Successful second hematopoietic cell transplantation in severe congenital neutropenia.

Allogeneic HCT is curative for SCN; however, a standard conditioning regimen or intensity has not been established. We describe a patient with SCN associated with c.1A>G (M1V) mutation in ELANE gene resulting in refractoriness to G-CSF, who received reduced-intensity HCT and developed secondary graft failure requiring a second myeloablative HCT. This case suggests that M1V mutation confers a poor G-CSF response and HCT using the best available donor is beneficial.

Characterization of the solubility of a poorly soluble hydroxylated metabolite in human urine and its implications for potential renal toxicity.

The solubility, in human urine, of the major hydroxylated metabolite (M1) of an experimental cognition enhancer was characterized through a series of in vitro experiments in an effort to estimate the probability of crystalluria occurring following oral administration of the parent compound. The aim of these experiments was to determine if a safety margin existed between clinically observed urine concentrations and the solubility of M1. The mean urine concentrations of M1 in young and elderly subjects following oral administration of the parent compound at the highest doses tested, were 4865 +/- 2368 ng/mL and 2764 +/- 791 ng/mL, respectively. In vitro solubility experiments with M1 were conducted in drug-free human urine (37 degrees C) from four male and four female healthy subjects under conditions of high and low urine osmolality. Mean concentrations (n = 16) of M1 in human urine to which solid M1 was added, were 3656 +/- 621 ng/mL, 4678 +/- 1169 ng/mL and 5378 +/- 2474 ng/mL after stirring for 24, 48 and 72 h, respectively, indicating that the ex vivo mean solubility of M1 in human urine is no greater then approximately 5 microg/mL. Addition of solid M1 to urine from human subjects dosed with the parent compound resulted in mean urine M1 concentrations 23.5% greater than those observed in vivo. The results from both experiments indicated a significant overlap between urine concentrations of M1 in vivo following the highest oral administration of the parent drug and M1 solubility measured in vitro, suggesting a high potential for in vivo saturation of urine with M1 with subsequent precipitation, crystalluria, and nephrotoxicity. Consequently, the results of these studies have placed restrictions on the dose that could be administered during clinical development of this compound.

Central administration of nicotine suppresses tracheobronchial cough in anesthetized cats.

We tested the hypothesis that nicotine, which acts peripherally to promote coughing, might inhibit reflex cough at a central site. Nicotine was administered via the vertebral artery [intra-arterial (ia)] to the brain stem circulation and by microinjections into a restricted area of the caudal ventral respiratory column in 33 pentobarbital anesthetized, spontaneously breathing cats. The number of coughs induced by mechanical stimulation of the tracheobronchial airways; amplitudes of the diaphragm, abdominal muscle, and laryngeal muscles EMGs; and several temporal characteristics of cough were analyzed after administration of nicotine and compared with those during control and recovery period. (-)Nicotine (ia) reduced cough number, cough expiratory efforts, blood pressure, and heart rate in a dose-dependent manner. (-)Nicotine did not alter temporal characteristics of the cough motor pattern. Pretreatment with mecamylamine prevented the effect of (-)nicotine on blood pressure and heart rate, but did not block the antitussive action of this drug. (+)Nicotine was less potent than (-)nicotine for inhibition of cough. Microinjections of (-)nicotine into the caudal ventral respiratory column produced similar inhibitory effects on cough as administration of this isomer by the ia route. Mecamylamine microinjected in the region just before nicotine did not significantly reduce the cough suppressant effect of nicotine. Nicotinic acetylcholine receptors significantly modulate functions of brain stem and in particular caudal ventral respiratory column neurons involved in expression of the tracheobronchial cough reflex by a mecamylamine-insensitive mechanism.

In-vitro metabolism of celecoxib, a cyclooxygenase-2 inhibitor, by allelic variant forms of human liver microsomal cytochrome P450 2C9: correlation with CYP2C9 genotype and in-vivo pharmacokinetics.

