Sensitive derivatization methods for the determination of genotoxic impurities in drug substances using hyphenated techniques.

Six sensitive derivatization methods for the determination of genotoxic impurities in selected drug substances were developed using hyphenated techniques. Some of the raw materials, reagents and reaction intermediates of the selected drug substances were identified as genotoxic impurities through DEREK software for windows. The genotoxic impurities which are amenable for derivatization were selected as substrates. Derivatizing agents were selected based on the functional groups of the genotoxic impurities. The chemistry involved in the derivatization was explained with suitable mechanisms. An appropriate hyphenated technique viz. LC-MS and GC-MS was opted based on the sensitivity and aromaticity of the derivatized genotoxic impurities. All the methods were validated as per International Conference on Harmonization guidelines. Correlation coefficient values were found about 0.99. The obtained % R.S.D values from replicate injections in the range of 2.3-4.8 and % recoveries of the added impurities in the range of 83.7-101.7 ensured the precision and accuracy, respectively.

Improving postgraduate clinical assessment tools: the introduction of video recordings to assess decision making.

BACKGROUND: Competency in the management of acutely unwell patients has not previously been formally assessed as part of an objective structured clinical examination (OSCE). AIM: The reliability of the paediatric postgraduate OSCE was calculated. An objective structured video examination was designed to assess candidates' clinical decision making ability when managing acutely unwell children. METHODS: The performance of 3522 postgraduate paediatric trainees was assessed (2006-2008). OSCE reliability was determined using Cronbach's alpha and mean inter-item correlation. Performance in the video station was compared with overall performance (not including video station; Mann-Whitney U) and video station scores correlated with individual station scores (Spearman's Rho correlation coefficient). RESULTS: Clinical examination pass rates for the 684 UK graduates, 1608 overseas candidates training in the UK and 1104 overseas candidates training overseas were 69.7%, 28% and 22.3%, respectively (graduation information not available for 126 candidates). Cronbach's alpha was 0.62. Mean inter-item correlation was 0.15. Candidates who passed the OSCE overall had significantly higher scores on the video station (t(3520) = 14.48); p < 0.001). There was significant positive correlation between scores on the video station, individual stations and overall total score (r's = 0.300; p = 0.001). CONCLUSIONS: The postgraduate paediatric OSCE provides a sound and valid means of assessing clinical skills at the postgraduate level. The video station provides an important new method of assessment. Its use in other postgraduate clinical examinations should be explored.

Development and validation of stability-indicating HPLC and UPLC methods for the determination of bicalutamide.

Six new process related impurities (Imp-08, Imp-09, Imp-10, Imp-12, Imp-13 and Imp-14) of bicalutamide (BCT) have been reported in this paper. BCT was subjected to oxidative, acid, alkaline, hydrolytic, thermal and photolytic degradation conditions and found to degrade in alkaline condition, yielding Imp-11. Stability-indicating high-performance liquid chromatography and ultra-performance liquid chromatography methods were developed for the determination of BCT in the presence of its 14 process-related impurities and 1 degradant by using Zorbax SB phenyl column (150 × 4.6 mm × 3.5 µm) and HSS T3 column (100 × 2.1 mm × 1.8 µm), respectively. Both the methods were validated as per International Conference on Harmonization guidelines. Quantitation limits (QL) were found be in the ranges of 0.02-0.03% for both the methods. Precision was evaluated by replicate analysis in which % relative standard deviation (RSD) values for areas were found below 2.0. Linearity for the impurities was established in the range of QL to 200% of the specification level and the correlation coefficients derived from of the respective calibration curves were approximately 0.999. The recoveries obtained for purity (90-100%) and assay (98-102%) ensured the accuracy of the developed methods.

Langerhans cell histiocytosis.

Langerhans cell histiocytosis is a rare disease. Depending on which organs are involved, the disease may prove rapidly fatal, develop a chronic reactivating but therapy-responsive pattern or resolve spontaneously. Understanding of the pathology of the disease is progressing rapidly, and while clinical trials of standard chemotherapy agents continue, it is likely that novel targeted therapy will become feasible in the next decade. Permanent consequences of the disease are more commoner than generally realised.

