Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.     

Zoonoses and potential zoonoses transmitted by bats

PURPOSE: The purpose of this study was to develop an analytical method for the quantitative determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF using Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFT) with Kubelka-Munk function analysis. METHODS: Carbopol 974P NF is a high molecular weight, chemically crosslinked polymer of acrylic acid, that has the C=O stretching band of the unionized carboxylic acid function at 1695 cm(-1). The quantitative determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF is based upon the asymmetrical C=O stretching of the carboxylate anion at 1570 cm(-1) measured by DRIFT spectroscopy. RESULTS: To overcome spectral differences arising from sample preparation (powders, granules and tablets) and in an effort to increase the precision of the analytical method, the following approaches were used: (1) an internal standard, (2) first derivative of the spectrum to eliminate the effect of baseline drift and (3) the ratio of the first derivative of the C=O stretch of the carboxylate anion peak (1570 cm(-1)) in the neutralized Carbopol 974P NF to that of the peak of the internal standard (866 cm(-1)). The above data treatment techniques proved to be superior to the usual methods of peak height or peak area. The calibration curve of the ratio of the first derivative (1570 cm(-1)/866 cm(-1)) was a linear function of the mass of sodium carboxylate over the range from 0.0% to 100.0% neutralization of the carboxylic acid function in Carbopol 974P NF (Fig. 1a). No particle size or sample preparation effects were noted within the experimental error. CONCLUSIONS: DRIFT spectroscopy using the Kubelka-Munk function is a powerful tool for the routine determination of the extent of neutralization of the carboxylic acid function in Carbopol 974P NF in complex pharmaceutical formulations.     

The use of oral contraceptives by adolescents for contraception, menstrual cycle problems or acne

BACKGROUND: Oral contraceptives are prescribed as contraception but also as therapy for menstrual cycle disturbances and acne. We studied the prevalence of oral contraceptive (OC) use and the indications to start OC use among adolescents. METHODS: A cohort consisting of ninth grade secondary school girls (mean age 15.3+/-0.6 (s.d.) years) answered a questionnaire on their menstrual cycle. OC users were asked about duration and reasons for OC-use and the name of the preparation they used. The influence of calendar age, gynecological age and level of education on the prevalence of OC was studied by multiple logistic regression analysis. RESULTS: The response on the questionnaire was 92%. Of 2248 responders 248 (11%) used oral contraceptives: 74% used low dose 'sub 50' preparations, 3% pills with 50 microg estrogen, 3% tri-phase preparations and 17% pills with antiandrogens. Of girls aged 14, 15 and 16 years 4%, 12% and 28% respectively, used OC. Of the 15-year-olds 31% mentioned contraception as most important reason for OC use, 18% menstrual cycle irregularity, 26% dysmenorrhea, 10% acne and 5% other reasons. Calendar age, gynecological age and level of education were independent variables for OC use in general and for OC use for contraception or dysmenorrhea, but less so for OC use for menstrual cycle irregularity or acne. CONCLUSIONS: During adolescence low dose OC's were frequently used. In The Netherlands OC use among girls aged 15 and 16 years doubled in comparison with 1982. One third of the adolescent OC-users mentioned contraception as most important reason to start OC. Gynecological age (a determinant of biological maturation), calendar age (a determinant of biological maturation and lifestyle in peer groups), and level of education (a determinant of lifestyle in peer groups) were associated with OC use.     

Simultaneous assessments of exocrine pancreatic function by cholesteryl-[14C]octanoate breath test and measurement of plasma p-aminobenzoic acid

Two noninvasive tests for assessing pancreatic exocrine function, the cholesteryl-[14C]octanoate breath test and the HPLCN-benzoyl-tyrosyl-p-aminobenzoic acid/p-aminosalicylic acid (NBT-PABA/PAS) test, were simultaneously performed in nine patients with pancreatic exocrine insufficiency due to chronic pancreatitis and in nine healthy volunteers. 14CO2 output in breath and plasma PABA concentration rose slowly in patients but increased rapidly in healthy subjects. The measurement time giving the best discrimination between both groups was 120 min for the cholesteryl-[14C]octanoate breath test and 90 min for the plasma PABA test. At these points, both single-sample tests had essentially identical diagnostic sensitivity. The diagnostic sensitivities of the two single-sample tests were equal to that of the cumulative 6-h urinary PABA recovery and the cumulative 6-h urinary PABA/PAS ratio. We conclude that, for both the cholesteryl-[14C]octanoate breath test and the plasma PABA test, a single test sample is sufficient for rapid detection of impaired exocrine pancreatic function.     

The impact of treatment time and smoking on local control and complications in T1 glottic cancer

Low frequency impedance measurements of pure egg lecithin (phosphatidylcholine) bilayers have revealed the presence of four layers which can be attributed to the acyl chain, carbonyl, glycerol bridge and phosphatidylcholine regions of the lecithin molecule. Measurements on bilayers formed in the presence of unoxidised-cholesterol revealed that cholesterol molecules were located in the hydrocarbon region of the bilayer with its hydroxyl groups aligned with the carbonyl region of the lecithin molecules. Measurements of oxidised-cholesterol lecithin bilayers revealed that these molecules protruded less into the hydrocarbon region and their polar hydroxyl group aligned with the glycerol bridge region of the lecithin molecule.     