In-vitro studies were conducted to assess the impact of CYP2C9 genotype on the metabolism (methyl hydroxylation) and pharmacokinetics of celecoxib, a novel cyclooxygenase-2 inhibitor and CYP2C9 substrate. When compared to cDNA-expressed wild-type CYP2C9 (CYP2C9*1), the Vmax/Km ratio for celecoxib methyl hydroxylation was reduced by 34% and 90% in the presence of recombinant CYP2C9*2 and CYP2C9*3, respectively. These data indicated that the amino acid substitution at position 359 (Ile to Leu) elicited a more pronounced effect on the metabolism of celecoxib than did a substitution at position 144 (Arg to Cys). The Vmax/Km ratio was also decreased in microsomes of livers genotyped CYP2C9*1/*2 (47% decrease, mean of two livers), or CYP2C9*1/*3 (59% decrease, one liver). In all cases, these changes were largely reflective of a decrease in Vmax, with a minimal change in Km. Based on simulations of the in-vitro data obtained with the recombinant CYP2C9 proteins, it was anticipated that the pharmacokinetics of celecoxib (as a much as a five-fold increase in plasma AUC) would be altered (versus CYP2C9*1/*1 subjects) in subjects genotyped heterozygous or homozygous for the CYP2C9*2 (Cys144) or CYP2C9*3 (Leu359) allele. In a subsequent clinical study, the AUC of celecoxib was increased (versus CYP2C9*1/*1 subjects) approximately 2.2-fold (range, 1.6-3-fold) in two CYP2C9*1/*3 subjects and one CYP2C9*3/*3 subject receiving a single oral dose (200 mg) of the drug. In contrast, there was no significant change in celecoxib AUC in two subjects genotyped CYP2C9*1/*2.

Dealing with doubt. How patients account for non-specific chronic low back pain.

OBJECTIVE: To explore the ways that persons with long standing chronic low back pain respond to the problem of medical doubt about the presence of organic pathology. METHOD: Qualitative analysis of accounts provided by 12 persons attending a back pain rehabilitation clinic in NW England. RESULTS: Subjects rejected the notion that they were culpable for their pain. They were not culpable for the onset of their pain. They argued that despite their cooperation, no sensible explanation of their pain was forthcoming from health professionals. Finally, they asserted that medical scepticism had been damaging and dispiriting. CONCLUSION: Patients dealt with clinical doubt by stressing their own expertise. They constituted their beliefs about the cause and trajectory of their pain and disability as accurate accounts of their disability. They resisted the suggestion that there might be psychological factors involved in their ill-health by locating culpability among clinicians, who were confused or uncertain about diagnosis and treatment.

Simultaneous determination of unlabeled and carbon-13-labeled etoricoxib, a new cyclooxygenase-2 inhibitor, in human plasma using HPLC-MS/MS.

A method for the simultaneous determination of etoricoxib and its carbon-13 analog ((13)C(6)-etoricoxib) from human plasma has been developed and used to support bioavailability studies. Plasma samples (0.5 mL) were extracted by using a 3M Empore 96-well plate (C(8)) and the resulting extracts were analyzed by using a PE-Sciex API-3000 HPLC-MS/MS with a heated nebulizer interface (500 degrees C). The method was validated with two different calibration curve ranges, one for etoricoxib (5 to 2500 ng/mL) determined in the presence of lower concentrations of (13)C(6)-etoricoxib (0.5 to 250 ng/mL), and a second curve for the quantitation of similar concentrations of both etoricoxib and (13)C(6)-etoricoxib (0.5 to 250 ng/mL). Extraction recoveries of etoricoxib, (13)C(6)-etoricoxib, and a methylated internal standard were >70% over the range of concentrations included in both calibration curves. Intraday precision and accuracy for the quantitation of etoricoxib were 7.8% relative standard deviation (RSD) or less and within 3.4% respectively over the range of 5 to 2500 ng/mL, and 10.8% RSD or less and within 4 % respectively over the range of 0.5 to 250 ng/mL. Within-batch precision and accuracy for the quantitation of (13)C(6)-etoricoxib over the range of 0.5 to 250 ng/mL were 8.3% RSD or less and within 2.3%, respectively. The validated assay was used in support of human clinical trials.

An application of the fear avoidance model to three chronic pain problems.

The Fear Avoidance Model of Exaggerated Pain Perception was developed in an attempt to explain how, and why, some individuals develop a more substantial psychological overlay to their low back pain problem than do others. The present paper describes a study in which three chronic pain groups, consisting of Post-Herpetic neuralgia patients, Reflex Sympathetic Dystrophy patients and chronic low back pain patients were compared with three pain-free comparison groups using the Fear Avoidance Model of Exaggerated Pain Perception. The results show statistically significant differences between the chronic groups and the recovered comparison groups. These results demonstrate the usefulness of the Fear Avoidance Model as an explanation of psychological overlay in chronic pain conditions regardless of pathology.

