Sustained Therapeutic Levels Research Articles

Nanosuspensions in Breast Cancer Therapy: A Comprehensive Overview

Nanosuspensions offer a promising avenue for enhancing breast cancer treatment through improved drug delivery, solubility, and targeting. These colloidal dispersions contain submicron drug particles, significantly increasing surface area and enhancing drug solubility and dissolution rates. This improvement can lead to increased drug bioavailability, enabling lower doses and reduced side effects. It is also possible to engineer nanosuspensions so that they are controlled-release drugs, providing sustained therapeutic levels at the tumor site. Nanosuspensions facilitate targeted drug delivery, which is one of their key advantages. In order to minimize systemic toxicity, nanosuspensions contain targeting ligands or antibodies that adhere to the surface of nanoparticles in order to deliver drugs specifically to breast cancer cells. The nanosuspension platform also allows a combination therapy approach to be used, allowing multiple drugs to be delivered simultaneously in order to achieve synergistic effects and combat drug resistance. Nanosuspension treatments can be beneficial both therapeutically and for imaging and diagnostic purposes. Using nanoparticles labeled with imaging agents, it is possible to visualize tumors and monitor treatment responses. Nanosuspensions may reduce side effects associated with traditional chemotherapy by improving drug targeting and reducing systemic exposure. A nanosuspension represents a promising treatment option for breast cancer. Nanosuspensions must be fully exploited for their full potential to improve outcomes and quality of life for patients.

Green Electrospun Nanofibers for Biomedicine and Biotechnology

Green electrospinning harnesses the potential of renewable biomaterials to craft biodegradable nanofiber structures, expanding their utility across a spectrum of applications. In this comprehensive review, we summarize the production, characterization and application of electrospun cellulose, collagen, gelatin and other biopolymer nanofibers in tissue engineering, drug delivery, biosensing, environmental remediation, agriculture and synthetic biology. These applications span diverse fields, including tissue engineering, drug delivery, biosensing, environmental remediation, agriculture, and synthetic biology. In the realm of tissue engineering, nanofibers emerge as key players, adept at mimicking the intricacies of the extracellular matrix. These fibers serve as scaffolds and vascular grafts, showcasing their potential to regenerate and repair tissues. Moreover, they facilitate controlled drug and gene delivery, ensuring sustained therapeutic levels essential for optimized wound healing and cancer treatment. Biosensing platforms, another prominent arena, leverage nanofibers by immobilizing enzymes and antibodies onto their surfaces. This enables precise glucose monitoring, pathogen detection, and immunodiagnostics. In the environmental sector, these fibers prove invaluable, purifying water through efficient adsorption and filtration, while also serving as potent air filtration agents against pollutants and pathogens. Agricultural applications see the deployment of nanofibers in controlled release fertilizers and pesticides, enhancing crop management, and extending antimicrobial food packaging coatings to prolong shelf life. In the realm of synthetic biology, these fibers play a pivotal role by encapsulating cells and facilitating bacteria-mediated prodrug activation strategies. Across this multifaceted landscape, nanofibers offer tunable topographies and surface functionalities that tightly regulate cellular behavior and molecular interactions. Importantly, their biodegradable nature aligns with sustainability goals, positioning them as promising alternatives to synthetic polymer-based technologies. As research and development continue to refine and expand the capabilities of green electrospun nanofibers, their versatility promises to advance numerous applications in the realms of biomedicine and biotechnology, contributing to a more sustainable and environmentally conscious future.

Gene therapy for cross-correction of somatic organs and the CNS in mucopolysaccharidosis II in rodents and non-human primates

Mucopolysaccharidosis II (MPS II) is a rare lysosomal storage disease characterized by deficient activity of iduronate-2-sulfatase (I2S), leading to pathological accumulation of glycosaminoglycans (GAGs) in tissues. We used iduronate-2-sulfatase knockout (Ids KO) mice to investigate if liver-directed recombinant adeno-associated virus vectors (rAAV8-LSP-hIDSco) encoding human I2S (hI2S) could cross-correct I2S deficiency in Ids KO mouse tissues, and we then assessed the translation of mouse data to non-human primates (NHPs). Treated mice showed sustained hepatic hI2S production, accompanied by normalized GAG levels in somatic tissues (including critical tissues such as heart and lung), indicating systemic cross-correction from liver-secreted hI2S. Brain GAG levels in Ids KO mice were lowered but not normalized; higher doses were required to see improvements in brain histology and neurobehavioral testing. rAAV8-LSP-hIDSco administration in NHPs resulted in sustained hepatic hI2S production and therapeutic hI2S levels in cross-corrected somatic tissues but no hI2S exposure in the central nervous system, perhaps owing to lower levels of liver transduction in NHPs than in mice. Overall, we demonstrate the ability of rAAV8-LSP-hIDSco to cross-correct I2S deficiency in mouse somatic tissues and highlight the importance of showing translatability of gene therapy data from rodents to NHPs, which is critical for supporting translation to clinical development.

