A framework for the biophysical screening of antibody mutations targeting solvent-accessible hydrophobic and electrostatic patches for enhanced viscosity profiles.

The formulation of high-concentration monoclonal antibody (mAb) solutions in low dose volumes for autoinjector devices poses challenges in manufacturability and patient administration due to elevated solution viscosity. Often many therapeutically potent mAbs are discovered, but their commercial development is stalled by unfavourable developability challenges. In this work, we present a systematic experimental framework for the computational screening of molecular descriptors to guide the design of 24 mutants with modified viscosity profiles accompanied by experimental evaluation. Our experimental observations using a model anti-IL8 mAb and eight engineered mutant variants reveal that viscosity reduction is influenced by the location of hydrophobic interactions, while targeting positively charged patches significantly increases viscosity in comparison to wild-type anti-IL-8 mAb. We conclude that most predicted in silico physicochemical properties exhibit poor correlation with measured experimental parameters for antibodies with suboptimal developability characteristics, emphasizing the need for comprehensive case-by-case evaluation of mAbs. This framework combining molecular design and triage via computational predictions with experimental evaluation aids the agile and rational design of mAbs with tailored solution viscosities, ensuring improved manufacturability and patient convenience in self-administration scenarios.

Micelle-Formulated Juglone Effectively Targets Pancreatic Cancer and Remodels the Tumor Microenvironment.

Pancreatic cancer remains a formidable challenge due to limited treatment options and its aggressive nature. In recent years, the naturally occurring anticancer compound juglone has emerged as a potential therapeutic candidate, showing promising results in inhibiting tumor growth and inducing cancer cell apoptosis. However, concerns over its toxicity have hampered juglone's clinical application. To address this issue, we have explored the use of polymeric micelles as a delivery system for juglone in pancreatic cancer treatment. These micelles, formulated using Poloxamer 407 and D-α-Tocopherol polyethylene glycol 1000 succinate, offer an innovative solution to enhance juglone's therapeutic potential while minimizing toxicity. In-vitro studies have demonstrated that micelle-formulated juglone (JM) effectively decreases proliferation and migration and increases apoptosis in pancreatic cancer cell lines. Importantly, in-vivo, JM exhibited no toxicity, allowing for increased dosing frequency compared to free drug administration. In mice, JM significantly reduced tumor growth in subcutaneous xenograft and orthotopic pancreatic cancer models. Beyond its direct antitumor effects, JM treatment also influenced the tumor microenvironment. In immunocompetent mice, JM increased immune cell infiltration and decreased stromal deposition and activation markers, suggesting an immunomodulatory role. To understand JM's mechanism of action, we conducted RNA sequencing and subsequent differential expression analysis on tumors that were treated with JM. The administration of JM treatment reduced the expression levels of the oncogenic protein MYC, thereby emphasizing its potential as a focused, therapeutic intervention. In conclusion, the polymeric micelles-mediated delivery of juglone holds excellent promise in pancreatic cancer therapy. This approach offers improved drug delivery, reduced toxicity, and enhanced therapeutic efficacy.

Lyotropic liquid crystals for parenteral drug delivery.

The necessity for long-term treatments of chronic diseases has encouraged the development of novel long-acting parenteral formulations intending to improve drug pharmacokinetics and therapeutic efficacy. Lately, one of the novel approaches has been developed based on lipid-based liquid crystals. The lyotropic liquid crystal (LLC) systems consist of amphiphilic molecules and are formed in presence of solvents with the most common types being cubic, hexagonal and lamellar mesophases. LC injectables have been recently developed based on polar lipids that spontaneously form liquid crystal nanoparticles in aqueous tissue environments to create the in-situ long-acting sustained-release depot to provide treatment efficacy over extended periods. In this manuscript, we have consolidated and summarized the various type of liquid crystals, recent formulation advancements, analytical evaluation, and therapeutic application of lyotropic liquid crystals in the field of parenteral sustained release drug delivery.

