Balancing the autonomic nervous system to reduce inflammation in rheumatoid arthritis.

Imbalance in the autonomic nervous system (ANS) has been observed in many established chronic autoimmune diseases, including rheumatoid arthritis (RA), which is a prototypic immune-mediated inflammatory disease (IMID). We recently discovered that autonomic dysfunction precedes and predicts arthritis development in subjects at risk of developing seropositive RA. In addition, RA patients with relatively high vagus nerve tone (higher parasympathetic parameters, measured by heart rate variability) respond better to antirheumatic therapies. Together, these data suggest that the ANS may control inflammation in humans. This notion is supported by experimental studies in animal models of RA. We have found that stimulation of the so-called cholinergic anti-inflammatory pathway by efferent electrical vagus nerve stimulation (VNS) or pharmacological activation of the alpha7 subunit of nicotinic acetylcholine receptors (α7nAChR) improves clinical signs and symptoms of arthritis, reduces cytokine production and protects against progressive joint destruction. Conversely, increased arthritis activity was observed in alpha7nAChR knockout mice. These studies together with previous work in animal models of sepsis and other forms of inflammation provided the rationale for an experimental clinical trial in patients with RA. We could for the first time show that an implantable vagus nerve stimulator inhibits peripheral blood cytokine production in humans. VNS significantly inhibited TNF and IL-6 production and improved RA disease severity, even in some patients with therapy-resistant disease. This work strongly supports further studies using a bioelectronic approach to treat RA and other IMIDs.

Toxicological results in a fatal and two non-fatal cases of scopolamine-facilitated robberies.

The use of scopolamine as an incapacitating drug, in sexual crimes and robberies, has been known for many decades. However, blood concentrations and doses of scopolamine in those cases are largely unknown. Here we present the toxicological results of one fatal and two non-fatal cases in a series of scopolamine-facilitated robberies. In the fatal case, the concentration of scopolamine in heart blood was 0.30mg/L, about 3000 times higher than the average therapeutic level of 0.0001mg/L (for one dermal patch). In femoral blood, the concentration of scopolamine was much lower (0.0048mg/L), but still 50 times higher than therapeutic levels. The scopolamine concentration in the stomach was very high (20mg/kg) as compared to the heart blood and femoral blood, which explains the very high concentration in heart blood by postmortem leakage from the stomach. In the non-fatal case, the scopolamine concentration in serum, obtained 23h after the incident, was 0.00035mg/L. The estimated concentration of scopolamine at the time of the incident is 0.0035mg/L. In the other non-fatal case, scopolamine was detected in urine and in hair.

Reproducibility of measurements of mid-upper arm circumference in older persons.

BACKGROUND: Mid-upper arm circumference (MUAC) is used as an alternative measure for body mass index to determine thinness in older persons. However, there are limited data on the reproducibility of this measurement in an older population. The present study examined the reproducibility of MUAC measurements in older persons, as well as the influence of different body positions and clothing. METHODS: A cross-sectional reproducibility study was performed in a nursing home (n = 43; age 65-96 years) and swimming pool facilities (n = 107; age 65-88 years). A different pair of observers independently measured the MUAC of each participant in the upright position on two occasions within 1 week. In the nursing home, measurements were also performed for each participant in the laying position and with clothes covering the upper arm. RESULTS: Mean differences and the 95% limit of agreement for inter-observer reproducibility of MUAC were 0.0 cm (-2.6 to 2.5 cm) for the swimming pool facilities and 0.3 cm (-0.6 to 1.3 cm) for the nursing home. Intra-class correlation coefficients (ICCs) were 0.89 and 0.92, respectively. Mean differences between laying and upright positions were 0.1 cm (-2.0 to 2.2 cm) and 0.0 cm (-1.9 to 2.0 cm) for each observer, respectively (ICC 0.96-0.97). Mean differences between clothes versus bare upper arm were -2.7 cm (-6.2 to 0.7) and -2.4 (-5.6 to 0.9 cm) (ICC 0.75 and 0.78). CONCLUSIONS: The reproducibility of the MUAC measurement in older persons is acceptable for group comparisons and, although borderline for the swimming pool facilities, remains acceptable for clinical purposes. The measurement can also be performed in the laying position but not with clothes covering the upper arm.