Implementation of RCGP guidelines for acute low back pain: a cluster randomised controlled trial.

BACKGROUND: The Royal College of General Practitioners (RCGP) has produced guidelines for the management of acute low back pain in primary care. AIM: To investigate the impact on patient management of an educational strategy to promote these guidelines among general practitioners (GPs). DESIGN OF STUDY: Group randomised controlled trial, using the health centre as the unit of randomisation. SETTING: Primary care teams in north-west England. METHOD: Twenty-four health centres were randomly allocated to an intervention or control arm. Practices in the intervention arm were offered outreach visits to promote national guidelines on acute low back pain, as well as access to fast-track physiotherapy and to a triage service for patients with persistent symptoms. RESULTS: Twenty-four centres were randomised. Two thousand, one hundred and eighty-seven eligible patients presented with acute low back pain during the study period: 1049 in the intervention group and 1138 in the control group. There were no significant differences between study groups in the proportion of patients who were referred for X-ray, issued with a sickness certificate, prescribed opioids or muscle relaxants, or who were referred to secondary care, but significantly more patients in the intervention group were referred to physiotherapy or the back pain unit (difference in proportion = 12.2%, 95% confidence interval [CI] = 2.8% to 21.6%). CONCLUSION: The management of patients presenting with low back pain to primary care was mostly unchanged by an outreach educational strategy to promote greater adherence to RCGP guidelines among GPs. An increase in referral to physiotherapy or educational programmes followed the provision of a triage service.

The prediction of chronicity in patients with an acute attack of low back pain in a general practice setting.

STUDY DESIGN: Three hundred patients, attending their general practitioners with attacks of acute low back pain, formed the subject population for a study of fear avoidance and other variables in the prediction of chronicity. Follow-up was at 2 and 12 months. OBJECTIVE: The hypothesis to be tested was that evidence of psychological morbidity, particularly fear-avoidance behavior, would be manifest from the outset of the presenting attack in susceptible subjects. SUMMARY OF BACKGROUND DATA: While back pain is an almost universal human experience, only about 5% of sufferers seek medical advice. Most of these respond to conservative treatment. However, approximately 10% of those who experience an acute attack of low back pain go on to become chronic sufferers. METHODS: Psychosocial and physiological data (including fear-avoidance measures) were collected from a sample of 300 acute low back pain patients within 1 week of presentation and at 2 months, to try to predict 12 month outcome. RESULTS: Data analysis showed that subjects who had not recovered by 2 months were those who went on to become chronic low back pain patients (7.3%). Using multiple regression analyses, fear-avoidance variables were the most successful in predicting outcome. Using multiple discriminant function analyses, the results suggest that the outcome in terms of the future course of low back pain can be correctly classified in 66% from fear-avoidance variables alone and in 88% of patients from all variables. CONCLUSIONS: The results suggest that, at the earliest stage of low back pain, fear of pain should be identified by clinicians and, where this is severe, pain confrontation should arguably form part of the approach to treatment.

Chronic low back pain rehabilitation programs: a study of the optimum duration of treatment and a comparison of group and individual therapy.

STUDY DESIGN: Eighty-four patients with chronic low back pain were treated using cognitive behavioral principles on a pain management program. Outcome data were collected at four points: 10 weeks before treatment, immediately before and immediately after treatment, and 6 months after treatment. In part 1 of the study, patients were assigned randomly to group or individual treatment contexts. In part 2 of the study, patients were assigned randomly to programs of 15, 30, or 60 hours duration. OBJECTIVES: To identify the differences in outcome between programs that treated patients as part of a group and those that treated patients individually and the effects of duration of treatment on outcome. SUMMARY OF BACKGROUND DATA: Cognitive behavioral programs have been shown to be an effective means of managing chronic low back pain. The literature is concerned with group programs, however, the duration of which vary widely. METHOD: Psychological and functional variables were measured before and after treatment and at the 6-month follow-up visit. Changes in these variables were measured, and comparisons were made between group and individual programs and between 15-, 30-, and 60-hour programs. RESULTS: Data analysis showed a significant, beneficial effect of intervention in terms of the majority of variables; however, these changes were generally independent of whether patients were treated as part of a group or individually and whether patients completed a 15-, 30-, or 60-hour program. CONCLUSIONS: Cognitive behavioral rehabilitation programs have been demonstrated to be an effective means of reducing psychological distress, of changing cognition, and of improving the function of patients with chronic low back pain; however, the length of program and whether patients were treated individually or as part of a group did not affect outcome. This finding has clinical and economic implications.

