Design of Experiments-Driven Optimization of Spray Drying for Amorphous Clotrimazole Nanosuspension.

This study employed a Quality by Design (QbD) approach to spray dry amorphousclotrimazole nanosuspension (CLT-NS) consisting of Soluplus® and microcrystallinecellulose. Using the Box-Behnken Design, a systematic evaluation was conducted toanalyze the impact of inlet temperature, % aspiration, and feed rate on the criticalquality attributes (CQAs) of the clotrimazole spray-dried nanosuspension (CLT-SDNS). In this study, regression analysis and ANOVA were employed to detect significantfactors and interactions, enabling the development of a predictive model for the spraydrying process. Following optimization, the CLT-SD-NS underwent analysis using Xraypowder diffraction (XRPD), Fourier transform infrared spectroscopy (FTIR), Dynamic Scanning Calorimetry (DSC), and in vitro dissolution studies. The resultsshowed significant variables, including inlet temperature, feed rate, and aspiration rate,affecting yield, redispersibility index (RDI), and moisture content of the final product. The models created for critical quality attributes (CQAs) showed statistical significanceat a p-value of 0.05. XRPD and DSC confirmed the amorphous state of CLT in theCLT-SD-NS, and FTIR indicated no interactions between CLT and excipients. In vitrodissolution studies showed improved dissolution rates for the CLT-SD-NS (3.12-foldincrease in DI water and 5.88-fold increase at pH 7.2 dissolution media), attributed torapidly redispersing nanosized amorphous CLT particles. The well-designed studyutilizing the Design of Experiments (DoE) methodology.

Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin.

Background: Luminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally certified reference standards. Therefore, there is an urgent need to characterize existing reference standards for the standardization of multiplex immunoassays (MIAs). Here, we report the development and validation of an MIA for the simultaneous estimation of levels of human serum immunoglobulin G (IgG) antibodies for pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), and tetanus toxoid (TT). Methods: The MIA was assessed using a panel of human serum samples and WHO reference standards. The WHO reference standards were also studied for suitability in the MIA. Purified antigens (PT, FHA, PRN, DT, and TT) were coupled to the spectrally unique magnetic carboxylated microspheres. The method was validated in accordance with the United States Food and Drug Administration (US FDA), European Medicines Agency (EMA), and the International Committee of Harmonization Multidisciplinary (ICH M10) guidelines, and parameters such as precision, accuracy, dilutional linearity, assay range, robustness, and stability were assessed. Method agreements with commercially available IgG enzyme-linked immunosorbent assay (ELISA) assays were also evaluated. In addition, the study assessed the level of correlation between the IgG levels estimated by the MIA and the cell-based neutralizing antibody assays for PT and DT. Results: We identified that an equimix of WHO international standards (i.e., 06/142, 10/262, and TE-3) afforded the best dynamic range for all the antigens in the MIA. For all five antigens, we observed that the back-fitted recoveries using the four-parameter logistic (4-PL) regression fits ranged between 80% and 120% for all calibration levels, and the percentage coefficient of variation (% CV) was < 20%. In addition, the difference in mean fluorescence intensity (MFI) between the monoplex and multiplex format was < 10% for each antigen, indicating no crosstalk among the beads. The MIA also showed good agreement with conventional and commercially available assays, and a positive correlation (> 0.75) with toxin neutralization assays for PT and DT was observed. Conclusion: The MIA that was calibrated in accordance with WHO reference standards demonstrated increased sensitivity, reproducibility, and high throughput capabilities, allowing for the design of robust studies that evaluate both natural and vaccine-induced immunity.

Strategizing Spray Drying Process Optimization for the Manufacture of Redispersible Indomethacin Nanoparticles Using Quality-by-Design Principles.

