Automated multifactorial design of experiment and Bayesian optimisation algorithm approaches to method development for the green analysis by supercritical fluid chromatography of a pharmaceutical ingredient.

During drug development, chromatography is frequently used for purity and stability testing of both drug substance and drug product. Reversed phase liquid chromatography (RPLC) is one of the most widely used methodologies due to its wide scope of application. In the later stages of drug development, the specified impurities and degradation products that define the critical quality attribute of the final API, also known as Key Predictive Sample Set (KPSS), are usually well defined and controlled. At this point, a method review enables selecting the most appropriate technique which should be the one providing optimal robustness (ICH-Q14[1]), with the support of Quality by Design (QbD) approaches. Supercritical Fluid Chromatography (SFC) is a preferred technique for its proven diversity in selectivity. The adoption of a technique which presents the most favourable environmental impact, such as, but not limited to, SFC, is also becoming increasingly important as laboratories strive to reduce carbon footprint. Re-developing a method requires high resource-demands in terms of staff, materials, and time. Any step of the process that can be automated can facilitate this approach, speeding up the delivery of the method whilst preserving robustness. In this article we describe how an SFC method was developed for the purity profiling of a late-stage oncology candidate, taking advantage of the superior selectivity of SFC towards structurally similar analytes, owed to the high orthogonality with R2 as low as 0.014 towards the KPSS. We describe two approaches to automate the method development. Firstly, a multifactorial design of experiments (DoE) and secondly, an optimization via a Bayesian algorithm, which was completed in one night, highlighting the potential and limitations, with an insight into the robustness. Both methods achieved baseline separation with varying levels of automation embedded into the process and a large reduction of the resource demands when compared to traditional optimisation methods. Finally, we describe the beneficial environmental impact that implementing SFC methods can yield, with a calculated green score reduced to a value between 17 and 30 % compared to RPLC, depending on the number of runs per sequence.

Transoral Parotidectomy.

Transoral parotidectomy allows for the management of parapharyngeal space tumors and accessory parotid gland tumors without the need for a transfacial/transparotid or mandible splitting procedure. It is a minimally invasive approach that permits a faster recovery and with a lesser risk of facial palsy.

Potential biomarkers for head and neck squamous cell carcinoma.

OBJECTIVE: The purpose of the study was twofold: 1) to search for potential biomarkers that were overexpressed in cell lines that could represent both a clinical premalignant (immortalized) and a malignant state, and 2) to attempt to correlate metallothionein gene expression with clinical outcome in laryngeal carcinoma. STUDY DESIGN: A series of in vitro experiments were used to unearth differentially expressed genes among normal, immortalized and tumorigenic cell lines. Secondarily, a retrospective analysis was undertaken. METHODS: Differential display analysis was conducted to identify differentially expressed genes between human papillomavirus-infected immortalized HOK16B and benzo[ ]pyrene-derived tumorigenic cell line, HOK16B-BaP-T. The cell-specific expressions were examined by Northern blot analysis and compared with other known immortalized and cancer cell lines. Immunohistochemical staining was also conducted to localize metallothionein (MT I/II) protein expression among the different cell lines studied. A retrospective analysis of laryngeal specimens from archival tissues of 29 cancer patients who underwent primary surgical resection was also undertaken after immunohistochemical staining. RESULTS: Twenty-one differentially expressed complementary cDNA clones, both novel and known, were identified using the differential display analysis. Northern blot analysis confirmed that clone 6 hybridized to a 1.6-kb RNA in HOK16B-Bap-T cell line. Clone 4 showed decreased expression in immortalized and cancer cell compared with NHOK. MT I/II transcript was observed in HOK16B, which was further elevated in HOK16B-Bap-T. Retrospective analysis showed that high immunoreactivity to MT I/II in surgically resected laryngeal cancer specimen correlated with increased frequency of recurrence within 2 years of surgery. CONCLUSION: These findings suggest that clone 4 may potentially function as a tumor suppressor gene, which may be significant in tumor progression and invasion. Clone 6 may participate in viral-mediated oncogenic transformation of normal cells. Clone 6 may also have potential as a tumor maker differentiating normal from malignant tissue, as in the determination of surgical resection margins. MT I/II gene product may serve as a prognostic biomarker for laryngeal squamous cell carcinoma. The differentially expressed genes and gene products may serve as sensitive biomarkers for improved early detection, diagnosis, and prognosis of head and neck squamous cell carcinoma.

Carcinoma of the oral pharynx: an analysis of subsite treatment heterogeneity.

The data indicate that SCC of the various subsites of the oropharynx can be treated successfully with acceptable locoregional control and survival rates by using either surgery or primary radiotherapy for TI or T2 primary lesions. Treatment success data for late-stage disease (T3 and T4) are less encouraging. regardless of which modality is used or which treatment center is administering treatment. This finding may suggest an intrinsic property of these lesions or the patient that may be going unnoticed.One problem is that the diversity of approaches to these lesions hinders any meaningful comparisons between series from different treatment centers. There exists heterogeneity in patient populations and approaches to staging and characterization of these diseases. This situation has ensured the same heterogeneity in treatment philosophy, which is largely institutionally based.

Phagocytosis of candida albicans by lymphatic tumour cells in vitro.

Experiments were carried out to investigate whether different lymphatic tumour cell lines have similar kinetic characteristics of phagocytosis of microorganisms. Six tumour cell lines were used. These were a human T-cell line (CEM), a mouse T-cell line (YAC-1), a human B-cell line (LAZ), and a human erythroleukemic tumour cells (K562), whereas 2 cell lines of professional phagocytosis were used as controls, a human macrophage cell line (THP1) and a mouse macrophage cell line (P388D1). Tumour cells were mixed with candida albicans at a ratio of 10:1 of candida to tumour cells and the percentage of tumour cells that had attached/phagocytosed candida was determined. After 4 h coculture with candida, tumour cells not of T-cell origin (LAZ and K562) showed moderate level of phagocytosis (28%), whereas tumour cells of T-cell origin (CEM and YAC-1) demonstrated low levels of phagocytosis (15%) as compared to macrophage cell lines (THP1 and P388D1) that showed maximum phagocytosis (64-78%). Acid phosphatase (AcPase) activity was increased by 33% during coculture of YAC-1 cells and yeast cells. In conclusion, the results suggest that lymphatic tumour cells of nonphagocytic origin acquire phagocytic properties during the course of malignancy, and digestion of phagocytosed yeast cells maybe related with AcPase activity, as well as that of other lysosomal enzymes. This phenomenon may represent one mechanism by which tumour cells downregulate immune surveillance.

Adult supraglottitis subsequent to smoking crack cocaine.

Supraglottitis is one possible complication of smoking crack cocaine. From 1992 through 2001, our institution treated nine patients for thermal supraglottitis secondary to crack cocaine inhalation. In this article, we describe two of these cases, and we briefly review what is known about this entity and the mechanism of injury. We also provide our recommendations for management.

Primary hyperparathyroidism secondary to simultaneous bilateral parathyroid carcinoma.

Parathyroid carcinoma is a rare malignancy, and experience with its management is limited. It is generally accepted that surgery is the treatment of choice, and the initial operative intervention might represent the only chance for long-term success. At the time of surgery, a high index of suspicion for carcinoma is vital to performing the appropriate surgical procedures, because frozen-section diagnosis of parathyroid carcinoma is notoriously unreliable. In this article, we describe our experience with this rare entity.