Simultaneous quantification of fludarabine and cyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

Fludarabine and cyclophosphamide are anticancer agents mainly used in the treatment of hematologic malignancies. We have developed and validated an assay using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry for the quantification of fludarabine in combination with cyclophosphamide in human heparin and human EDTA plasma. Sample pre-treatment consisted of a protein precipitation with cold acetonitrile (-20 degrees C) using 250 microL of plasma. Separation was performed on an Extend C18 column (150 x 2.1 mm i.d.; 5 microm) with a stepwise gradient using 1 mM ammonia solution and acetonitrile at a flow rate of 400 microL/min. The analytical run time was 12 min. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over a concentration range of 1 to 100 ng/mL for fludarabine and cyclophosphamide in human heparin and human EDTA plasma. The coefficients of variation were <13.9% for inter- and intra-day precisions. Mean accuracies were also within the designated limits (+/-15%). The analytes were stable in plasma, processed extracts and in stock solution under all relevant conditions.

Oral bioavailability of docetaxel in combination with OC144-093 (ONT-093).

OBJECTIVE: Docetaxel given orally as monotherapy results in low bioavailability of <10%. Previous studies have indicated that the intestinal efflux pump P-glycoprotein (P-gp) prevents uptake from the gut resulting in low systemic exposure to docetaxel. The purpose of this study was to determine the degree of enhancement of the oral uptake of docetaxel on combination with orally administered OC144-093, a potent P-gp inhibitor. Furthermore, the safety of combined treatment was determined and whether known functional genetic polymorphisms of the MDR1 gene could be associated with variability in docetaxel pharmacokinetics was also investigated. PATIENTS AND METHODS: A proof of concept study was carried out in 12 patients with advanced solid tumors. Patients were randomized to receive one course of 100 mg oral docetaxel combined with 500 mg OC144-093 followed 2 weeks later by a second i.v. course of docetaxel at a flat dose of 100 mg, without OC144-093, or vice versa. This was followed by standard i.v. docetaxel treatment if indicated. RESULTS: The apparent relative oral bioavailability of docetaxel was 26+/-8%. Orally administered docetaxel combined with oral OC144-093 resulted in a Cmax of 415+/-255 ng ml(-1), an AUC0-infinity of 844+/-753 ng h ml(-1) and kel of 0.810+/-0.296 h(-1). These values differed significantly from those following i.v. administration of docetaxel, with a Cmax of 2124+/-1054 ng ml(-1), an AUC0-infinity of 2571+/-1598 ng h ml(-1) and a kel of 1.318+/-0.785 h(-1) (P=0.003, P=0.006, P=0.016). The study medication was well tolerated and most of the adverse events possibly or probably related to OC144-093 and docetaxel were of CTC grade 1 and 2. One patient had a homozygous 3435T/T mutation, which is associated with low intestinal P-gp expression, and two other patients had a homozygous mutation on exon 21. CONCLUSION: The relative apparent bioavailability of 26% was most likely caused by a significant effect of OC144-093 on the oral uptake of docetaxel. Large intrapatient and interpatient (pharmacokinetic) variation was found after oral as well as after i.v. administration of docetaxel. Higher plasma levels were observed after 100 mg i.v. docetaxel than after 100 mg oral docetaxel plus 500 mg oral OC144-093. The safety of the oral combination was good. More patients should be evaluated to determine the effect of P-gp single nucleotide polymorphisms on oral pharmacokinetic values of docetaxel.

Quantitative analysis of docetaxel in human plasma using liquid chromatography coupled with tandem mass spectrometry.

An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid-liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25--1000 ng/mL using 200 microL plasma aliquots. The method requires only a limited volume (200 microL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel.

Urinary excretion of thioTEPA and its metabolites in patients treated with high-dose cyclophosphamide, thioTEPA and carboplatin.

The urinary excretion of N,N',N"-triethylenethiophosphoramide (thioTEPA), and its metabolites N,N',N"-triethylenephosphoramide (TEPA), N,N'-diethylene,N"-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA--mercapturate was determined in patients receiving thioTEPA as part of a high-dose combination chemotherapy regimen with cyclophosphamide and carboplatin. The thioTEPA dose was 40 or 60 mg/m(2) in short infusions, twice daily, during 4 days. Urine samples were collected after each voiding on each day of drug administration until 24--48 h after the last thioTEPA infusion. ThioTEPA, TEPA and monochloroTEPA concentrations were determined with gas chromatography and thioTEPA--mercapturate with liquid chromatography--mass spectrometry with direct sample injection. ThioTEPA was present in urine 30 min after infusion and was still excreted 18 h after the last infusion. All metabolites were detected in urine 1 h after infusion. Patients with a creatinine clearance above 140 ml/minl showed higher excretion of TEPA than patients with a creatinine clearance below 140 ml/min (12.8 versus 4.9%, p=0.01). The excretion of monochloroTEPA relative to the excreted amount of TEPA increased at lower pH values of the urine. The excretion of thioTEPA--mercapturate relative to the dose was higher in patients treated with 60 mg/m(2). Excretion of thioTEPA and monochloroTEPA both accounted for only 0.5% of the dose, while TEPA and thioTEPA--mercapturate both accounted for 11.1%.