Development and validation of LC methods with visible detection using pre-column derivatization and mass detection for the assay of voglibose.

Two sensitive and selective liquid chromatographic methods were developed for the assay of voglibose (VB) and validated as per International Conference on Harmonization (ICH) guidelines. First method is based on the pre-column derivatization of VB followed by visible detection (LC-VD) and second method involves mass spectrometric detection (LC-MS). In LC-VD method, VB was derivatized with sodium metaperiodate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH). The derivatized color product of VB (DCPVB) was run through Novapak C18 (300 x 3.9 mm, 4 microm) column using the mobile phase containing buffer (0.01 M mixture of sodium di hydrogen orthophosphate and disodium hydrogen orthophosphate, pH 6.0) and acetonitrile in 35:65 v/v ratio. The eluted DCPVB was monitored at 667 nm. The fixation of optimum conditions in LC-VD method is described. DCPVB structure was confirmed by mass spectral analysis. In LC-MS method, VB was passed through Venusil XBPPH (150 x 4.6 mm, 5 microm) column using a 95:5 v/v mixture of 0.01% formic acid and methanol as mobile phase. The assay concentrations of VB in pure form and in tablets for LC-VD and LC-MS methods are 25 and 5 ngml(-1), respectively.

Incidence and clinical features of Langerhans cell histiocytosis in the UK and Ireland.

OBJECTIVES: There are few published studies on the epidemiology of Langerhans cell histiocytosis (LCH). We undertook a survey to ascertain all newly diagnosed cases aged 0-16 years in the UK and Republic of Ireland. DESIGN: Three methods of ascertainment were used: the British Paediatric Surveillance Unit (BPSU) system, a survey by Newcastle University, and the Children's Cancer and Leukaemia Group (CCLG) registry. Deaths data were obtained from the UK Office for National Statistics and the Central Statistics Office in Ireland. Clinicians who reported cases were sent a questionnaire to obtain demographic and clinical details. RESULTS: Over the 2-year period, 94 cases were identified. The age-standardised incidence rate of LCH in children aged 0-14 years was 4.1 per million per year. The sex ratio (M:F) was 1.5:1 and the median age at diagnosis was 5.9 years. Single system disease (predominantly bony involvement) accounted for 73% of cases and 27% had multisystem disease of whom 7% had involvement of "risk organs" (liver, lung, spleen and bone marrow). Three children died, two of whom were diagnosed after death. CONCLUSIONS: This is the first study of LCH to use an active surveillance method with additional sources of ascertainment. Our incidence is comparable with those in other national reports, although it is likely to be an underestimate as each method may have missed some cases, either diagnosed or undiagnosed.

Biochemical approaches to the synthesis of ethyl 5-(s)-hydroxyhexanoate and 5-(s)-hydroxyhexanenitrile.

Three different biochemical approaches were used for the synthesis of ethyl 5-(S)-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2. In the first approach, ethyl 5-oxo-hexanoate 3 and 5-oxo-hexanenitrile 4 were reduced by Pichia methanolica (SC 16116) to the corresponding (S)-alcohols, ethyl (S)-5-hydroxyhexanoate 1 and 5-(S)-hydroxyhexanenitrile 2, with an 80-90% yield and >95% enantiomeric excess (e.e). In the second approach, racemic 5-hydroxyhexanenitrile 5 was resolved by enzymatic succinylation, leading to the formation of (R)-5-hydroxyhexanenitrile hemisuccinate and leaving the desired alcohol 5-(S)-hydroxyhexanenitrile 2 with a yield of 34% (50% maximum yield) and >99% e.e. In the third approach, enzymatic hydrolysis of racemic 5-acetoxy hexanenitrile 6 resulted in the hydrolysis of the R-isomer to provide 5-(R)-hydroxyhexanenitrile, leaving 5-(S)-acetoxyhexanenitrile 7 with a 42% yield (50% maximum yield) and >99% e.e.