Microfluidics Formulated Liposomes of Hypoxia Activated Prodrug for Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) presents as an unmet clinical challenge for drug delivery due to its unique hypoxic biology. Vinblastine-N-Oxide (CPD100) is a hypoxia-activated prodrug (HAP) that selectively converts to its parent compound, vinblastine, a potent cytotoxic agent, under oxygen gradient. The study evaluates the efficacy of microfluidics formulated liposomal CPD100 (CPD100Li) in PDAC. CPD100Li were formulated with a size of 95 nm and a polydispersity index of 0.2. CPD100Li was stable for a period of 18 months when freeze-dried at a concentration of 3.55 mg/mL. CPD100 and CPD100Li confirmed selective activation at low oxygen levels in pancreatic cancer cell lines. Moreover, in 3D spheroids, CPD100Li displayed higher penetration and disruption compared to CPD100. In patient-derived 3D organoids, CPD100Li exhibited higher cell inhibition in the organoids that displayed higher expression of hypoxia-inducible factor 1 alpha (HIF1A) compared to CPD100. In the orthotopic model, the combination of CPD100Li with gemcitabine (GEM) (standard of care for PDAC) showed higher efficacy than CPD100Li alone for a period of 90 days. In summary, the evaluation of CPD100Li in multiple cellular models provides a strong foundation for its clinical application in PDAC.

A clinically relevant formulation for direct administration of nerve specific fluorophores to mitigate iatrogenic nerve injury.

Iatrogenic nerve injury significantly affects surgical outcomes. Although intraoperative neuromonitoring is utilized, nerve identification remains challenging and the success of nerve sparing is strongly correlated with surgeon experience levels. Fluorescence guided surgery (FGS) offers a potential solution for improved nerve sparing by providing direct visualization of nerve tissue intraoperatively. However, novel probes for FGS face a long regulatory pathway to achieve clinical translation. Herein, we report on the development of a clinically-viable, gel-based formulation that enables direct administration of nerve-specific probes for nerve sparing FGS applications, facilitating clinical translation via the exploratory investigational new drug (eIND) guidance. The developed formulation possesses unique gelling characteristics, allowing it to be easily spread as a liquid followed by rapid gelling for subsequent tissue hold. Optimization of the direct administration protocol with our gel-based formulation enabled a total staining time of 1-2 min for compatibility with surgical procedures and successful clinical translation.

Early Experience of High-dose Intravenous Mycobacterium w in Critically Ill Patients of COVID-19.

BACKGROUND: Immune dysregulation is one of the main reasons for mortality and morbidity in coronavirus disease 2019 (COVID-19). Mycobacterium w (Mw) is recently approved for gram-negative sepsis. Moreover, it is also found effective in COVID-19 patients in previous studies. The traditional route of administration for Mw is intradermal, which has a limitation of administering 0.1 mL per injection and local injection site reaction. Intravenous (IV) administration of Mw has not been explored in COVID-19. We report the retrospective analysis of six critically ill COVID-19 patients who received Mw (IV). PATIENTS AND METHODS: At baseline, all patients in this case series required O2 supplementation, and their inflammatory biomarkers were elevated. All patients received 0.6 mL Mw (high-dose) in normal saline along with the standard-of-care treatment. RESULTS: After Mw administration, gradual improvement in O2 requirement was observed and patients were discharged from the hospital with no mortality. A reduction in mean C-reactive protein (CRP) (51.48-18.52 mg/dL), interleukin-6 (IL-6) (260.22-14.47 pg/mL), and FiO2 (81.67-43.33) was also observed. No side effects were observed with the use of Mw by IV route. CONCLUSION: Use of 0.6 mL Mw by IV route in this case series was associated with decreased O2 supplementation without any side effects in critically ill patients of COVID-19. HOW TO CITE THIS ARTICLE: Patel PS, Patel S, Shah V, Aswani V, Narwaria M. Early Experience of High-dose Intravenous Mycobacterium w in Critically Ill Patients of COVID-19. Indian J Crit Care Med 2021;25(9):1066-1068.

Clinically translatable formulation strategies for systemic administration of nerve-specific probes.