Influence of co-medicated drugs on the biotransformation of thioTEPA to TEPA and thioTEPA-mercapturate.

BACKGROUND: The combination of cyclophosphamide, thioTEPA and carboplatin is used in our Institute for the treatment of breast or germ cell cancer. ThioTEPA inhibits the bioactivation of cyclophosphamide, and platinum drugs are known to interfere with the hepatic metabolism of several anticancer drugs. Of the co-administered drugs to prevent unwanted side effects, some are enzyme inducers, cytochrome P450 inhibitors or substrates. The aim of this study was to investigate the influence of co-medicated drugs on the biotransformation of thioTEPA. METHODS: The possible inhibition of the metabolism of thioTEPA to TEPA was investigated in human microsomes. Influences on the conversion of thioTEPA to monoglutathionylthioTEPA, was studied by the incubation of thioTEPA with glutathione and glutathione S-transferase. RESULTS: No inhibition of the metabolism of thioTEPA to form TEPA was observed for cyclophosphamide and carboplatin, or any other co-medicated drug (ciproflocaxin, amphotericin B, itraconazol, fluconazol, ondansetron, dexamethasone, granisetron, aciclovir, ranitidine, lorazepam). The conversion of thioTEPA to monoglutathionylthioTEPA was inhibited by cyclophosphamide, itraconazol, amphotericin B and ondansetron with IC50 values of 58, 256, 55 and 40 mM, respectively, which are far higher than therapeutic drug levels. CONCLUSION: No clinically relevant drug-drug interactions occur in the CTC regimen as applied in our Institute.

Simultaneous determination of thioTEPA, TEPA and a novel, recently identified thioTEPA metabolite, monochloroTEPA, in urine using capillary gas chromatography.

An assay for the simultaneous quantitative determination of thioTEPA, TEPA and the recently identified metabolite N,N'-diethylene-N"-2-chloroethylphosphoramide (monochloroTEPA) in human urine has been developed. MonochloroTEPA was synthesized by incubation of TEPA with sodium chloride at pH 8. Thus, with this assay monochloroTEPA is quantified as TEPA equivalents. Analysis of the three analytes in urine was performed using gas chromatography with selective nitrogen-phosphorous detection after extraction with a mixture of 1-propanol and chloroform from urine samples. Diphenylamine was used as internal standard. Recoveries ranged between 70 and 100% and both accuracy and precision were less than 15%. Linearity was accomplished in the range of 25-2500 ng/ml for monochloroTEPA and 25-5000 ng/ml for thioTEPA and TEPA. MonochloroTEPA proved to be stable in urine for at least 4 weeks at -80 degrees C. ThioTEPA, TEPA and monochloroTEPA cummulative urinary excretion from two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples and that the excreted amount of monochloroTEPA exceeded that of thioTEPA on day 2 to 5 of urine collection.

Stability of thioTEPA and its metabolites, TEPA, monochloroTEPA and thioTEPA-mercapturate, in plasma and urine.

The degradation of N,N',N"-triethylenethiophosphoramide (thioTEPA) and its metabolites N,N',N"-triethylenephosphoramide (TEPA), N, N'-diethylene,N"-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA-mercapturate in plasma and urine has been investigated. ThioTEPA, TEPA and monochloroTEPA were analyzed using a gas chromatographic (GC) system with selective nitrogen/phosphorous detection; thioTEPA-mercapturate was analyzed on a liquid chromatography-mass spectrometric (LC-MS) system. The influences of pH and temperature on the stability of thioTEPA and its metabolites were studied. An increase in degradation rate was observed with decreasing pH as measured for all studied metabolites. In urine the rate of degradation at 37 degrees C was approximately 2.5+/-1 times higher than at 22 degrees C. At 37 degrees C thioTEPA and TEPA were more stable in plasma than in urine, with half lives ranging from 9-20 h for urine and 13-34 h for plasma at pH 6. Mono- and dichloro derivatives of thioTEPA were formed in urine and the monochloro derivative was found in plasma. Degradation of TEPA in plasma and urine resulted in the formation of monochloroTEPA. During the degradation of TEPA in plasma also the methoxy derivative of TEPA was formed as a consequence of the applied procedure. The monochloro derivative of thioTEPA-mercapturate was formed in urine, whereas for monochloroTEPA no degradation products could be detected.

Characterization of mouse glycogenin-1 cDNA and promoter region.

