm excitation and 410 nm emission. Methylmercaptopurine and methylthioguanine were separated on a cyanopropylsilane column using methanol-40 mM sodium phosphate (22:78), pH 2.7, and detected by ultraviolet absorbance at 314 and 290 nm, respectively.