The present study adopted a Quality by Design (QbD) approach to spray dry indomethacin nanosuspension (IMC-NS) consisting of HPC-SL, poloxamer 407, and lactose monohydrate. The Box-Behnken Design was used to systematically evaluate the effects of inlet temperature, aspiration rate, and feed rate on the critical quality attributes (CQAs) [redispersibility index (RDI; minimize), % yield (maximize), and % release at 15 min (maximize)] of the indomethacin spray dried nanosuspension (IMC-SD-NS). To identify significant main and quadratic effects, two-way interactions, and create a predictive model for the spray drying process, regression analysis and ANOVA were utilized. Following optimization, the IMC-SD-NS was analyzed for its physicochemical properties using X-ray powder diffraction (XRPD), Fourier transform infrared spectroscopy (FTIR), and in vitro dissolution studies. Statistical analysis revealed significant independent variables, including inlet temperature, feed rate, and aspiration rate, that critically impacted the solidified end product's RDI, % yield, and % release at 15 min. The models developed for critical quality attributes (CQAs) were significant at a p-value of 0.05. The crystalline state of IMC was maintained in the solidified product, as confirmed by XRPD, and no interactions were observed between IMC and the excipients as evaluated by FTIR. In vitro dissolution studies showed improved dissolution rate for the IMC-SD-NS (3.82-fold increase in overall drug release), which may be attributed to the readily redispersible nanosized drug particles. The implementation of a well-designed study, utilizing Design of Experiments (DoE) methodology, played a crucial role in the development of a highly effective spray drying process.

Optimized L-SNEDDS and spray-dried S-SNEDDS using a linked QbD-DM3 rational design for model compound ketoprofen.

A QbD-DM3 strategy was used to design ketoprofen (KTF) optimized liquid (L-SNEDDS) and solid self-nanoemulsifying drug delivery systems (S-SNEDDS). Principal component analysis was used to identify the optimized L-SNEDDS containing Capmul® MCM NF, 10 % w/w; Kolliphor® ELP, 60 % w/w; and propylene glycol, 30 % w/w. The S-SNEDDS was manufactured by spray-drying a feed dispersion prepared by dissolving the optimized KTF-loaded L-SNEDDS in an ethanol-Aerosil® 200 dispersion. A Box Behnken design was employed to evaluate the effect of drug concentration (DC), Aerosil® 200 concentration (AC) and feed rate (FR) on maximizing percent yield (PY) and loading efficiency (LE). The optimal levels of DC, AC, and FR were 19.9 % w/w, 30.0 % w/w, and 15.0 %, respectively. The optimized S-SNEDDS was amorphous, and its dissolution showed a 2.37-fold increase in drug release compared to KTF in 0.1 HCl. An optimized independent spray-dried S-SNEDDS verification batch showed that the predicted and observed PY and LE were 70.49 % and 92.49 %, and 70.02 % and 91.27 %, respectively. The optimized L-SNEDDS and S-SNEDDS also met their quality target product profile criteria for globule size <100 nm, polydispersity index < 0.400, emulsification time < 30 s, and KTF L-SNEDDS solubility 100-fold greater than its water solubility.

Rare Presentation of a Patient with Cardiac Arrest Due to Cerebral Fat Embolization Following Polytrauma.

Cerebral fat embolism (CFE) syndrome is a known complication that can occur following polytrauma, particularly in cases involving fractures of long bones, but cardiac arrest is a rare presentation following cerebral fat embolization.1 Our patient met with a road traffic accident (RTA), sustaining multiple long bones injuries with hypovolemic shock. After 10 hours of admission and achieving hemodynamic stability, the patient developed cerebral fat embolization. He developed sudden cardiac arrest and was resuscitated. We instituted ventilator support, inotropic infusion, antibiotics, and intravenous (IV) fluids. Our patient regained consciousness without neurological deficit over a period of 10 days and underwent surgery for all three major fractures with due precautions. The patient was discharged after 3 weeks of treatment from the hospital. How to cite this article: Rathod N, Rathod V, Parikh B, et al. Rare Presentation of a Patient with Cardiac Arrest Due to Cerebral Fat Embolization Following Polytrauma. J Assoc Physicians India 2023;71(11):89-93.

Effect of temperature on growth performance and immuno-biochemical changes in endangered golden mahseer, Tor putitora (Hamilton, 1923).