Nerves are extremely difficult to identify and are often accidently damaged during surgery, leaving patients with lasting pain and numbness. Herein, a novel near-infrared (NIR) nerve-specific fluorophore, LGW01-08, was utilized for enhanced nerve identification using fluorescence guided surgery (FGS), formulated using clinical translatable strategies. Formulated LGW01-08 was examined for toxicology, pharmacokinetics (PK), and pharmacodynamics (PD) parameters in preparation for future clinical translation. Optimal LGW01-08 imaging doses were identified in each formulation resulting in a 10x difference between the toxicity to imaging dose window. Laparoscopic swine surgery completed using the da Vinci surgical robot (Intuitive Surgical) demonstrated the efficacy of formulated LGW01-08 for enhanced nerve identification. NIR fluorescence imaging enabled clear identification of nerves buried beneath ~3 mm of tissue that were unidentifiable by white light imaging. These studies provide a strong basis for future clinical translation of NIR nerve-specific fluorophores for utility during FGS to improve patient outcomes.

Metabolic convergence on lipogenesis in RAS, BCR-ABL, and MYC-driven lymphoid malignancies.

BACKGROUND: Metabolic reprogramming is a central feature in many cancer subtypes and a hallmark of cancer. Many therapeutic strategies attempt to exploit this feature, often having unintended side effects on normal metabolic programs and limited efficacy due to integrative nature of metabolic substrate sourcing. Although the initiating oncogenic lesion may vary, tumor cells in lymphoid malignancies often share similar environments and potentially similar metabolic profiles. We examined cells from mouse models of MYC-, RAS-, and BCR-ABL-driven lymphoid malignancies and find a convergence on de novo lipogenesis. We explore the potential role of MYC in mediating lipogenesis by 13C glucose tracing and untargeted metabolic profiling. Inhibition of lipogenesis leads to cell death both in vitro and in vivo and does not induce cell death of normal splenocytes. METHODS: We analyzed RNA-seq data sets for common metabolic convergence in lymphoma and leukemia. Using in vitro cell lines derived in from conditional MYC, RAS, and BCR-ABL transgenic murine models and oncogene-driven human cell lines, we determined gene regulation, metabolic profiles, and sensitivity to inhibition of lipogenesis in lymphoid malignancies. We utilize preclinical murine models and transgenic primary model of T-ALL to determine the effect of lipogenesis blockade across BCR-ABL-, RAS-, and c-MYC-driven lymphoid malignancies. Statistical significance was calculated using unpaired t-tests and one-way ANOVA. RESULTS: This study illustrates that de novo lipid biogenesis is a shared feature of several lymphoma subtypes. Using cell lines derived from conditional MYC, RAS, and BCR-ABL transgenic murine models, we demonstrate shared responses to inhibition of lipogenesis by the acetyl-coA carboxylase inhibitor 5-(tetradecloxy)-2-furic acid (TOFA), and other lipogenesis inhibitors. We performed metabolic tracing studies to confirm the influence of c-MYC and TOFA on lipogenesis. We identify specific cell death responses to TOFA in vitro and in vivo and demonstrate delayed engraftment and progression in vivo in transplanted lymphoma cell lines. We also observe delayed progression of T-ALL in a primary transgenic mouse model upon TOFA administration. In a panel of human cell lines, we demonstrate sensitivity to TOFA treatment as a metabolic liability due to the general convergence on de novo lipogenesis in lymphoid malignancies driven by MYC, RAS, or BCR-ABL. Importantly, cell death was not significantly observed in non-malignant cells in vivo. CONCLUSIONS: These studies suggest that de novo lipogenesis may be a common survival strategy for many lymphoid malignancies and may be a clinically exploitable metabolic liability. TRIAL REGISTRATION: This study does not include any clinical interventions on human subjects.

Thermoresponsive liquid crystalline formulation of Exemestane: Design and structural characterization.