Engineering Bifunctional Calcium Alendronate Gene‐Delivery Nanoneedle for Synergistic Chemo/Immuno‐Therapy Against HER2 Positive Ovarian Cancer

Ovarian cancer is the most lethal gynecological malignancy. Most patients are diagnosed at an advanced stage with widespread peritoneal dissemination and ascites. Bispecific T-cell engagers (BiTEs) have demonstrated impressive antitumor efficacy in hematological malignancies, but the clinical potency is limited by their short half-life, inconvenient continuous intravenous infusion, and severe toxicity at relevant therapeutic levels in solid tumors. To address these critical issues, the design and engineering of alendronate calcium (CaALN) based gene-delivery system is reported to express therapeutic level of BiTE (HER2×CD3) for efficient ovarian cancer immunotherapy. Controllable construction of CaALN nanosphere and nanoneedle is achieved by the simple and green coordination reactions that the distinct nanoneedle-like alendronate calcium (CaALN-N) with a high aspect ratio enabled efficient gene delivery to the peritoneum without system in vivo toxicity. Especially, CaALN-N induced apoptosis of SKOV3-luc cell via down-regulation of HER2 signaling pathway and synergized with HER2×CD3 to generate high antitumor response. In vivo administration of CaALN-N/minicircle DNA encoding HER2×CD3 (MC-HER2×CD3) produces sustained therapeutic levels of BiTE and suppresses tumor growth in a human ovarian cancer xenograft model. Collectively, the engineered alendronate calcium nanoneedle represents a bifunctional gene delivery platform for the efficient and synergistic treatment of ovarian cancer.

Development of a B cell protein factory to produce sustained therapeutic levels of alpha-galactosidase A for the treatment of Fabry disease

Development of a B cell protein factory to produce sustained therapeutic levels of alpha-galactosidase A for the treatment of Fabry disease

PLGA-The Smart Biocompatible Polimer: Kinetic Degradation Studies and Active Principle Release.

The aim of our research was the development of prolonged delivery systems for therapeutic agents with various properties (prevention and treatment of bone diseases, anti-neoplastic, anti-inflammatory, antioxidant) that would ensure sustained therapeutic levels of the active principle, above the minimum inhibitory concentration, without reaching toxic levels over a long period of time as alternatives to conventional routes of administration. PLGA (poly lactic-co-glycolic acid), a biodegradable and biocompatible synthetic polymer, FDA approved, with a 65:35 lactic acid (LA): glycolic acid (GA) copolymer ratio, was chosen as delivery system. Our studies have shown that in PBS it undergoes two simultaneous degradation processes, hydrolysis and autohydrolysis, degrading completely in about 40 days. The release of the active principle is determined by the diffusion from inside the polymer matrix to the outside, which occurs simultaneously with the erosion of the polymer, during 35 days.

Re-Sensitization of Allogeneic T Cell Resistant Murine AML with Pegylated IFN-Gamma