Growth and endocrine disorders in multisystem Langerhans' cell histiocytosis.

INTRODUCTION: Langerhans' cell histiocytosis is a rare disorder, with diabetes insipidus occurring in up to half of patients. Causes of growth failure include the illness itself, treatments used and growth hormone insufficiency. PATIENTS AND METHODS: We identified all patients with an endocrinopathy secondary to Langerhans' cell histiocytosis (LCH). Growth data were analysed from all patients with multisystem involvement. RESULTS: Of 144 patients with multisystem LCH, 50 had an endocrinopathy, 49 of whom had diabetes insipidus. Growth hormone insufficiency (GHI) was present in 21 patients, seven of whom had other anterior pituitary deficiencies as well (gonadotrophin deficiency + GHI n = 2, gonadotrophin deficiency + TSH deficiency + GHI n = 2, panhypopituitarism n = 3). GH insufficiency, the development of which appeared to be independent of pituitary radiation, occurred at a median age of 8.3 years (4.7-18 years) and at a median interval of 3.5 years (0-11.8 years) after diagnosis of LCH. The median height SDS at diagnosis of growth hormone insufficiency was -2.9. Thirteen of the patients with growth hormone insufficiency attained final height with a median height SDS of -1.2. The final height SDS of 15 patients without GH insufficiency was closer to target height SDS, but not statistically different from that of the GH insufficient group. CONCLUSIONS: GH therapy significantly improves growth in GH insufficient patients with Langerhans' cell histiocytosis. Early institution of GH therapy may further improve height outcome. However, most children with Langerhans' cell histiocytosis regardless of endocrine function, failed to reach target height.

Enzymatic acetylation of 10-deacetylbaccatin III to baccatin III by C-10 deacetylase from Nocardioides luteus SC 13913.

Baccatin III is a polycyclic diterpene which can be used for the semi-synthesis of paclitaxel and analogs. An enzymatic process was developed for the conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III without requiring protection of the 7-hydroxyl group. A C-10 deacetylase from Nocardioides luteus SC 13912 was immobilized on diethylaminoethyl cellulose (DEAE-Cellulose) and the immobilized enzyme was used in the biotransformation process. The reaction was catalyzed using vinyl acetate as acyl donor at ambient temperature and at pH 7. A reaction yield of 51% was obtained.

Long term follow up of topical mustine treatment for cutaneous langerhans cell histiocytosis.

BACKGROUND AND OBJECTIVES: Skin lesions in Langerhans cell histiocytosis (LCH) are often painful and difficult to treat. Topical application of nitrogen mustard (0.02% mechlorethamine hydrochloride, mustine), an alkylating cytostatic agent, has been shown to be effective. There is, however, concern about potentially harmful long term side effects. STUDY DESIGN: In a retrospective study 20 children with LCH (average extent of initial skin involvement: 16.4% body surface) were followed up for an average of 8.3 years after completion of topical mustine therapy. They had received a total of 34 courses (mean duration 14.2 weeks) of topical mustine. Disease status on follow up was assessed according to the Histiocyte Society classification. RESULTS: After mustine was introduced, 16 patients were able to discontinue systemic steroids and/or chemotherapy. Topical mustine was well tolerated in 18 patients, but two developed irritant dermatitis. On follow up, the disease was inactive in 10 patients. Among the children with active disease, six had mild skin disease and four had progressive disease, two of them with skin lesions unresponsive to mustine treatment. Scars confined to areas of formerly active skin disease were found in six patients. There was no evidence of premalignant or malignant skin disease in the treated areas. CONCLUSION: Topical mustine is an effective and safe treatment for skin disease in most children with LCH. Residual scarring was probably a result of the disease itself rather than to mustine. Although no evidence of skin cancer was found in this study, continued long term follow up is advisable.