The present study to evaluated the effect of different thermal regimes on growth performance, oxidative stress, antioxidant enzymes, and immuno-biochemical responses of endangered golden mahseer, Tor putitora. A total of 144 healthy fingerlings were randomly distributed into four experimental groups (13 °C, 16 °C, 19 °C, 22 ± 0.5 °C) in triplicate rectangular fiberglass reinforced plastics tanks. The highest and lowest Growth rate, Specific growth rate, Daily growth index, Thermal growth coefficient, Viscera-somatic index, and Growth hormone were observed at 19 °C and 13 °C, respectively. The lowest SGR and VSI were observed at 22 °C. The feed conversion ratio was significantly higher in the control group (p < 0.05), whereas there was a significant decrease in all the treatment groups. The highest and lowest hepato-somatic index was observed at a temperature of 13 °C and 16 °C, respectively, while no significant (p > 0.05) impact of temperature regimes were observed on the length-weight relationship. Regarding hematological indices results, the hematocrit, hemoglobin, total erythrocyte count, and total leucocyte count were significantly higher at 13 °C, whereas the lowest was observed in all the treatment groups (p < 0.05). Higher myeloperoxidase activity and lysozyme activity were observed at 13 °C, whereas lowest at 22 °C. Anti-oxidant potential, SOD, and catalase (muscle) were significantly higher in the control group, whereas they gradually decreased with an increasing temperature. Catalase (liver) level was significantly higher at 22 °C. GST content was significantly higher at 22 °C compared to 13 °C, and GST content increased gradually with an increased temperature. The lipid peroxidation level in the liver and muscle was significantly higher at 22 °C and 13 °C, respectively. The present study concluded that the different phenotypic, antioxidative, and immuno-biochemical modulations of golden mahseer fingerlings in response to different temperature regimes could be used for better management and propagation.

Asymptomatic Bilateral Giant Multilocular Epididymal Cyst in a Middle-Aged Patient.

Bilateral multilocular huge epididymal cysts are a rare entity with few reports in the literature. Epididymal cysts are mostly found in middle-aged men with or without symptoms. We present the case of a 45-year-old man with asymptomatic bilateral scrotal swelling, which was clinically diagnosed as a right epididymal cyst with left hydrocele. However, an ultrasound of the scrotum revealed bilateral epididymal cysts with normal testes. Intraoperatively, it demonstrated bilateral huge epididymal cysts for which the patient underwent excision of bilateral epididymal cysts. Postoperatively, the patient is doing well on follow-up. Thus, it is concluded that when the epididymal cyst is larger than 10 mm or 1 cm and does not involute with time, surgery is indicated. In comparison, epididymal cysts smaller than 10 mm or 1 cm are managed conservatively.

Understanding the Impact of Multi-factorial Composition on Efficient Loading of the Stable Ketoprofen Nanoparticles on Orodispersible Films Using Box-Behnken Design.

The purpose of the present study was to prepare Orodispersible films (ODFs) loaded with ketoprofen nanoparticles (KT-NP). The Box-Behnken design was constructed in developing and optimizing the KTF-NP-ODFs. The effect of independent variables: Soluplus® concentration (X1, stabilizer), Tween 80 concentration (X2, surfactant), and KTF concentration (X3, drug) were studied on the dependent variables: particle size (PS, Y1), zeta potential (ZP, Y2), and the polydispersity index (PDI, Y3) of the NPs, as well as on the tensile strength (TS, Y4) and permeability coefficient (PC, Y5) of the KTF-NP-ODFs. Hydroxypropyl methylcellulose (HPMC E15) and polyethylene glycol (PEG 400) were used as the film former polymer and plasticizer, respectively, and their concentrations were kept constant for all formulations. KTF-NPs were prepared by antisolvent precipitation technology. This was followed by the addition of HPMC E15 and PEG 400 to prepare the ODFs using the solvent-casting method. The PS, PDI, and ZP for all the formulations were found in the range of 94 nm to 350 nm, 0.09 to 0.438, and -21.83 mV to -8.03 mV, respectively. The TS and PC of the prepared KTF-NP-ODFs were found between 1.21 MPa to 3.93 MPa and 3.12 × 10-4 cm/h to 34.23 × 10-4 cm/h, respectively. The amorphous nature of the KTF-NP in the ODFs was confirmed by the absence of characteristic crystalline peaks and endothermic events of KTF in X-ray diffraction (XRD) and modulated differential scanning calorimetry (mDSC), respectively. The optimized formulation showed Ì´ 4 times higher permeability as compared to the pure KTF. In addition, the dissolution of pure KTF and the optimized KTF-NP-ODF in pH 1.2 at the end of 60 min was found to be Ì´ 30% and Ì´ 95%, respectively. Conclusively, KTF-NP-ODFs can be a promising drug delivery system to counter the issues related to dysphagia and bypass the common side effects, such as the gastric irritation associated with NSAIDs like KTF.

Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells.

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the ß(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

Hybridized nanoamorphous micellar dispersion using a QbD-DM3 linked rational product design strategy for ritonavir: A BCS IV drug.

A QbD-DM3 linked rational product design strategy was adopted to create a hybridized ritonavir (RTV, BCS Class IV) nanoamorphous micellar dispersion (RTV-NAD). A DM3 research strategy was employed in conjunction with the quality-by-design spaces, and quality target product profile to link the critical material attributes and critical process parameters to the quality target product profile's critical product attributes QbD elements. A Box-Behnken design and multivariate analysis using multiple linear regression and partial least squares provided data analysis. The hybridized strategy leveraged three different mechanisms to increase RTV's solubility and four mechanisms to increase its dissolution rate. Statistically significant models were generated for critical product attributes: particle size (p = 0.0000, R2 adjusted = 0.9513), polydispersity index (p = 0.0002, R2 adjusted = 0.6398), zeta potential (p = 0.0000, R2 adjusted = 0.9744), and drug loading on a dry basis (p = 0.0000, R2 adjusted = 0.9951). The impact of drug concentration, Soluplus® concentration, and solvent:antisolvent ratio, their interactions and square effects on the critical product attributes were assessed by multivariate analysis. The QbD optimal formulation was determined for RTV-NAD. Multiple linear regression and partial least squares computational predictability was evaluated using three verification batches. The prediction error for critical product attributes was <5%. RTV-NAD and ritonavir microsuspension were characterized by x-ray diffraction and in-vitro dissolution studies. X-ray diffraction confirmed the amorphous nature of the RTV-NAD. RTV-NAD exhibited a 'spring-hover' dissolution profile at pH 4.5. At pH 6.8, a classic 'spring-parachute' dissolution behavior was observed.

Systematic implementation of quality-by-design (QbD) to develop NSAID-loaded nanostructured lipid carriers for ocular application: preformulation screening studies and statistical hybrid-design for optimization of variables.

The objective of the present project was to develop and optimize the Ibuprofen (IBU)-loaded nanostructured lipid carrier (IBU-NLCs) for sustained-release ocular drug delivery using a quality-by-design (QbD) approach. The BCS class II drug IBU was selected as the model drug for the preparation of IBU-NLCs by melt-emulsification and ultrasonication technique. Extensive preformulation screening of the components of NLC dispersion (i.e. solid and liquid lipid, surfactants, and osmolality agents) was performed. From the various lipids screened, Dynasan®114 and Miglyol®840 were selected as the most suitable solid and liquid lipid, respectively. These lipids, at a matrix ratio of 6:4, demonstrated a lower melting-point and crystallinity-index based on DSC, XRD, and compatibility studies. Various surfactants were evaluated, and among them, Kolliphor®HS15 demonstrated lower z-average particle size (PS) and polydispersity index (PDI), while Kolliphor®P188 resulted in a zeta potential (ZP) <-20 mV. Glycerol was selected from various osmolality agents due to its negligible effects on physicochemical properties of the optimized formulation. A Plackett-Burman design (PBD) was used for the initial screening of the critical variables, followed by a Box-Behnken design (BBD) for further optimization of the NLC dispersion. The optimized formulation demonstrated the PS of 147 nm, with narrow PDI (0.159), ZP of -25.7 mV, and an entrapment efficiency (EE) of 97.89%. In vitro diffusion of IBU from the optimized IBU-NLC dispersion showed a sustained-release of ∼51% for up to 12 h. Preformulation studies and a statistical hybrid-design approach was effectively applied to incorporate IBU in NLCs, resulting in a robust ophthalmic formulation with superior physicochemical properties.

Human volunteer, in vitro, and molecular level evaluation of an optimized taste-masked isoniazid-chitosan spray-dried microparticle matrix.