Background: The prognosis of AML relapsing after allogeneic hematopoietic stem cell transplant (alloHSCT) is dismal with very few salvage therapy options. Knowing the biological changes that occur at relapse could lead to potential treatment strategies. Using murine AML cells (Dnmt3aR878H/FLT3-ITD model, kind gift from Dr. Ley's lab), we generated allogeneic T cell resistant murine AML cells (alloTr AML1) via sequential MHC-mismatched alloHSCTs. As shown in Figure 1A, mice injected with the AML1 tumor and allogeneic T cells displayed a significantly decreased median survival time (MST) from >250 days (undefined) to 26 days during progression from the primary alloHSCT to the tertiary alloHSCT, respectively (P<0.0001, 1° HSCT vs. 3° HSCT). Genomic analysis using WES showed no mutations in MHC I, MHC II or genes associated in immune functions in the alloTr AML1 cells. Preliminary transcriptome analysis of the alloTr AML1 after the tertiary transplant shows downregulation of genes mediating cell-cycle regulation and activation of T cells, including regulation of IFN-gamma (IFNg) production pathways. Our group previously showed that human AML cells relapsing after alloHSCT downregulated MHC class II and that IFNg could re-induce MHC-II expression and restore allogeneic T cell-mediated cytotoxicity (Christopher et al, NEJM, 2019). Since native IFNg has a very short terminal half-life of approximately 1h in vivo, it necessitates frequent administration to maintain sustained therapeutic levels. Our collaborators at Bolder Biotech have generated site-specific PEGylated IFNg that exhibits a terminal half-life of 26h in rats and can be detected in the plasma for at least 6 days after a single injection (Fam et al, J Interferon & Cytokine Research, 2014). Here, we evaluated if PEGylated murine (PEG-muIFNg) from Bolder Biotech could re-sensitize alloTr AML1 cells to T cell killing after allo-HSCT. Method: Lethally irradiated B6 recipients were reconstituted with 129 SVJ bone marrow (BM) alone on day 0. On day 10 after alloHSCT, mice were injected with 1.5E6 alloTr AML cells and divided into the following groups: (1) untreated controls (BM only), (2) PEG-muIFNg only, (3) 5 x 106 allogeneic 129 SVJ T cells (allo-T) only on day 10 or (4) 5 x 106allogeneic 129 SVJ T cells on day 10 plus PEG-muIFNg. Two doses of PEG-muIFNg (15 ug or 3.8 ug per injection) were tested in these experiments and treatment was started on day 10 post-alloHSCT at the time of allo-T injection. A total of 6 PEG-muIFNg doses were given (3 doses/week x 2 weeks). Mice were monitored for survival and graft-versus-host disease (GVHD). AML1 tumor burden was assessed by FACS for GFP. Results: The untreated BM only controls died of AML within 18 days of tumor injection (Figure 1B). Treatment with PEG-muIFNg alone, slightly extended the median survival time (MST) of mice in a dose dependent manner with 15 ug PEG-muIFNg dosing providing a MST of 38 days while the 3.8ug dose level exhibited a MST of 32 days. Recipients treated with 5 x 106 allo-T cells alone succumbed to leukemia similar to the untreated controls (MST = 26 days), confirming that these cells were resistant to allo-T cell killing. However, mice treated with allo-T + 15 ug PEG-muIFNgexhibited prolonged survival (MST = 49 days) compared to the T cell only (MST= 26 days; p=0.0001) and the 15 ug PEG-muIFNg only controls (MST =38 days; p=0.0001). The 15 ug dose level provided greater graft-versus-leukemia (GVL) activity (only 9/15 mice relapsed vs. 13/15 with 3.8 ug dosing) but was associated with slightly higher GVHD (6/15 mice developed lethal GVHD). In contrast, the 3.8 ug dose had fewer GVHD related deaths (2/15) but provided slightly less GVL activity (13/15 mice succumbed to AML). Conclusion: Treatment with PEG-muIFNg beginning 10 days after allo-HSCT can re-sensitize the alloTr AML1 tumors to allogeneic T cell-mediated killing and provide a significant survival advantage. Ongoing studies are also examining the candidate IFNg signaling pathway genes dysregulated in our model to determine if they are upregulated after PEG-muIFNg treatment. Furthermore, future studies will explore if extending PEG-muIFNgtreatment at the 3.8 ug concentration beyond 2 weeks can enhance GVL activity while limiting lethal GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model.FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period.We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway.In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. DisclosuresNo relevant conflicts of interest to declare.

Intra-articular administration of vancomycin and tobramycin during primary cementless total knee arthroplasty : determination of intra-articular and serum elution profiles.

Intra-articular administration of antibiotics during primary total knee arthroplasty (TKA) may represent a safe, cost-effective strategy to reduce the risk of acute periprosthetic joint infection (PJI). Vancomycin with an aminoglycoside provides antimicrobial cover for most organisms isolated from acute PJI after TKA. However, the intra-articular doses required to achieve sustained therapeutic intra-articular levels while remaining below toxic serum levels is unknown. The purpose of this study is to determine the intra-articular and serum levels of vancomycin and tobramycin over the first 24 hours postoperatively after intra-articular administration in primary cementless TKA. A prospective cohort study was performed. Patients were excluded if they had poor renal function, known allergic reaction to vancomycin or tobramycin, received intravenous vancomycin, or were scheduled for same-day discharge. All patients received 600 mg tobramycin and 1 g of vancomycin powder suspended in 25 cc of normal saline and injected into the joint after closure of the arthrotomy. Serum from peripheral venous blood and drain fluid samples were collected at one, four, and 24 hours postoperatively. All concentrations are reported in µg per ml. A total of 22 patients were included in final analysis. At one, four, and 24 hours postoperatively, mean (95% confidence interval (CI)) serum concentrations were 2.4 (0.7 to 4.1), 5.0 (3.1 to 6.9), and 4.8 (2.8 to 6.9) for vancomycin and 4.9 (3.4 to 6.3), 7.0 (5.8 to 8.2), and 1.3 (0.8 to 1.8) for tobramycin; intra-articular concentrations were 1,900.6 (1,492.5 to 2,308.8), 717.9 (485.5 to 950.3), and 162.2 (20.5 to 304.0) for vancomycin and 2,105.3 (1,389.9 to 2,820.6), 403.2 (266.6 to 539.7), and 98.8 (0 to 206.5) for tobramycin. Intra-articular administration of 1 g of vancomycin and 600 mg of tobramycin as a solution after closure of the arthrotomy in primary cementless TKA achieves therapeutic intra-articular concentrations over the first 24 hours postoperatively and does not reach sustained toxic levels in peripheral blood. Cite this article: Bone Joint J2021;103-B(11):1702-1708.

