SDH-B, INSM1, and GATA3 expression in cauda equina neuroendocrine tumors: report on 21 cases from a North indian tertiary care center.

Primary neuroendocrine tumors (NETs) of the central nervous system are rare, primarily seen in the cauda equina region, known as cauda equina NETs. This study was carried out to evaluate the morphological and immunohistochemical characteristics of cauda equina NETs. All cases of histologically proven NETs that originated within the spinal cord from 2010 to 2021 were retrieved from the surgical pathology electronic database. For each case, the clinical presentation, site, radiological features, functional status, and preoperative diagnosis were recorded. Immunohistochemical stains for GFAP, synaptophysin, chromogranin A, cytokeratin 8/18, INSM1, Ki-67, GATA3, and SDH-B were performed for every case using an automated immunostainer. GATA3 immunohistochemistry was repeated manually. A retrospective probe of records revealed 21 cases of NETs having a mean age of 44 years and slight male dominance (M:F ratio: 1.2:1). Cauda equina was the most prevalent site of involvement (19, 90.5%). The most typical presentation was lower backache and weakness of bilateral lower limbs. The histopathological features were similar to NETs seen at other sites. Reactivity for at least one neuroendocrine marker was seen in all cases while GFAP was negative. Cytokeratin 8/18 was expressed in the majority (88.9%) of cases. INSM1 and GATA3 expression was seen in 20 (95.2%) and 3 (14.3%) cases, respectively. All cases retained SDH-B cytoplasmic staining. Higher Ki-67 index (≥3%) was associated with a higher risk of recurrence. Cauda equina NETs rarely express GATA3 and are unlikely to be associated with SDH mutations. Recurrent cases may be negative for synaptophysin, chromogranin, and cytokeratin; thus, INSM1 immunohistochemistry is helpful.

A Two-in-One Tumor in the Adrenal: A Functional Adrenocortical Adenoma with Myelolipomatous Differentiation.

BACKGROUND: Adrenocortical adenoma (ADA) and myelolipoma are two common benign neoplasms of the adrenal cortex that have been reported to occur together. CASE REPORT: A 14-year-old girl presented with the features of ACTH-independent endogenous Cushing syndrome. Abdominal CECT revealed a left adrenal 2.3 × 1.8 × 1.5 cm arterially enhancing nodular lesion with central hypodensity. Histologically, this was an ADA with oncocytic change and myelolipomatous differentiation/metaplasia. DISCUSSION/CONCLUSION: ADA with myelolipomatous differentiation/metaplasia can occur in the pediatric age group.

Assessment of risk of malignancy by application of the proposed Sydney system for classification and reporting lymph node cytopathology.

BACKGROUND: Fine-needle aspiration cytology (FNAC) is one of the most commonly used techniques for evaluating lymphadenopathy. Recently, the Sydney system was proposed for assessing the performance, classification, and reporting of lymph node (LN) cytopathology. The present study was conducted to assess the risk of malignancy associated with each of the diagnostic categories of the proposed Sydney system. METHODS: This was a 2-year retrospective study of LN-FNAs; cytologic diagnoses were categorized by the proposed Sydney system. Cytological diagnoses were correlated with the corresponding histopathological diagnoses to assess diagnostic accuracy and risk of malignancy for each diagnostic category. RESULTS: Of 23,335 FNAs during the study period, 6983 (30%) were performed on LNs. Of these, 289 (4.1%) cases were reported as nondiagnostic/inadequate (L1); 3397 (48.6%) were reported as benign (L2); 33(0.5%) as atypical cells of undetermined significance (L3), 96 (1.4%) as suspicious for malignancy (L4) and 3168 (45.4%) as malignant (L5). Subsequent histopathology was available for 618 (8.8%) cases. On cytohistopathologic correlation, 552 (89.3%) were concordant and 66 (10.7%) discordant. The overall sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy of LN-FNA were 79.9%, 98.7%, 98.4%, 83.1%, and 89.3%, respectively. The risk of malignancy was 27.5% for the nondiagnostic category, 11.5% for the benign, 66.7% for the atypical, 88% for the suspicious, and 99.6% for the malignant categories. CONCLUSIONS: FNAC has high diagnostic accuracy for the diagnosis of various LN pathologies. Application of the proposed Sydney system can help in achieving uniformity and reproducibility in cytologic diagnoses and also help in risk-stratification on cytology.