Exemestane (EXE), a drug used for the treatment of breast cancer, has limited aqueous solubility of 0.08 mg/mL and log P∼ 4.22. The only available marketed formulation in form of tablets possess limitations of poor oral absorption (∼ 42 %), low solubility, extensive hepatic metabolism and numerous adverse effects due to its peripheral absorption. In order to address these issues, an alternative route of topical application is attempted through a lamellar liquid crystal based formulation. Pluronic® was used as stabilizer due to its higher surface activity and gelling properties. The solubility enhancement of EXE was achieved using liquid crystal formulation. We have investigated the effect of concentration of oil, Smix (surfactant - cosurfactant mixture) and EXE on lattice parameter, rheology and drug release for various combinations of the formulation. The small angle x-ray scattering (SAXS) measurement demonstrated an evidence of a lamellar structure with lattice parameter ∼15 nm, which increases with corresponding increase in oil and EXE due to increase in hydrophobic interactions leading to an expansion of lamella. The inter lamellar distance decreases at higher surfactant concentration, due to the distribution of the same amount of oil and drug within larger concentration of surfactant molecules. The rheology measurement exhibited gel like properties at low shear rate indicating soft gel formation, which converts to Newtonian type flowing liquid at higher shear rate. At constant Smix with increasing oil content, the viscosity decreases, which is attributed to the dilution of the lamellar structures with oil. The temperature sweep rheology reveals a change in the viscosity near physiological temperature, which may be attributed to the structural transition of lamellae. The formulation remains gel like at room temperature, which aids in proper application to skin and converts it to free flowing liquid above 37 °C. The invitro drug release of optimized formulation for 24 h was ∼ 38 % at 37 °C, which increased to 50 % at 42 °C. Accordingly, this formulation containing thermoresponsive lamellar liquid crystal gels of EXE represents a viable option for hyperthermia induced enhanced drug release. The characteristic and advantageous features offered by this formulation includes improved bioavailability of EXE due to enhanced solubility, permeability and absorption.

Prematurity and postnatal alterations in intermittent hypoxaemia.

Intermittent hypoxaemia (IH) events are well described in extremely preterm infants, but the occurrence of IH patterns in more mature preterm infants remains unclear. The objective of this study was to characterise the effect of gestational age on early postnatal patterns of IH in extremely (<28 weeks), very (28-<32 weeks) and moderately (32-<34 weeks) preterm infants. As expected, extremely preterm infants had a significantly higher frequency of IH events of longer durations and greater time with hypoxaemia versus very and moderately preterm infants. In addition, the postnatal decrease in IH duration was comparable in the very and moderately preterm infants. This progression of IH events should assist clinicians and families in managing expectations for resolution of IH events during early postnatal life.

Visual Rehabilitation With Contact Lenses Following Open Globe Trauma.

OBJECTIVES: To describe visual outcomes with various contact lens modalities in patients with a history of ocular trauma who underwent surgical open globe repair. METHODS: Records of all patients with a history of open globe injury and repair at a tertiary care hospital between January 1, 2010, and December 31, 2016, were reviewed. Demographics, type of injury, and visual acuity were assessed before and after contact lens evaluation. RESULTS: Of 214 patients who underwent open globe repair, 29 (13.6%) were evaluated with a contact lens. Visual acuity improved in 97% (28 of 29) of patients from 1.47±0.75 to 0.67±0.71 logarithm of the minimal angle of resolution (logMAR) with manifest refraction to 0.28±0.45 logMAR with contact lenses (n=29; P<0.0001). Corneal opacity was the most common clinical indication (20 of 29) for fitting followed by aphakia (13 of 29). A range of contact lens modalities, including corneal rigid gas-permeable (12 of 28), soft (9 of 28), hybrid (3 of 28), scleral gas-permeable (2 of 28), and piggyback (2 of 28) lenses were prescribed. CONCLUSION: In this study, patients with a history of trauma and open globe repair with good neurosensory visual potential had improvements in visual acuity with contact lens greater than manifest refraction. Soft and gas-permeable lenses were used to improve visual acuity in patients with a history of open globe repair and corneal scarring, aphakia, iris abnormalities, or other ocular sequelae. Although corneal rigid gas-permeable lenses were prescribed most often, additional consideration should be given to other contact lens modalities, including soft, piggyback, hybrid, and scleral gas-permeable lenses.

The Relationship between Oxidative Stress, Intermittent Hypoxemia, and Hospital Duration in Moderate Preterm Infants.