Current Updates in Transdermal Therapeutic Systems and Their Role in Neurological Disorders.

The transdermal therapeutic system plays an important role in the treatment of neuropathic pain. Transdermal drug delivery is considered an ideal therapeutic approach for the management of chronic neurological disorders in the elderly population. It is a simple to use, non-invasive and painless drug delivery system, which provides sustained therapeutic plasma levels of drug for an extended period. Moreover, it bypasses the first-pass metabolism of the active agent, improves bioavailability and reduces undesired adverse effects, which in turn improves patient compliance. Several transdermal delivery systems are currently under investigation for the treatment of Parkinson's syndrome, Alzheimer's disorders, and Neurological pain. Drug delivery via the transdermal route is proposed as an alternative remedy to overcome the drawbacks associated with the conventional dosage forms for chronic neurological disorders. The management of Alzheimer's disease via transdermal drug administration exhibits the greatest therapeutic improvements in the treatment of cognition and global functioning among neuropathic patients. Technological breakthroughs in transdermal drug administration such as the microreservior system, microneedles, metered-dose transdermal spray (MDTS), needle-free injections, and ultrasound-based transdermal therapeutic systems have been successfully used to treat neurological disorders. For example, microneedle (MN) is a highly efficient and versatile device due to its distinctive properties. Ultrasounds have been very popular for the delivery of bioactive agents across the skin barrier. This review focuses on the recent advances of various technologies employed in the transdermal therapeutic systems and their applications on neurological disorders for achieving therapeutic outcomes on patients.

Pediatric Antibiotic Stewardship: Optimization of Vancomycin Therapy Based on Individual Pharmacokinetics.

Vancomycin has been a first-line treatment for Gram-positive infections for decades. However, strategies for therapeutic drug monitoring (TDM) and dose-optimization in pediatrics remain controversial. In this study, we analyzed the impact of specific antibiotic stewardship interventions on efficacy and safety of vancomycin therapy. From September 2014 to May 2017, we conducted a prospective study to compare a control and a TDM intervention group in our tertiary care center. As part of an antibiotic stewardship program, we implemented internal guidelines on correct vancomycin dosing, TDM timing, as well as targeted trough level range and installed a pharmacokinetic (PK) consultation service to adapt vancomycin dosing to individually calculated PK parameters. As primary clinical outcomes, the percentage of patients with sustained therapeutic vancomycin trough levels and treatment days with therapeutic vancomycin trough levels, that is, 10-15 mg/L were analyzed. Secondary outcomes included nephrotoxicity, readmission rate and mortality. Median daily dose required to achieve therapeutic trough levels was examined. Clinical outcomes for 90 control patients were compared with outcomes for 19 patients guided by a PK consultation service. Percentage of patients with sustained therapeutic vancomycin trough levels increased from 17.8% to 94.7% (P < 0.001) and percentage of treatment days with therapeutic vancomycin trough levels increased from 18.4% (117/637) to 665% (155/233, P < 0.001). Readmission rate decreased from 24.4% to 5.3% (P = 0.07). No differences in nephrotoxicity or mortality rate were observed between groups. A median daily dose of 72 mg/kg/d was required to achieve therapeutic trough levels. Our data demonstrate that implementation of internal guidelines and a PK consultation service was associated with a profound improvement of vancomycin therapy and, therefore, patient safety.

In Vivo Electroporation-Mediated, Intrahepatic Alpha1 Antitrypsin Gene Transfer Reduces Pulmonary Emphysema in Pallid Mice.