A fatal case of enterovirus infection with secondary hemophagocytosis-case report with review of literature.

Enterovirus is a common viral infection, which can affect multiple organ systems with an array of clinical presentation such as meningitis, encephalitis, myocarditis, and disseminated infections. The illness is usually asymptomatic and self-limited but few cases can be severe and life-threatening especially when associated with hemophagocytosis. We discuss a fatal case of disseminated enterovirus infection and the histomorphological features of the infection.

Mitigating Cocrystal Physical Stability Liabilities in Preclinical Formulations.

Poor aqueous solubility of a majority of new small molecule chemical entities is a significant challenge in drug discovery since considerably high exposures are often required to enable pharmacokinetic, pharmacology, and toxicology studies. Pharmaceutical cocrystals have received considerable attention in recent years owing to their potential to improve the physicochemical properties and in vivo performance of poorly soluble drugs. However, physical instability in supersaturated solution/suspension formulations is a major concern for their use in preclinical studies. This review will present an overview of the thermodynamic and kinetic contributions impacting physical stability of cocrystals in preclinical formulations with a focus on the role of surfactants, polymeric excipients, and pH. Finally, the in vivo performance of cocrystals will be discussed. The article will conclude with a perspective on strategies to develop physically stable preclinical cocrystal formulations.

A canine biorelevant dissolution method for predicting in vivo performance of orally administered sustained release matrix tablets.

Preclinical species are a crucial component of drug development, but critical differences in physiology and anatomy need to be taken into account when attempting to extrapolate to humans or between species. The same is true when trying to develop oral formulations for preclinical species, especially unconventional formulations, such as sustained release tablets. During the evaluation of such specialized dosage forms, dissolution can be a critical in vitro tool used to rank-order formulations and ultimately choose the desired release rate. Here, the development of a canine biorelevant dissolution method for the prediction of the in vivo performance of sustained release matrix tablets in beagle dogs is described. The method accounts for differences in physiology between humans and dogs such as gastrointestinal fluid composition, gastric emptying forces, and gastric residence time. The most critical dissolution method parameters were found to be the paddle speed used to simulate the gastric emptying forces as well as the time spent in simulated gastric fluid. The resulting differences in method conditions are further explored through in silico models of the hydrodynamic forces applied to a dosage form. Two case studies are reported showing that the method was able to obtain excellent in vitro-in vivo relationships (slopes ranging from 1.08-1.01) which are significantly (p < 0.01-0.05) improved compared to human biorelevant dissolution used to predict in vivo performance in humans (slopes ∼1.5-1.75). The quality of the method's predictive ability allows for it to help drive the development of matrix sustained release formulations intended for preclinical studies.

Modulating the dehydration conditions of adefovir dipivoxil dihydrate to obtain different physical forms of anhydrate.

The physical form of anhydrous adefovir dipivoxil (AD), obtained following the dehydration of AD dihydrate, was governed by the kinetics of water removal. The rate and extent of water removal following the dehydration of AD dihydrate was manipulated by altering the sample size, pan configuration, and heating rate in a differential scanning calorimeter. Interestingly, when there was moderate resistance to water removal, a new anhydrous polymorph (melting point 80°C) was obtained. High resistance to water removal resulted in amorphous AD. Variable temperature XRD of AD provided direct and unambiguous evidence of this new polymorph. We have prepared and characterized this new anhydrous polymorph as well as amorphous AD. Based on HPLC, AD dihydrate heated under different conditions in the DSC was observed to be chemically stable. When exposed to water vapor (RH ≥ 80%; 25°C), the new polymorph had a stronger propensity to convert to AD dihydrate than the amorphous anhydrate or AD form I.

Mechanism of amorphous itraconazole stabilization in polymer solid dispersions: role of molecular mobility.