Glycogenin-1 is an autocatalytic, self-glucosylating protein which acts as the primer for glycogen synthesis in mammalian skeletal muscle. In this study, we have cloned the glycogenin-1 cDNA from mouse skeletal muscle. Mouse glycogenin-1 has a predicted molecular mass of 37¿ omitted¿399 Da, and the deduced amino acid sequence exhibited 87% homology with human glycogenin-1. Northern blot analysis specifically detected mouse glycogenin-1 transcript in skeletal muscle and heart, and to a lesser extent in kidney, lung and brain. 5'-RACE analysis revealed the major transcription start site to be localized 47 bp upstream of the initiation of translation codon. Sequence analysis of approximately 2 kb of the 5'-flanking region revealed potentially important regions of homology between the mouse and human glycogenin-1 promoters. Several conserved but putative elements, including a TATA box, Sp1 site, and a cyclic AMP responsive element, were observed proximal to the transcription start site. Significantly, Northern blot analysis revealed dibutyryl-cAMP treatment of cultured mouse C2C12 myotubes markedly reduced the levels of glycogenin-1 mRNA in a dose-dependent manner.

Liquid chromatographic-mass spectrometric determination of the novel, recently identified thioTEPA metabolite, thioTEPA-mercapturate, in urine.

An assay for the quantitative determination of the mercapturic acid conjugate of N,N',N"-triethylenethiophosphoramide (thioTEPA-mercapturate) in human urine has been developed. ThioTEPA-mercapturate, a recently identified metabolite of the alkylating anticancer agent thioTEPA, was analyzed using LC-MS and with direct sample injection. Sulphadiazine was used as internal standard. Linearity was accomplished in the therapeutic relevant range of 1-25 microg/ml; recovery was 84% and both accuracy and precision were less than 20% for the lower limit of quantification (1.0 microg/ml) and less than 10% for the other concentration levels. The stability of thioTEPA-mercapturate proved to be satisfactory over a period of 2 months, when kept at -80 degrees C. ThioTEPA-mercapturate urine concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples.

A search for new metabolites of N,N',N''-triethylenethiophosphoramide.

An attempt was made to unravel the metabolic profile of the alkylating agent N,N',N''-triethylenethiophosphoramide (thioTEPA). thioTEPA and its metabolite N,N',N-triethylenephosphoramide (TEPA) were quantified in urine of treated patients by gas chromatography with selective nitrogen/phosphorous detection. Total alkylating activity was assessed by p-nitrobenzylpyridine reactivity. The total alkylating activity exceeded the amount of thioTEPA and TEPA, indicating the presence of other alkylating metabolites. Solid-phase extraction and liquid-liquid extractions followed by gas chromatography-mass spectrometry analysis revealed the conversion of an aziridinyl function of TEPA into a beta-chloroethyl moiety. This metabolite, N,N'-diethylene-N''-2-chloroethylphosphoramide, was quantified by gas chromatography with selective nitrogen/phosphorous detection and accounted for only 0.69% of the administered dose. Large volumes of urine were concentrated with solid-phase extraction and fractionated with high-performance liquid chromatography. Alkylating activity was determined for each 2-ml fraction and showed the presence of an alkylating compound eluting between 8 and 12 ml. The fractions with alkylating activity were collected, evaporated under a stream of nitrogen at room temperature to dryness, reconstituted in methanol, and subjected to fast atom bombardment-mass spectrometry and fast atom bombardment-tandem mass spectrometry. A new metabolite was found with a molecular mass of 352 Da, the same as that of thioTEPA-mercapturate. thioTEPA-mercapturate is likely the result of glutathione conjugation, after which the glutathione adduct loses two amino acid residues in separate stages. The fragmentation pattern and chromatographic properties of this new metabolite were identical to those of the reference, thioTEPA-mercapturate, which was obtained by incubation of thioTEPA with N-acetylcysteine at pH 11 and 95 degrees C for 30 min. Quantification of thioTEPA-mercapturate was carried out by liquid chromatography-mass spectrometry. The thioTEPA-mercapturate levels in urine accounted for 12.3% of the administered dose and exceeded the amount of TEPA, which was previously assumed to be the main metabolite of thioTEPA. The total excreted amount of thioTEPA and its metabolites accounts for 54-100% of the total alkylating activity, indicating the presence of still other alkylating metabolites.

Characterization of the human glycogenin-1 gene: identification of a muscle-specific regulatory domain.