INTRODUCTION: Lipid peroxidation products are present following oxidation of polyunsaturated fatty acids in the eye, brain, and various cell membranes. Elevated levels of lipid peroxidation products and increased intermittent hypoxemia (IH) events have been associated with adverse outcomes in extremely preterm infants. The moderate preterm newborn has a still-developing oxidant defense system and immature respiratory control, but little is known about lipid peroxidation levels and IH in this larger and more common preterm population. OBJECTIVE: To determine the association between oxidative stress and IH in moderate preterm infants. METHOD: Oxygen saturation was continuously monitored in 51 moderate preterm infants (i.e., 31 + 0/7 to 33 + 6/7 weeks' gestation). Urine samples were collected at the end of the first and second weeks of life. Samples were analyzed for total lipid peroxidation products (neurofurans, isofurans, neuroprostanes, isoprostanes, and di-homo-isofurans). RESULT: At week 1, there was a correlation between increased IH frequency and neurofurans (p < 0.04) and di-homo-isofurans (p < 0.003). At week 2, there was no correlation between IH and lipid peroxidation markers. Ele-vations in neurofurans, isofurans, neuroprostanes, and di-homo-isofurans in the first and/or second week of life were associated with a longer stay in hospital. CONCLUSION: Elevations in lipid peroxidation biomarkers in moderate preterm infants during their first weeks of life are associated with a higher frequency of IH and prolonged hospitalization.

Hypoxia: Friend or Foe for drug delivery in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors with an overall five-year survival rate of that has only just reached 10%. The tumor microenvironment of PDAC is characterized by desmoplasia, which consist of dense stroma of fibroblasts and inflammatory cells, resulting in a hypoxic environment due to limited oxygen diffusion through the tumor. Hypoxia contributes to the aggressive tumor biology by promoting tumor progression, malignancy, and promoting resistance to conventional and targeted therapeutic agents. In depth research in the area has identified that hypoxia modulates the tumor biology through hypoxia inducible factors (HIFs), which not only are the key determinant of pancreatic malignancy but also an important target for therapy. In this review, we summarize the recent advances in understanding hypoxia driven phenotypes, which are responsible for the highly aggressive and metastatic characteristics of pancreatic cancer, and how hypoxia can be exploited as a target for drug delivery.

Select Stabilization of a Tumor-Suppressive PP2A Heterotrimer.

In cancer, suppression of protein phosphatases, such as protein phosphatase 2A (PP2A), that normally counteract kinases, contributes to aberrant signaling. Leonard et al. recently demonstrated that a novel small-molecule activator of PP2A, DT-061, selectively stabilizes a specific PP2A holoenzyme responsible for dephosphorylating critical oncogenic targets, including MYC. The 3.6-Å cryo-electron microscopy map of the heterotrimer assembly provides insight into the druggable structure of PP2A, guiding future phosphatase therapeutics.

Development of probiotic yogurt: effect of strain combination on nutritional, rheological, organoleptic and probiotic properties.

Seven combinations of yogurt; C1 [yogurt starter culture (YSC)], T1, [YSC + Lactobacillus acidophilus (LA)], T2 [YSC + Bifidobacterium bifidum (BB)], T3 [YSC + Lactobacillus plantarum (LP)], T4 [YSC + Lactobacillus casei (LC)], T5 [YSC + LA + BB] and T6 [YSC + LP + LC] were developed. Nutritional [proximate and minerals], rheological [total soluble solids (TSS), pH, titratable acidity (TA), water holding capacity, synersis, viscosity] organoleptic and probiotic properties [viability, acid tolerance, bile salt tolerance] were assessed with standard methods. Nutritional composition differed significantly among samples except for the iron and zinc (P < 0.05). Yogurt containing LP as single or in combination with LC resulted in significantly higher ash, protein, calcium and phosphorous level. Probiotic combination also significantly affected the rheological properties of yogurts (P < 0.05). Yogurt with LP and LC as single or in combination lead to significantly higher TSS and viscosity while significantly low syneresis, whereas yogurt with LA as single or in combination resulted in low pH and high TA (P < 0.05). Interestingly, combination of LA and BB increased TSS, reduced pH and syneresis as compare to these bacteria as single probiotic source. Panel experts found yogurt with LP more flavourful. Combination of multi-strain and multi-species probiotic resulted in improved texture but we found no significant difference in overall acceptability. Combination of probiotic strains also resulted in better probiotic potential with multi-species combination found to be even more effective. BB seemed more stable than three other probiotic strains. The present study can be helpful to dairy industry in developing new probiotic products and may provide a rational for selecting a combination of probiotic strains.