Physical instability of amorphous solid dispersions can be a major impediment to their widespread use. We characterized the molecular mobility in amorphous solid dispersions of itraconazole (ITZ) with each polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose acetate succinate (HPMCAS) with the goal of investigating the correlation between molecular mobility and physical stability. Dielectric spectra showed two mobility modes: α-relaxation at temperatures above the glass transition temperature (Tg) and ß-relaxation in the sub-Tg range. HPMCAS substantially increased the α-relaxation time, with an attendant increase in crystallization onset time and a decrease in crystallization rate constant, demonstrating the correlation between α-relaxation and stability. The inhibitory effect on α-relaxation as well as stability was temperature dependent and diminished as the temperature was increased above Tg. PVP, on the other hand, affected neither the α-relaxation time nor the crystallization onset time, further establishing the link between α-relaxation and crystallization onset in solid dispersions. However, it inhibited the crystallization rate, an effect attributed to factors other than mobility. Interestingly, both of the polymers acted as plasticizers of ß-relaxation, ruling out the latter's involvement in physical stability.

Molecular motions in sucrose-PVP and sucrose-sorbitol dispersions-II. Implications of annealing on secondary relaxations.

PURPOSE: To determine the effect of annealing on the two secondary relaxations in amorphous sucrose and in sucrose solid dispersions. METHODS: Sucrose was co-lyophilized with either PVP or sorbitol, annealed for different time periods and analyzed by dielectric spectroscopy. RESULTS: In an earlier investigation, we had documented the effect of PVP and sorbitol on the primary and the two secondary relaxations in amorphous sucrose solid dispersions (1). Here we investigated the effect of annealing on local motions, both in amorphous sucrose and in the dispersions. The average relaxation time of the local motion (irrespective of origin) in sucrose, decreased upon annealing. However, the heterogeneity in relaxation time distribution as well as the dielectric strength decreased only for ß1- (the slower relaxation) but not for ß2-relaxations. The effect of annealing on ß2-relaxation times was neutralized by sorbitol while PVP negated the effect of annealing on both ß1- and ß2-relaxations. CONCLUSIONS: An increase in local mobility of sucrose brought about by annealing could be negated with an additive.

Controlling the physical form of mannitol in freeze-dried systems.

A potential drawback with the use of mannitol as a bulking agent is its existence as mannitol hemihydrate (MHH; C6H14O6·0.5H2O) in the lyophile. Once formed during freeze-drying, MHH dehydration may require secondary drying under aggressive conditions which can be detrimental to the stability of thermolabile components. If MHH is retained in the lyophile, the water released by MHH dehydration during storage has the potential to cause product instability. We systematically identified the conditions under which anhydrous mannitol and MHH crystallized in frozen systems with the goal of preventing MHH formation during freeze-drying. When mannitol solutions were cooled, the temperature of solute crystallization was the determinant of the physical form of mannitol. Based on low temperature X-ray diffractometry (using both laboratory and synchrotron sources), MHH formation was observed when solute crystallization occurred at temperatures ≤ -20 °C, while anhydrous mannitol crystallized at temperatures ≤ -10 °C. The transition temperature (anhydrate - MHH) appears to be ∼-15 °C. The use of a freeze-dryer with controlled ice nucleation technology enabled anhydrous mannitol crystallization at ∼-5 °C. Thus, ice crystallization followed by annealing at temperatures ≤ -10 °C can be an effective strategy to prevent MHH formation.

Molecular mobility as a predictor of the water sorption by annealed amorphous trehalose.

PURPOSE: The work aims at investigating the correlation of water sorption potential with different measures of molecular mobility in an annealed amorphous model compound (trehalose). METHODS: Amorphous trehalose, prepared by freeze-drying, was annealed at 100°C (17°C < T (g)) for up to 120 h. Global molecular mobility was studied using a broadband dielectric spectrometer in the frequency range of 10(6)-10(-2) Hz. Enthalpic recovery was measured by differential scanning calorimetry and water sorption profiles were obtained using an automated vapor sorption balance. RESULTS: As a function of annealing time, there was an increase, both in average α-relaxation time and enthalpic recovery and a decrease in the amount of sorbed water. A strong linear correlation was observed between the water sorption potential and the dielectric relaxation time, indicating a common underlying mechanism of the effect of annealing time on these properties. Enthalpic recovery, which is widely used as a measure of structural relaxation, did not correlate well with the extent of water sorption. CONCLUSIONS: The α-relaxation time can be used as a predictor of the water sorption potential of amorphous trehalose. It will be of interest and value to develop such predictive models for other amorphous compounds of pharmaceutical interest.

Correlation between molecular mobility and physical stability of amorphous itraconazole.