Psychopathology in young adults with congenital heart disease. Follow-up results

Non-steroidal anti-inflammatory drugs inhibit constitutive (COX-1) and induced cyclooxygenase (COX-2), blocking prostaglandin production. We have compared the effects on nociceptive reflexes of meloxicam, which is COX-2 selective, with indomethacin, which is non-selective, using an in vitro spinal cord preparation. Cords were taken from naive rats, and from rats with carrageenan-induced hyperalgesia of one hindpaw. Reflex thresholds were lower in carrageenan preparations. Superfusion with meloxicam (10-100 microM) dose-dependently inhibited baseline reflexes and wind-up in normal and carrageenan preparations, whereas indomethacin (100-300 microM) had no effect. Thus meloxicam inhibits spinal reflexes, whereas indomethacin does not, despite its high affinity for both COX isoforms. We conclude that meloxicam has spinal antinociceptive actions which cannot be explained by the current concept of COX inhibition.     

Evidence that C1q binds specifically to CH2-like immunoglobulin gamma motifs present in the autoantigen calreticulin and interferes with complement activation

Calreticulin (CRT) is located predominantly in the endoplasmic reticulum (ER) of cells, where it functions as a quality control controller of protein folding. However, CRT is also a prevalent autoantigen in patients with systemic lupus erythematosus (SLE), where its release from the cell may arise as a results of dysfunctional apoptosis and inefficient removal of ER vesicles, which are an abundant source of CRT and other autoantigens. Indicative of this is the presence of autoantibodies against CRT in the sera of 40-60% of all SLE patients. Once released into the circulation, CRT might bind directly to C1q and we have suggested that this association may result in a defect in C1q-mediated clearance of antigen-antibody complexes. It has been previously shown that CRT under physiological salt conditions binds to the globular head of C1q. It is known that the globular head region of C1q binds to the CH2 domain in the Fc portion of immunoglobulin gamma (IgG). The N-terminal half of CRT contains a number of short regions of 7-10 amino acids that show sequence similarity to the putative C1q binding region in the CH2 domain of IgG. By use of a series of 92 overlapping CRT synthetic peptides, a number of C1q binding sites on the CRT molecule have been identified, including several containing a CH2-like motif similar to the ExKxKx C1q binding motif found in the CH2 domain of IgG. A number of these peptides were shown to inhibit binding of C1q to IgG and reduce binding of native CRT to C1q. Moreover, several of the peptides were capable of inhibiting the classical pathway of complement activation. These studies have identified specific binding sites on the CRT molecule for C1q and lend support to the hypothesis that interaction of CRT with C1q may interfere with the ability of C1q to associate with immune complexes in autoimmune-related disorders.     

A phase I/II trial of transurethral surgery combined with concurrent cisplatin, 5-fluorouracil and twice daily radiation followed by selective bladder preservation in operable patients with muscle invading bladder cancer

PURPOSE: We describe a protocol designed to evaluate the use of twice daily radiation used together with cisplatin and 5 fluorouracil (5-FU) in the treatment of operable transitional cell carcinoma of the bladder with potential bladder preservation. MATERIALS AND METHODS: A total of 18 consecutive patients with T2-T4a bladder tumors underwent as complete a transurethral resection as possible, which was visibly complete in 14 cases. They then received twice daily radiation and infusion cisplatin and 5-FU during an induction phase. No therapy was given for 3 weeks, following which patients were reevaluated cystoscopically. Cases of clinical complete response by biopsy and cytology were consolidated with further chemotherapy/radiation using the same chemotherapeutic agents and radiation schedule. Patients who had incomplete responses were advised to undergo an immediate radical cystectomy. Of the 18 patients 15 subsequently received 3 cycles of adjuvant chemotherapy, consisting of methotrexate, cisplatin and vinblastine. Median followup for the entire group is 32 months. RESULTS: Of the 18 patients 14 had no detectable tumor after induction therapy. Of the 4 patients with persistent tumor 2 underwent radical cystectomy and 2 refused cystectomy, 1 of whom was treated with partial cystectomy and the other with consolidation chemotherapy/radiation. The actuarial overall survival at 3 years was 83%. The chance of a patient being alive at 3 years with a native bladder was 78%. No patient required cystectomy for hematuria or bladder shrinkage. Three patients in whom superficial tumors developed were treated successfully with bacillus Calmette-Guerin. Small bowel obstruction in 1 case was corrected surgically. CONCLUSIONS: This pilot study demonstrates a high rate of response to this combined chemotherapy/radiation regimen in conjunction with a visibly complete transurethral resection. Reevaluation after a short induction phase allows for the early selection of patients with persistent disease for radical cystectomy.