Selective fluorogenic derivatization of a peptide nucleic acid trimer with naphthalene-2,3-dicarboxaldehyde.

The reversed-phase high-performance liquid chromatography of a Peptide Nucleic Acid (PNA) trimer has been studied after its preseparation fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA/CN). Trace levels of the PNA trimer were determined in cell homogenate samples containing the PNA trimer at prederivatization concentrations as low as 48.9 ng ml-1. The sample pretreatment operations included a deproteination step, achieved by ultra-filtration, followed by fluorogenic derivatization (NDA/CN). Subsequently, to achieve adequate selectivity, the fluorescently labeled PNA was subjected to high performance anion exchange chromatography prior to quantitation via fluorescence detection. The various problems encountered during sample pretreatment and separation of derivatized PNA trimer in biological samples are presented and discussed.

High-throughput simultaneous determination of the HIV protease inhibitors indinavir and L-756423 in human plasma using semi-automated 96-well solid phase extraction and LC-MS/MS.

A method for the simultaneous determination of the HIV protease inhibitors indinavir and L-756423, in human plasma has been developed. Plasma samples (0.5 ml) were extracted using a 3M Empore 96-well plate in the mixed phase cation exchange (MPC) format. The extraction method was automated through the application of both the Packard 204DT and TOMTEC Quadra 96 work stations, and the resulting extracts were analyzed using a PE-Sciex API-3000 LC-MS/MS with a heated nebulizer interface (500 degrees C). The assay was linear in the concentration range 1-2500 ng/ml for indinavir and 5 2500 ng/ml for L-756423 when 0.5-ml aliquots of plasma were extracted. Recoveries of indinavir and L-756423 were greater than 76 and 80%, respectively, over the calibration curve range when using the described sample preparation method. Within-batch precision and accuracy for the quantitation of indinavir over the range 1-2500 ng/ml were 5.4% R.S.D. or less and within 4.0%, respectively. Within-batch precision and accuracy for the quantitation of L-756423 over the range 5-2500 ng/ml were 5.3% R.S.D. or less and within 3.4%, respectively. Interbatch variability for the analysis of indinavir QC samples at low (3 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) were 3.2, 2.9, and 1.9%, respectively. Interbatch variability for the analysis of L-756423 QC samples at low (15 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) concentration were 2.0, 2.5, and 3.3%, respectively. The validated assay was used in support of human clinical trials.

Diagnosis and reconstruction of the human temporomandibular joint after trauma or internal derangement.

This study reviewed the standardized records of 1100 patients with the symptoms of temporomandibular joint syndrome. Of these patients, only 4.5 percent required surgical intervention. The remaining patients were found to have masticatory muscle spasm and were treated by conservative dental methods. Over half the surgical patients had significant macrotrauma to the jaws in their past history. In addition, electromyographic measurement of the masseteric silent period duration in these patients did not reveal muscle spasm. These factors further serve to differentiate the surgical patient from the patient with myofascial pain dysfunction. The patients selected for surgery demonstrated moderate to severe joint disease and required arthroplasty with partial meniscectomy. A surgical technique is presented demonstrating the reconstruction of the meniscus with silicone implant. This same surgical technique is studied in 10 monkeys, and their joints are examined histologically. The results of surgery reveal that 87 percent of the patients reported improvement 1 year after surgery. In all patients complaining of temporomandibular joint clicking or crepitus, surgery produced complete alleviation of these symptoms. The results of surgery were also associated with a 62 percent increase of jaw opening. Histologic evaluation of the human meniscal resections revealed that in addition to an anatomic displacement of the meniscus, there are also significant cellular changes. These changes consisted of calcification, a decrease in cellularity, hyperemia, and a decrease in elastin content.