The goal was to investigate the correlation between molecular mobility and physical stability in amorphous itraconazole and identify the specific mobility mode responsible for its instability. The molecular mobility of amorphous itraconazole, in the glassy as well as the supercooled liquid state, was comprehensively characterized using dynamic dielectric spectroscopy. Isothermal frequency sweeps in the 5-40 °C temperature range revealed a ß-relaxation which exhibited Arrhenius temperature dependence. As the temperature approached T(g), ß-relaxation became progressively less resolved due to interference from the high frequency tail of the α-relaxation and then transformed into an excess wing. Above T(g), nonlinear temperature dependence of the α-relaxation was described by the Vogel-Tammann-Fulcher (VTF) model. Itraconazole was found to be a fragile glass former with a VTF strength parameter of ∼4. Isothermal crystallization kinetics, at several temperatures over the range of 75 to 95 °C, was best described by the 3-dimensional nucleation and growth model. Primary relaxation appeared to be the mobility responsible for the observed physical instability at temperatures above T(g) as indicated by the linear correlation of α-relaxation with both crystallization onset and kinetics (represented by the inverse of the crystallization rate constant). A strong coupling between global mobility and crystallization onset was evident. However, for growth kinetics, the coupling was less pronounced, indicating the involvement of factors other than global mobility.

Molecular mobility as an effective predictor of the physical stability of amorphous trehalose.

Amorphous trehalose was prepared by different methods, viz., freeze-drying, spray-drying and dehydration of trehalose dihydrate. The different molecular relaxations were characterized by dynamic dielectric spectroscopy. The preparation method significantly affected the structural relaxation time and its temperature dependence in the glassy region. The order of activation energy for α-relaxation was spray-dried > freeze-dried > dehydrated. However, the secondary relaxation times remained essentially unaffected by the preparation method. Isothermal crystallization kinetics was studied at several temperatures above the glass transition temperature (T(g)). A linear correlation was observed between the crystallization time (inverse of crystallization rate constant) and the average α-relaxation time, suggesting a similar molecular origin for these two processes. There was also strong coupling of the crystallization onset time with global molecular mobility in the supercooled liquid region, enabling the development of predictive models. The observed experimental onset times were in excellent agreement with the predicted values at temperatures around and below T(g).

Use of dielectric spectroscopy to monitor molecular mobility in glassy and supercooled trehalose.

Dielectric spectroscopy was used to comprehensively characterize the molecular mobility in amorphous trehalose, an extensively used bioprotective agent. Isothermal frequency sweeps were carried out at different temperatures in the glassy and supercooled liquid states of freeze-dried trehalose. Two previously reported secondary relaxations were observed at temperatures far below its glass transition temperature (T(g)). At temperatures close to T(g), removal of dc conductivity contribution revealed a relaxation peak. The origin of this peak was evaluated using several diagnostic tests and determined to be the α-relaxation. There was also an excess wing in the high-frequency tail of α-relaxation. Sub-T(g) annealing caused the primary relaxation to shift to lower frequencies, enabling resolution of the excess wing, which was characterized to be the true Johari-Goldstein (JG) relaxation. A qualitatively similar effect of annealing on JG relaxation was also observed. The average relaxation times of the two previously reported secondary relaxations were unaffected by annealing.

Subtraction of DC conductivity and annealing: approaches to identify Johari-Goldstein relaxation in amorphous trehalose.

Amorphous trehalose finds extensive use as a stabilizer of biomolecules including proteins and phospholipids. Hypothesizing that molecular mobility is a determinant of its stability, dynamic dielectric spectroscopy (DDS) was used to map the different modes of mobility. Isothermal dielectric relaxation profiles of amorphous trehalose were obtained, over the frequency range of 10(-1)-10(7) Hz, and at temperatures ranging from 30-170 °C. At temperatures close to the glass transition (T(g)), the α-relaxation was not readily discernible due to interference from dc conductivity. We used Kramers-Kronig transformation that enabled not only the complete resolution of α-relaxation but also the identification of an excess wing, in the high frequency tail of α-relaxation. On annealing, this excess wing developed into a partially resolved and hitherto unidentified ß-relaxation peak. This peak, due to its position in the dielectric spectrum, its annealing time dependence and the good agreement with the calculated independent relaxation time, was assigned to the Johari-Goldstein process. This work demonstrates the utility of conductivity subtraction coupled with sub-T(g) annealing to successfully study all the modes of mobility in amorphous trehalose. This approach can potentially be extended to situations wherein dc conductivity impedes the complete characterization of molecular mobility.