Basilar invagination as a sequela of multisystem Langerhans' cell histiocytosis.

We report the presence of basilar invagination, an unexpected and previously undescribed abnormality of the skull base, in 7 of 38 long-term survivors of multisystem Langerhans' cell histiocytosis. The abnormality is acquired, but its pathogenesis is uncertain.

Purification, cloning, and characterization of an arylsulfotransferase from the anaerobic bacterium Eubacterium rectale IIIH.

A bacterium, Eubacterium rectale IIIH, which possessed arylsulfotransferase (ASST) activity was isolated from human feces. The ASST gene (astA) was cloned and the corresponding protein partially characterized. This gene shows only moderate homology to the previously sequenced ASST genes of Klebsiella and Enterobacter, which are very closely related to each other.

Colon involvement in Langerhans' cell histiocytosis.

BACKGROUND: Langerhans' cell histiocytosis (LCH), a granulomatous disorder of unknown cause, most often affects the bony skeleton and skin. Obvious gut involvement is uncommon, and colon involvement has been cited in only a small number of case reports, probably because most patients who have LCH with diarrhoea and/or failure to thrive are not investigated by colonoscopy and biopsy. The current study was conducted to determine the incidence of symptomatic colon involvement among patients with multisystem LCH treated at a single institution. METHOD: A retrospective review of cases in hospital records and the literature. RESULTS: Of the 275 children with LCH in the database, 5 were identified as having biopsy-confirmed colonic involvement. Another 14 cases were identified by a literature review. CONCLUSION: Colonic involvement in patients with multisystem LCH is probably more common than currently recognised. In addition to the other investigations recommended by the Writing Group of the Histiocyte Society, it is recommended that patients with any symptoms suggestive of gut involvement undergo colonoscopy and biopsy of the colonic mucosa.

Failure of IGF-I and IGFBP-3 to diagnose growth hormone insufficiency.

BACKGROUND: Growth hormone insufficiency (GHI) is diagnosed conventionally by short stature and slow growth, and is confirmed by diminished peak GH response to a provocation test. Insulin-like growth factor I (IGF-I) and IGF binding protein 3 (IGFBP-3) have previously been considered individually OBJECTIVE: To test the hypothesis that the combined analysis of IGF-I and IGFBP-3 could act as a surrogate marker for the diagnosis of GHI. DESIGN: Reference ranges for IGF-I and IGFBP-3 were calculated using 521 normal individuals. A retrospective analysis was performed on 318 children referred for investigation of short stature. RESULTS: No significant difference was found between either the IGF-I or IGFBP-3 standard deviation scores (SDSs) in children with and without GHI. If the requirement were for both tests to be positive (< -2 SDS) for a diagnosis of GHI, then 99% of children without GHI would be correctly identified; however, the sensitivity of the test was only 15%. CONCLUSIONS: Neither IGF-I nor IGFBP-3 alone is a marker for GHI. In addition, they cannot be used as an effective screening test in combination.

Why is the retention of gonadotrophin secretion common in children with panhypopituitarism due to septo-optic dysplasia?

Septo-optic dysplasia (De Morsier syndrome) is a developmental anomaly of mid-line brain structures and includes optic nerve hypoplasia, absence of the septum pellucidum and hypothalamo-pituitary abnormalities. We describe seven patients (four female, three male) who had at least two out of the three features necessary for the diagnosis of septo-optic dysplasia. Four patients had hypopituitarism and yet normal gonadotrophin secretion: one of these also had anti-diuretic hormone insufficiency; three had isolated GH deficiency and yet had premature puberty, with the onset of puberty at least a year earlier than would have been expected for their bone age. In any progressive and evolving anterior pituitary lesion it is extremely unusual to lose corticotrophin-releasing hormone/ACTH and TRH/TSH secretion and yet to retain gonadotrophin secretion. GnRH neurons develop in the nasal mucosa and migrate to the hypothalamus in early fetal life. We hypothesise that the arrival of GnRH neurons in the hypothalamus after the development of a midline hypothalamic defect may explain these phenomena. Progress in spontaneous/premature puberty in children with De Morsier syndrome may have important implications for management. The combination of GH deficiency and premature puberty may allow an apparently normal growth rate but with an inappropriately advanced bone age resulting in impaired final stature. GnRH analogues may be a therapeutic option. In conclusion, some patients with De Morsier syndrome appear to retain the ability to secrete gonadotrophins in the face of loss of other hypothalamic releasing factors. The migration of GnRH neurons after the development of the midline defect may be an explanation.