Keyboard operating posture and actuation force: implications for muscle over-use.

A substantial proportion of the problems in keyboard operator 'Over-use Syndromes' occur in the wrist and finger extensor muscle group. Biomechanical analysis shows these muscles to be subject to substantial sustained static (isometric) muscle contraction during the work task. This study measured the maximum relaxed finger press forces for 60 subjects in three arm support methods, in order to predict what the minimum keypress force should be to permit finger support sufficient to facilitate relaxation of finger extensor muscles. It was postulated that the minimum key activation force should accommodate the 95 percentile predicted population relaxed finger weights. The predicted force of 0.8 newton is within limits previously proposed for performance criteria, and which have been found practical commercially.

Development and validation of a method for the determination of a therapeutic peptide with affinity for the human B1 receptor in human plasma using HPLC-MS/MS.

The peptide described in this report (MW 1180 Da; 10-amino acid synthetic peptide) is a potent and selective antagonist of the human B1 receptor (B1) that has been investigated for the treatment of chronic pain. A method to quantitate this peptide in human plasma has been developed to support human clinical trials designed to evaluate the safety, pharmacokinetics, and efficacy of this compound. Plasma samples (0.2 mL) were extracted using a Waters Oasis MAX (10 mg) 96-well plate and the resulting samples were analyzed using an Applied Biosystems API-5000 HPLC-MS/MS with an electrospray ionization (ESI) source. The method was validated for the determination of the B1 peptide in human plasma over the concentration range of 1-50 ng/mL. Isotopically labeled B1 peptide ((13)C6(15)N(2)-B1 peptide) was used as an internal standard. Interday precision and accuracy, determined from analysis of quality control (QC) samples, yielded coefficients of variation (CV) of less than 5.3% and accuracy within a 2.4%. Within batch precision and accuracy determinations provided CV values of less than 7.3% and accuracy within a 6.0% bias. Precautions had to be taken to prevent B1 peptide loss to container surfaces and contamination of the HPLC-MS/MS. The validated assay was used in support of human clinical trials.

Determination of prednisolone in human adipose tissue incubation medium using LC-MS/MS to support the measurement of 11beta-hydroxysteroid dehydrogenase activity.

A method for the determination of prednisolone in human adipose tissue incubation medium has been developed, validated and used to support studies designed to measure the activity of 11beta-hydroxysteroid dehydrogenase in human adipose tissue. After incubation, samples (80 microL) were extracted using Oasis HLB microElute SPE plates and the resulting extracts were analyzed using reversed-phase chromatography coupled to an Applied Biosystems Sciex PE API-4000 mass spectrometer with a TurboIonSpray interface (400 degrees C). The method was validated over the calibration range of 0.5-100 ng/mL. Intraday precision and accuracy were 6.1% R.S.D. or less and within 6.3%, respectively. Interday precision and accuracy were 4.2% R.S.D. or less and within 3.6%, respectively. Extraction recovery of prednisolone was greater than 84% over the range of low to high quality control sample concentrations. The validated assay was used to support studies designed to estimate ex vivo 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme activity in human adipose tissue.

Influence of microinjections of D,L-homocysteic acid into the Botzinger complex area on the cough reflex in the cat.

Microinjections of D,L-homocysteic acid (DLH) were used to test the hypothesis that neuronal activation within the Botzinger complex area can modify the spatiotemporal characteristics of the cough reflex in 17 spontaneously breathing pentobarbitone anesthetized cats. DLH (50 mM, 1.25-1.75 nmol, 9 cats) reduced the number (P<0.01) of coughs and expiratory amplitude of abdominal electromyographic activity (P<0.01), and also esophageal pressure (P<0.001) during mechanically induced tracheobronchial cough. The duration of cough abdominal activity was shortened by 48% (P<0.05). DLH microinjections also temporarily reduced the respiratory rate (P<0.01) and increased the mean arterial blood pressure (P<0.001), baseline of esophageal pressure (P<0.01), and end tidal CO(2) concentrations (P<0.01). Lower doses of DLH (0.27-0.35 nmol, 7 cats) or vehicle (25-35 nl, 8 cats) induced few alterations in cardiorespiratory or cough characteristics. The results support predominantly inhibitory effects of neurons in the region of the Bötzinger complex on cough abdominal activity and cough number.