Rationale: Mutation in the alpha1 antitrypsin (AAT) gene leads to low circulating levels of AAT, which is associated with several disease processes including pulmonary emphysema. The standard of care relies on substitution with plasma-purified AAT. We studied a novel approach to obtain sustained therapeutic levels of circulating AAT using nonviral in vivo electroporation-mediated gene transfer to the liver. Methods: In vivo intrahepatic electroporation-mediated human AAT gene transfer was performed in C57 Bl/6J mice carrying a genetic deficiency of murine AAT (pallid mice) and suffering from pulmonary emphysema. The animals were evaluated for lung function using flexiVent and detailed stereological assessments. Lung neutrophilic burden was assessed. Results: Pallid mice showed morphologically detectable pulmonary emphysema. Thirty days after in vivo electroporation-mediated gene transfer directly aimed at the liver, circulating human AAT was elevated and lung function was significantly improved compared to non-treated pallid mice. Stereological analysis revealed a reduction in pulmonary emphysema. Conclusion: Our data indicate that in vivo intrahepatic electroporation-mediated gene transfer of AAT is a safe and efficient procedure resulting in reduction of pulmonary emphysema in pallid mice.

Carbamazepine bilayer tablets combining hydrophilic and hydrophobic cyclodextrins as a quick/slow biphasic release system

This work aimed to explore the combination of hydrophilic and hydrophobic cyclodextrins to prepare bilayer tablets that can perform as quick/slow biphasic release systems of a poorly soluble drug. This formulation approach is of particular interest for treatments that require a rapid action followed by sustained therapeutic levels. Carbamazepine (CBZ), an antiepileptic and anticonvulsant agent, was used as a model of a BCS Class 2 drug. Hydroxypropyl-β-cyclodextrin (HPβCD) was chosen as complexing agent in the rapid release layer. Differently, triacetyl-β-cyclodextrin (TAβCD) was tested as controlling release agent in the sustained release layer. Croscarmellose sodium was utilized as superdisintegrant in the rapid release layer, and sodium stearyl fumarate was applied as anti-adherent lubricant in both layers. Bilayer tablets were characterized through several techniques. The results highlighted the feasibility of the combination of HPβCD/CBZ inclusion complex with croscarmellose sodium in the rapid release layer to achieve fast dissolution for the first 30–45 min, and TAβCD as controlling agent in the sustained release layer of the bilayer tablets to obtain a prolonged release during 720 min. In conclusion, combinations of hydrophilic and hydrophobic cyclodextrins may help addressing the formulation of poorly soluble drugs in bilayer tablets as a fast/slow biphasic release system.

Formulation and Development of Extended Release Ocusert for Gemifloxacin Mesylate with Dexamethsaone

Ocular drug delivery is one of the most challenging and fascinating task faced by the Pharmaceutical Researchers. The challenges to the formulators are to circumvent the protective barriers of the eye without causing permanent tissue damage, maintain a stable therapeutic level at the site of action for a prolonged period of time and reduce the frequency of instillation thereby maintaining a continuous sustained therapeutic level. The objective of the present investigation is to formulate a novel drug delivery system for ophthalmic use for the treatment of ocular conjunctivitis. Ocuserts of Gemifloxacin mesylate and Dexamethasone were formulated and evaluated. Materials and Methods: Initially preformulation studies of Gemifloxacin mesylate, Dexamethasone, polymer and excipients were performed to check their compatibility. Different combinations of Gemifloxacin mesylate, Dexamethasone, Carbopol 974, 980 981, PEG 400 and glycerine were formulated by solvent cast method and evaluated. Results and Discussion: The formulated ocusert was evaluated for clarity, smoothness, surface pH, drug content, and in-vitro drug release study. Conclusion: Formula GD 74 fulfilled the needs of all organoleptic parameters and also the in vitro release study. These Ocusert will be surely an alternative for conventional eye drops and boon to patients in future.

Correcting Rare Blood Disorders Using Coagulation Factors Produced In Vivo By Shielded Living TherapeuticsTM Products