For the first time, isoniazid (INH) bitterness value, threshold, and sensitivity (low, moderate, high, and extremely high) was determined in six human volunteers. INH demonstrated a large range in bitterness sensitivity. The current work demonstrates the design of a taste-masked isoniazid (INH)-loaded chitosan microspheres (INH-LCM) using an ionic-gelation and spray drying technique. A 24 full factorial design with three center points was employed to optimize and study the independent variables (chitosan concentration, sodium tripolyphosphate (TPP)-volume, feed rate, and air inlet temperature) effects on the critical quality attributes (percent yield [PY] and entrapment efficiency [EE]). Statistically significant models were developed for PY (p = 0.0357; adjusted R2 = 0.6078) and EE (p = 0.0190; adjusted R2 = 0.6713). A multicriteria prediction profiler was utilized to determine the optimum formulation and process parameters. Two verification batches confirmed excellent predictability and lot-to-lot consistency. In vitro dissolution was used to evaluate the taste masking ability of INH-LCM. The in vitro dissolution test of the optimized INH-LCM suggested that taste masking would be accomplished for the "low" and "moderate" bitterness taste sensitivity groups. Further in vitro and human volunteer taste panel studies with INH-LCM are required for better understand the potential taste masking capability for the "high" and "extremely high" bitterness taste sensitivity groups. The in vitro dissolution method and FTIR data analysis support that TPP crosslinked chitosan may provide taste masking by two mechanisms: (1) acts as a physical barrier and delays INH dissolution; and (2) provides a chemical barrier by forming hydrogen bonds between INH's bitter tasting amino group and chitosan.

Forgotten long-term indwelling double "J" stent.

A poor, uneducated patient from a rural background presented to us with burning micturition and colicky pain in the loin. He had undergone surgery for pelvi-ureteric junction (PUJ) obstruction on the right side four years earlier. Following surgery, the patient was irregular in his follow-up and, as such, he did not get the double "J" (DJ) stent, which was placed during surgery, removed. Ultrasonography performed during the present admission revealed mild hydronephrosis of the right kidney with a tiny calculus in the urinary bladder. Intravenous urography revealed mild hydronephrosis with the DJ stent in situ in the right kidney. After an unsuccessful attempt with cystoscopy, the stent was removed successfully by suprapubic cystostomy. The post-operative course was uneventful and the patient was discharged in seven days.

Asymptomatic infantile cystic nephroma: a diagnostic dilemma.

Cystic nephroma, also called multilocular cystic nephroma, is a relatively rare, nongenetic, benign, unilateral, renal multicystic lesion. The non-specific clinical findings and the poor contribution of imaging examinations make the preoperative diagnostic dilemma from other cystic renal neoplasia; thus nephrectomy seems to be the most preferable treatment. We report a case of cystic nephroma in 11 months old male child presented with asymptomatic lump in abdomen. After a series of examinations including abdominal ultrasound, intravenous pylography and computed tomography, he underwent radical nephrectomy and diagnosis is confirmed on histopathology.

Rural centre based management of the carotid body tumour.

A 40 year old male patient reported to our rural based hospital with a complaint of discomfort associated with a swelling on the left side of the neck since 8 years. A provisional diagnosis of a carotid body tumour was made based on clinical examination and ultrasound examination. Higher investigations could not be performed due to unavailability at the rural setup and referral to a specialty centre was not possible due to financial constraints of the patient. Even with advances in diagnostic and surgical techniques, surgery still presents a major threat of injury to the cranial nerves. Nevertheless, it remains the preferred method of treatment for these tumours. Our case shows that such masses can be removed successfully and that, with care, the cranial nerves and the carotid arteries can be preserved at the rural hospital.

An omental teratoma in a young girl.

Teratoma is the most commonly encountered germ cell tumour among the most common ovarian tumours; however, teratomas of the omentum and mesentery are extremely rare. They are usually asymptomatic or can produce compressive symptoms. The imaging features are suggestive. The present report describes such a case of primary omental teratoma encountered in a young patient, which was managed by surgical resection. The histopathological examination confirmed the diagnosis of mature cystic teratoma. Germ cell tumors are congenital tumors containing derivatives of all the three germinal layers, frequently seen in gonads. But their occurrence in extragonadal sites, such as omental teratoma, is relatively rare.