Labyrinthine involvement in Langerhans' cell histiocytosis.

BACKGROUND: Langerhans' cell histiocytosis, a rare condition caused by the proliferation of abnormal Langerhans' cells ('LCH cells') and an accompanying granulomatous infiltrate, can affect several organs including the ear. External and middle ear involvement are common with a reported incidence as high as 61%. The bony labyrinth is resistant to erosion by the granulation tissue, thereby protecting the cochlea and vestibular structures. Probably for this reason, involvement of the inner ear is rare, with few case reports in the literature. PATIENTS: We report two girls, one with bilateral and the other with unilateral mastoid involvement, in whom there was invasion of the labyrinth. The first girl had 'single system' LCH affecting only bone and developed an acute hearing loss due to invasion of the cochlea, while the second had both bone and skin involvement and labyrinthine involvement was diagnosed on imaging prior to the onset of labyrinthine symptoms. CONCLUSION: Inner ear involvement can lead to permanent deafness, which may be prevented by early institution of treatment. Threatened inner ear involvement requires urgent systemic medical therapy with steroids, possibly combined with chemotherapy.

Fermentation and isolation of C10-deacetylase for the production of 10-deacetylbaccatin III from baccatin III.

10-Deacetylabaccatin III (10 DAB), an important precursor for paclitaxel semisynthesis, is enhanced in yew extracts using C10-deacetylase and C13-deacylase enzymes.(4) C10-deacetylase is an intracellular enzyme produced by the fermentation of a soil microorganism, Nocardioides luteus (SC 13912). During the fermentation of Nocardioides luteus, the growth of cells reaches a maximum growth at 28 h. C10-deacetylase enzyme activity starts at 26 h and peaks at 38 h of the fermentation. The cells are recovered by centrifugation. The C10-deacetylase enzyme was purified from the Nocardioides luteus cells. The enzyme was purified 190-fold to near homogeneity. The purified enzyme appeared as a single band on 12.5% SDS-PAGE analysis with a molecular weight of 40,000 daltons. (c) 1995 John Wiley & Sons, Inc.

Site-specific enzymatic hydrolysis of taxanes at C-10 and C-13.

The production of large amounts of paclitaxel for use as an anticancer treatment has been a challenging problem because of the low concentration of the compound in yew trees and its occurrence as part of a mixture of other taxanes. Two novel enzymes were isolated to facilitate the production of 10-deacetylbaccatin III, a precursor used for semisynthesis of paclitaxel and analogs. A strain of Nocardioides albus (SC13911) was isolated from soil and found to produce an extracellular enzyme that specifically removed the C-13 side chain from paclitaxel, cephalomannine, 7-beta-xylosyltaxol, 7-beta-xylosyl-10-deacetyltaxol, and 10-deacetyltaxol. The enzyme was purified to near homogeneity to give a polypeptide with 47,000 M(r) on a sodium dodecyl sulfate gel. A strain of Nocardioides luteus (SC13912) isolated from soil was found to produce an intracellular 10-deacetylase that removed the 10-acetate from baccatin III and paclitaxel. The 10-deacetylase was purified to give a polypeptide with 40,000 M(r) on a sodium dodecyl sulfate gel. Treatment of extracts prepared from a variety of yew cultivars with the C-13-deacylase and C-10-deacetylase converted a complex mixture of taxanes primarily to 10-deacetylbaccatin III and increased the amount of this key precursor by 4-24 times.