The de-novo synthesis of glycogen is now known to involve a novel class of self-glucosylating protein primers. In mammalian skeletal muscle, glycogenin-1 is the protein responsible for this initiation step. Northern blot analysis revealed that glycogenin-1 gene transcription is differentially regulated in the C2C12 mouse muscle cell line. To define the regulatory elements that control expression of the glycogenin-1 gene, we have cloned and characterized the genomic structure of the human glycogenin-1 gene and its promoter region. This gene consists of seven exons and six introns, and spans over 13kb. Transcription of human glycogenin-1 is initiated at two major sites, 80 and 86bp upstream from the initiation of translation codon. Nucleotide sequence analysis of 2.1kb of the 5'-flanking region revealed the proximal promoter contains both a TATA box and two putative Sp1 binding sites located in a CpG island. There are numerous binding sites for developmental and cell-type-specific transcription factors, including AP-1, AP-2, GATA, and several potential Oct 1 binding domains. There are also nine consensus E-boxes that bind the basic helix-loop-helix family of muscle-specific transcription factors. The transcriptional activity of the glycogenin-1 gene was investigated by transient transfection of the 5'-flanking region in HepG2 cells and C2C12 myoblasts and myotubes. These results permitted the definition of a minimal 232bp promoter fragment that is responsible for basal level transcription in a cell-type-independent manner. Furthermore, we have identified a regulatory region located between -2076 and -1736 of the 5'-flanking region of the human glycogenin-1 gene that allows myotube-specific expression in C2C12 cells.

Degradation study of thiotepa in aqueous solutions.

The degradation of N,N',N"-triethylenethiophosphoramide (thiotepa) in aqueous solutions has been investigated over the pH range 1-14. Samples were analyzed using a high-performance liquid chromatographic system with UV detection. The degradation kinetics were studied as a function of pH, sodium chloride concentration and temperature. The degradation of thiotepa follows pseudo first order kinetics. The pH-log kobs profile shows that thiotepa is most stable in the pH range 7-11. At pH?11 chloride has no influence on the degradation rate. The degradation products were isolated and the structures identified by mass spectrometry. Chloro adducts of thiotepa are generated in the presence of sodium chloride and in acidic medium. In the pH range 7-11 only the mono-chloro adduct of thiotepa could be found. No detectable degradation products were formed at pH?11.

Molecular characterization of virus-specific RNA produced in the brains of flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitis virus.

Natural resistance to flaviviruses in mice is controlled by a single genetic locus, FIv, on chromosome 5. Although the mechanism of this resistance is not fully understood, it is believed to operate at the level of virus replication rather than the immune response. It has been hypothesized that enhanced production of viral defective interfering (DI) particles is responsible for a substantial reduction in the titres of infectious virus in resistant mice. However, this has never been established at the molecular level since such particles have not been isolated and characterized. We have studied the products of virus replication in the brains of flavivirus-susceptible C3H/HeJ (Flv(s)) and -resistant congenic C3H/RV (Flv(r)) mice after an intracerebral challenge (i.c.) with Murray Valley encephalitis (MVE) virus and have found no evidence for the accumulation of truncated viral RNA in the brains of resistant mice. All three major viral RNA species, the replicative intermediate (RI), replicative form (RF) and virion RNA (vRNA) together with a subgenomic RNA species of 0.6 kb, which has not been previously described, were present in the brains of both mouse strains. However, the viral RF and RI RNA forms preferentially accumulated in the brains of resistant mice. Thus, we confirm that the resistance allele Flv(r) interferes with discrete steps in flavivirus replication, although the precise mechanism remains to be determined.

Synergistic effects of platelet-activating factor and other platelet agonists in human platelet aggregation and release: the role of ADP and products of the cyclooxygenase pathway.

The synergistic effects of platelet-activating factor (PAF) with ADP, collagen, thrombin, A23187, adrenaline, sodium arachidonate and ristocetin in human platelet aggregation and beta-thromboglobulin (beta-TG) release were investigated in citrated platelet-rich plasma (PRP). Synergism in both aggregation and release was present with all agonists except ristocetin. Upon oral intake of aspirin (ASA) the PAF-induced irreversible aggregation as well as the synergistic irreversible aggregation became reversible. Both prior to and after ASA ingestion ADP removal by creatine phosphate/creatine phosphokinase (CP/CPK) resulted in a reduced, reversible platelet aggregation induced by PAF alone or in combination with the other agonists. The ADP-removal and ASA-ingestion also strongly inhibited the beta-TG release. The synergistic aggregation and release were also inhibited by ASA and indomethacin in vitro as well as by the competitive ADP-inhibitor ATP. It is concluded that not only the activation of human platelets by low doses of PAF itself, but also the synergism of PAF and other platelet agonists is highly dependent upon ADP and products of the cyclooxygenase pathway.