Hemophilia A arises from mutations in the F8 gene, affecting ~ 1/5000 males. Treatment options include frequent intravenous factor and subcutaneous non-factor therapies. While these approaches have been widely used, they have significant limitations, such as breakthrough bleeds and joint disease due to suboptimal adherence, non-ideal factor kinetics, inhibitor generation, (Weyand, Blood 2018) as well as risk of thrombotic events and coagulation test interference with newer non-factor therapies. (Peters, Nat Rev Drug Discov 2018) Alternative modalities such as cell therapies with genetically modified, ready-made human cells are being investigated. To avoid a cytotoxic immune response by the host, allogeneic cells either need to be physically shielded and/or the host immunosuppressed. Various biomaterials, e.g. hydrogels, could serve as the physical barrier that prevents host immune cells from accessing the allogeneic cells, avoiding the need for immunosuppression altogether. However, the host can still activate a foreign body response (FBR), targeting the biomaterial, which significantly limits cell survival and durability of cell therapies. (Anderson, Semin Immunol 2008) We have successfully identified a library of proprietary small molecules, which when conjugated to alginate used to create encapsulating spheres, limit the FBR (Bochenek, Nat Biomed Eng 2018). In addition, we further reduced the FBR using two-compartment design, 1.5 mm diameter spheres, in which the cells are encapsulated in an inner compartment surrounded by an outer, acellular compartment. Using this innovative technology, we aimed to create a novel product that will deliver long-term, sustained human coagulation factor VIII (hFVIII) in vivo. First, we selected a human epithelial cell line with optimal properties for encapsulation within the spheres; considerations included safety, contact inhibition and longevity. We genetically modified this cell line using a non-viral vector and an optimized the coding sequence for a B-domain deleted hFVIII to create a proprietary engineered cell line that constitutively expresses high levels of this protein. Second, we optimized the inner compartment matrix by modulating cell density/sphere and by the addition of a novel modified alginate; these changes maximized cell viability and protein production in vivo. Finally, we further optimized the acellular outer compartment with a proprietary mixture of small-molecule-modified and unmodified alginates. The resulting SIG-001 product candidate consists of two-compartment, 1.5 mm spheres encapsulating hFVIII-expressing human cells. The spheres are sufficiently porous to allow gasses, nutrients, and secreted proteins to freely diffuse, while limiting FBR and prohibiting cell contact with the host's tissues including immune cells. Our in vitro studies demonstrated similar secretion of hFVIII protein by non-encapsulated and encapsulated engineered cells, along with viability of the same cell line after encapsulation. Several doses of SIG-001 were administered intraperitoneally to mice. Stable hFVIII production and good cell viability was shown for spheres retrieved after long-term placement in immunocompromised mice (up to 6 months). Furthermore, our data showed dose-responsive hFVIII activity and efficacious correction of the bleeding phenotype in immunocompetent Hemophilia A mice (Carmona, ISTH 2019). In conclusion, SIG-001 can deliver sustained therapeutic plasma levels of hFVIII in vivo. Our technology could eliminate the need for regular factor injections, lowering the patient burden and providing consistent factor levels without the peaks and troughs observed with factor and non-factor therapies. It also has the potential for expanded use in pediatric patients, and allows for re-dosing if needed. Additionally, there is no concern about the pre-existing antibodies to viral capsids which limit eligibility for gene therapies. We aim to use our technology platform to develop a new category of medicines for severe chronic diseases including rare blood disorders such as Hemophilia A, and to advance their development into clinical testing. Disclosures Carmona: Sigilon Therapeutics: Employment. Barney:Sigilon Therapeutics: Employment. Sewell:Sigilon Therapeutics: Employment. Newman:Sigilon Therapeutics: Employment. Carroll:Sigilon Therapeutics: Employment. Beauregard:Sigilon Therapeutics: Employment. Huang:Sigilon Therapeutics: Employment. Heidebrecht:Sigilon Therapeutics: Employment. Corzo:Sigilon Therapeutics: Employment. Moller:Sigilon Therapeutics: Employment. Smith:Sigilon Therapeutics: Employment. Peritt:Sigilon Therapeutics: Employment. Vivaldi:Sigilon Therapeutics: Employment.

Nongenotoxic antibody-drug conjugate conditioning enables safe and effective platelet gene therapy of hemophilia A mice

Gene therapy offers the potential to cure hemophilia A (HA). We have shown that hematopoietic stem cell (HSC)-based platelet-specific factor VIII (FVIII) (2bF8) gene therapy can produce therapeutic protein and induce antigen-specific immune tolerance in HA mice, even in the presence of inhibitory antibodies. For HSC-based gene therapy, traditional preconditioning using cytotoxic chemotherapy or total body irradiation (TBI) has been required. The potential toxicity associated with TBI or chemotherapy is a deterrent that may prevent patients with HA, a nonmalignant disease, from agreeing to such a protocol. Here, we describe targeted nongenotoxic preconditioning for 2bF8 gene therapy utilizing a hematopoietic cell-specific antibody-drug conjugate (ADC), which consists of saporin conjugated to CD45.2- and CD117-targeting antibodies. We found that a combination of CD45.2- and CD117-targeting ADC preconditioning was effective for engrafting 2bF8-transduced HSCs and was favorable for platelet lineage reconstitution. Two thirds of HA mice that received 2bF8 lentivirus-transduced HSCs under (CD45.2+CD117)-targeting ADC conditioning maintained sustained therapeutic levels of platelet FVIII expression. When CD8-targeting ADC was supplemented, chimerism and platelet FVIII expression were significantly increased, with long-term sustained platelet FVIII expression in all primary and secondary recipients. Importantly, immune tolerance was induced and hemostasis was restored in a tail-bleeding test, and joint bleeding also was effectively prevented in a needle-induced knee joint injury model in HA mice after 2bF8 gene therapy. In summary, we show for the first time efficient engraftment of gene-modified HSCs without genotoxic conditioning. The combined cocktail ADC-mediated hematopoietic cell-targeted nongenotoxic preconditioning that we developed is highly effective and favorable for platelet-specific gene therapy in HA mice.