Spectral Doppler Ultrasound Can Help Diagnose Children With Hepatic Sinusoidal Obstructive Syndrome After Hematopoietic Stem Cell Transplantation.

BACKGROUND: Hepatic sinusoidal obstruction syndrome (SOS) is a potentially fatal complication after hematopoietic stem cell transplantation (HSCT). Current clinical guidelines state that spectral Doppler changes occur late in the disease, and imaging is only useful for confirming SOS diagnosis. OBJECTIVE: Our primary objective was to examine abdominal ultrasound variables as predictors of development of SOS in pediatric HSCT patients. MATERIALS AND METHODS: A single-center cohort retrospective study was conducted on patients aged 0 to 21 years who underwent HSCT between September 2001 and May 2017 at our institution. Patients were excluded if they did not have abdominal ultrasounds after HSCT. Clinical, demographic, grayscale, and spectral Doppler liver ultrasound findings were evaluated. We modeled the odds of SOS diagnosis within 100 days after HSCT as a function of each of the 15 ultrasound variables. RESULTS: A total of 333 patients received an HSCT. One hundred forty subjects had ultrasound data available. Thirty-two patients developed SOS, and 9 of these patients died. Sinusoidal obstruction syndrome odds more than double per 1-SD increase in peak systolic velocity in common hepatic artery or left hepatic artery and more than triple per 1-SD decrease in main portal vein velocity or change in ascites severity. Several ultrasound variables were statistically significant predictors in the Cox models for time to SOS diagnosis. CONCLUSION: Several ultrasound variables can be used as predictors for a patient's risk of developing SOS. The strongest predictors are ascites severity, main portal vein velocity, common hepatic artery peak systolic velocity, and left hepatic artery peak systolic velocity.

Chemosensitization and mitigation of Adriamycin-induced cardiotoxicity using combinational polymeric micelles for co-delivery of quercetin/resveratrol and resveratrol/curcumin in ovarian cancer.

This work looks to improve the efficacy of Adriamycin (ADR) while mitigating its cardiotoxicity using combinations of micellar resveratrol (R): quercetin (Q) (mRQ) or R: curcumin (C) (mRC) in healthy mice and ovarian cancer xenograft models. Ovarian cancer cells, ES2-Luc, or A2780ADR are inoculated in mice (n =4/group) and sorted into eight cohorts. Mice are treated weekly for 4 weeks with ADR, ADR+mRQ, ADR+mRC, or controls (saline, empty micelles, ADR+EM, mRQ, or mRC). To evaluate the degree of cardioprotection, serum is collected to determine the cardiac Troponin I (cTnI). Cardiac tissue is collected for morphological evaluation and evaluation of creatine kinase levels. Our results indicate that mRQ+ADR is statistically significant in tumor reduction in xenograft models. In healthy mice, the left ventricular ejection fraction and fractional shortening in the ADR treated group is most compromised. Co-administration of mRQ with ADR can reduce ADR dosing through chemosensitization while being cardioprotective.

Liposomes produced by microfluidics and extrusion: A comparison for scale-up purposes.

Successful liposomal formulations in the clinic are severely limited due to poor translational capability of the traditional bench techniques to clinical production settings. The gold standard for liposome bench manufacturing is a multi-step and parameter dependent extrusion method. Moreover, these parameters need re-optimization for clinical production. The microfluidics technique utilizes vigorous mixing of fluids at a nanoliter scale to produce liposomes in batches from milliliters to a couple liters. The fine control of process parameters results in improved reproducibility between batches. It is inherently scalable; however, the characteristics of liposomes produced by microfluidics both in vitro and in vivo have never been compared to those produced using extrusion. In this manuscript, we describe the comparison between the traditional extrusion method to microfluidics, the new paradigm in liposome production and scale-up.