Monitoring phase transformations in intact tablets of trehalose by FT-Raman spectroscopy.

The purpose of this study is to monitor phase transformations in intact trehalose tablets using FT-Raman spectroscopy. Tablets of trehalose dihydrate, amorphous trehalose (obtained by freeze-drying aqueous trehalose solutions), and anhydrous trehalose (beta-trehalose) were prepared. The tablets were exposed to different conditions [11% and 0% RH (60 degrees C); 75% RH (25 degrees C)] and monitored periodically over 96 h using Raman spectroscopy. Within 96 h of storage, the following phase transformations were observed: (1) trehalose dihydrate-->beta-trehalose (11% RH, 60 degrees C), (2) trehalose dihydrate-->alpha-trehalose (0% RH, 60 degrees C), (3) beta-trehalose-->trehalose dihydrate (75% RH, 25 degrees C), and (4) amorphous trehalose-->trehalose dihydrate (75% RH, 25 degrees C). FT-Raman spectroscopy was a useful technique to identify the solid form and monitor multiple-phase transformations in intact trehalose tablets stored at different conditions.

Is relief from diabetes just a breath away?

Pulmonary insulin delivery is steadily emerging as a promising solution for the treatment of diabetes mellitus. The large as well as thin absorptive area of the lungs has not been explored until now for the treatment of systemic disease like diabetes. With an understanding of the lung anatomy and physiology and the transport mechanism of insulin through lungs, diabetic treatment through the pulmonary route may well become the reality of the 21(st) century. Though the transport of insulin through the lungs itself appears quite encouraging, potential problems concerning the formulation of a peptide like insulin in the form of an aerosol seem to be the most challenging. Stability aspects, stringent control of Mass Median Aerodynamic Diameter, antigenicity, insulin losses due to the device and impaction, sedimentation and diffusion in the nonabsorptive areas of the airway system (especially in the oropharynx) emerge as major concerns. This is in addition to the problems of lack of reproducibility of dose delivery by an inhaler where individual variations due to inspiratory differences and method of use of device come into play. Lung diseases and smoking may alter lung mechanisms and dose alterations are to be studied in such cases. Though almost equally effective, if not more, than the subcutaneous insulin route, even with proved short-term efficacy, insulin delivery through lungs is a potential but not a wholly proven means for blood glucose control.

Study of forced degradation behavior of enalapril maleate by LC and LC-MS and development of a validated stability-indicating assay method.

In the present study, comprehensive stress testing of enalapril maleate was carried out according to ICH guideline Q1A(R2). The drug was subjected to acid (0.1N HCl), neutral and alkaline (0.1N NaOH) hydrolytic conditions at 80 degrees C, as well as to oxidative decomposition at room temperature. Photolysis was carried out in 0.1N HCl, water and 0.1N NaOH at 40 degrees C. Additionally, the solid drug was subjected to 50 degrees C for 60 days in a dri-bath, and to the combined effect of temperature and humidity, with and without light, at 40 degrees C/75% RH. The products formed under different stress conditions were investigated by LC and LC-MS. The LC method that could separate all degradation products formed under various stress conditions involved a C18 column and a mobile phase comprising of ACN and phosphate buffer (pH 3). The flow rate and detection wavelength were 1 ml min(-1) and 210 nm, respectively. The developed method was found to be precise, accurate, specific and selective. It was suitably modified for LC-MS studies by replacing phosphate buffer with water, where pH was adjusted to 3.0 with formic acid. The drug showed instability in solution state (under acidic, neutral, alkaline and photolytic stress conditions), but was relatively stable in the solid-state, except formation of minor products under accelerated conditions. Primarily, maximum degradation products were formed in acid conditions, though the same were also produced variably under other stress conditions. The LC-MS m/z values and fragmentation patterns of two of the five products matched with enalaprilat and diketopiperazine derivative, previously known degradation products of enalapril. Also, m/z value of another product matched with an impurity listed in the drug monograph in European Pharmacopoeia. Rest two were hitherto unknown degradation products. The products were characterized through LC-MS fragmentation studies. Based on the results, a more complete degradation pathway for the drug could be proposed.