Photothermal-responsive nanosized hybrid polymersome as versatile therapeutics codelivery nanovehicle for effective tumor suppression

Effective cancer therapies often demand delivery of combinations of drugs to inhibit multidrug resistance through synergism, and the development of multifunctional nanovehicles with enhanced drug loading and delivery efficiency for combination therapy is currently a major challenge in nanotechnology. However, such combinations are more challenging to administer than single drugs and can require multipronged approaches to delivery. In addition to being stable and biodegradable, vehicles for such therapies must be compatible with both hydrophobic and hydrophilic drugs, and release drugs at sustained therapeutic levels. Here, we report synthesis of porous silicon nanoparticles conjugated with gold nanorods [composite nanoparticles (cNPs)] and encapsulate them within a hybrid polymersome using double-emulsion templates on a microfluidic chip to create a versatile nanovehicle. This nanovehicle has high loading capacities for both hydrophobic and hydrophilic drugs, and improves drug delivery efficiency by accumulating at the tumor after i.v. injection in mice. Importantly, a triple-drug combination suppresses breast tumors by 94% and 87% at total dosages of 5 and 2.5 mg/kg, respectively, through synergy. Moreover, the cNPs retain their photothermal properties, which can be used to significantly inhibit multidrug resistance upon near-infrared laser irradiation. Overall, this work shows that our nanovehicle has great potential as a drug codelivery nanoplatform for effective combination therapy that is adaptable to other cancer types and to molecular targets associated with disease progression.

In Vivo Enrichment of Genetically Manipulated Platelets for Murine Haemophilia B Gene Therapy

Hemophilia B (HB) is an X chromosome-linked bleeding disorder resulting from a deficiency of factor FIX (FIX). Our previous studies have demonstrated that platelet-targeted FIX gene therapy can provide sustained therapeutic levels of FIX expression in platelets without inhibitory antibody (inhibitor) development, indicating platelet gene therapy could be a promising strategy for the treatment of HB patients. In this study, we aimed to enhance platelet-FIX expression in HB mice with MGMT P140K-mediated in vivo selection of hematopoietic stem cells (HSCs) under a nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT LV), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the drug-resistance MGMT P140K gene. Platelet-FIX expression was introduced in nonmyeloablative preconditioned HB mice after 2bF9/MGMT LV-mediated HSC transduction and transplantation. After 8 weeks of bone marrow reconstitution, recipients were administered with O6-benzylguanine/1, 3-bis-2-chloroethyl-1- nitrosourea (BG/BCNU) once every 4 weeks for a total of 3 treatments. Animals were analyzed starting at 4 weeks after transplantation by PCR, quantitative real-time PCR (qPCR), FIX assays, inhibitor assays, and tail bleeding test.PCR results showed that 2bF9 proviral DNA was detected in all recipients that received 2bF9/MGMT LV-transduced HSCs. Quantitative PCR results confirmed that 2bF9/MGMT LV-transduced cells were effectively enriched after each round of BG/BCNU selective treatment in vivo. The average copy number of the 2bF9 cassette per cell after three BG/BCNU treatments was 0.50 ± 0.04, which was significantly higher than pre-BG/BCNU treatment (0.13 ± 0.01). There was an approximately 2.9-fold higher FIX antigen (FIX:Ag) (4.11 ± 0.76 vs. 1.43 ± 0.06 mU/108 platelets) and a 3.7-fold FIX activity (FIX:C) (2.56 ± 0.50 vs. 0.69 ± 0.04 mU/108 platelets) level, respectively, post-treatment compared to pre-treatment. There was a small amount of FIX:Ag detected in the 2bF9/MGMT LV-transduced recipient plasma. When we normalized FIX:Ag levels to total whole blood FIX content, the results demonstrated that 96.32 ± 0.32% of whole blood FIX in the BG/BCNU treated animals was stored in platelets. To assess whether the bleeding phenotype was rescued in HB mice after receiving 2bF9/MGMT LV-transduced HSCs, we used a 6-hour tail bleeding test. All 2bF9/MGMT LV-transduced recipients stopped bleeding within 6 hours with a clotting time of 2.6 ± 0.5 hours and a remaining hemoglobin level of 65.1 ± 4.9%, which were not significantly different from those of the wild-type control group (1.7 ± 0.9 hours and 70.6 ± 13.9%). In contrast, none of the FIXnull mice stopped bleeding within 6 hours with a remaining hemoglobin level of 38.8 ± 6.7%.Notably, none of the recipients developed anti-FIX inhibitory antibodies as measured by a modified Bethesda assay. To investigate whether immune tolerance was induced in 2bF9/MGMT LV-transduced recipients, all the recipients were challenged with 200U/kg recombinant human FIX (rhFIX) in the presence of Incomplete Freund's adjuvant (IFA) at 9 months after transplantation. Only one out of four recipients developed a low titer of inhibitors (1.3 BU/ml). In contrast, all of the HB controls developed inhibitors ranging from 17-120 BU/ml after the same challenge (n = 4). When we used the ELISA assay for the quantification of total anti-FIX IgG antibodies, low titer antibodies were found in the recipients after they were challenged with rhFIX plus IFA. However, the antibody levels in the HB control mice under the same conditioning were 213-fold higher than those developed in 2bF9 MGMT LV-transduced recipients.In conclusion, our data demonstrated that using the MGMT-mediated drug-selection system in 2bF9 gene therapy can significantly enhance therapeutic platelet-FIX expression, resulting in sustained phenotypic correction and immune suppression in HB mice. Our studies suggest that platelet-targeted FIX gene therapy together with in vivo enrichment of engineered cells may provide a promising strategy for the treatment of HB patients. DisclosuresNo relevant conflicts of interest to declare.

Pegylated poly(anhydride) nanoparticles for oral delivery of docetaxel

The aim of this work was to investigate the potential of pegylated poly(anhydride) nanoparticles to enhance the oral bioavailability of docetaxel (DTX). Nanoparticles were prepared after the incubation between the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN), poly(ethylene glycol) (PEG2000 or PEG6000) and docetaxel (DTX). The oral administration of a single dose of pegylated nanoparticles to mice provided sustained and prolonged therapeutic plasma levels of docetaxel for up 48–72 h. In addition, the relative oral bioavailability of docetaxel was around 32%. The organ distribution studies revealed that docetaxel underwent a similar distribution when orally administered encapsulated in nanoparticles as when intravenously as Taxotere®. This observation, with the fact that the clearance of docetaxel when loaded into the oral pegylated nanoparticles was found to be similar to that of intravenous formulation, suggests that docetaxel would be released at the epithelium surface and then absorbed to the circulation.

Topical nepafenac for prevention of post-cataract surgery macular edema in diabetic patients: patient selection and perspectives.

Since its first description, the prevention of pseudophakic cystoid macular edema (PCME) continues to pose challenges for ophthalmologists. Recent evidence suggests that prophylaxis is unnecessary in patients without risk factors. Diabetes mellitus is generally considered as a risk factor for the development of PCME after cataract surgery since it causes breakdown of the blood–retinal barrier. Diabetic retinopathy (DR) increases the risk even further. Therefore, prophylactic nonsteroidal anti-inflammatory drugs (NSAIDs) should be considered in diabetic patients, especially if they have DR. NSAIDs block the cyclooxygenase enzymes responsible for prostaglandin production and reduce the incidence of PCME after cataract surgery. Nepafenac seems superior to other NSAIDs in terms of ocular penetration allowing higher and sustained therapeutic levels in retina and choroid. Topical steroids are less effective and may cause intraocular pressure increase limiting their long-term use. Nepafenac is cost effective, when the burden of PCME prevention is compared with the burden of treatment. Prevention is much cheaper and less harmful than invasive treatments like periocular or intravitreal injections. Overall, both nepafenac 0.1% and nepafenac 0.3% are well tolerated. They should be used carefully in patients with compromised corneas such as those with severe dry eye or penetrating grafts. If otherwise healthy cataract patients have ≥2 risk factors, like PCME in the other eye or posterior capsule rupture during surgery, treatment should be considered. Once-daily nepafenac 0.3% dosing may improve postoperative outcomes through increased patient compliance and may reduce treatment burden further. Every patient should be assessed in terms of risks/benefits of the treatment, in individual basis, before cataract surgery.