Thermostability-assisted limited proteolysis-coupled mass spectrometry for capturing drug target proteins and sites.

BACKGROUND: Identifying drug-binding targets and their corresponding sites is crucial for drug discovery and mechanism studies. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a sophisticated method used for the detection of compound and protein interactions. However, in some cases, LiP-MS cannot identify the target proteins due to the small structure changes or the lack of enrichment of low-abundant protein. To overcome this drawback, we developed a thermostability-assisted limited proteolysis-coupled mass spectrometry (TALiP-MS) approach for efficient drug target discovery. RESULTS: We proved that the novel strategy, TALiP-MS, could efficiently identify target proteins of various ligands, including cyclosporin A (a calcineurin inhibitor), geldanamycin (an HSP90 inhibitor), and staurosporine (a kinase inhibitor), with accurately recognizing drug-binding domains. The TALiP protocol increased the number of target peptides detected in LiP-MS experiments by 2- to 8-fold. Meanwhile, the TALiP-MS approach can not only identify both ligand-binding stability and destabilization proteins but also shows high complementarity with the thermal proteome profiling (TPP) and machine learning-based limited proteolysis (LiP-Quant) methods. The developed TALiP-MS approach was applied to identify the target proteins of celastrol (CEL), a natural product known for its strong antioxidant and anti-cancer angiogenesis effect. Among them, four proteins, MTHFD1, UBA1, ACLY, and SND1 were further validated for their strong affinity to CEL by using cellular thermal shift assay. Additionally, the destabilized proteins induced by CEL such as TAGLN2 and CFL1 were also validated. SIGNIFICANCE: Collectively, these findings underscore the efficacy of the TALiP-MS method for identifying drug targets, elucidating binding sites, and even detecting drug-induced conformational changes in target proteins in complex proteomes.

Combination of PEGylation and Cationization on Phospholipid-Coated Cyclosporine Nanosuspensions for Enhanced Ocular Drug Delivery.

Strong precorneal clearance mechanisms including reflex blink, constant tear drainage, and rapid mucus turnover constitute great challenges for eye drops for effective drug delivery to the ocular epithelium. In this study, cyclosporine A (CsA) for the treatment of dry eye disease (DED) was selected as the model drug. Two strategies, PEGylation for mucus penetration and cationization for potent cellular uptake, were combined to construct a novel CsA nanosuspension (NS@lipid-PEG/CKC) by coating nanoscale drug particles with a mixture of lipids, DSPE-PEG2000, and a cationic surfactant, cetalkonium chloride (CKC). NS@lipid-PEG/CKC with the mean size ∼173 nm and positive zeta potential ∼+40 mV showed promoted mucus penetration, good cytocompatibility, more cellular uptake, and prolonged precorneal retention without obvious ocular irritation. More importantly, NS@lipid-PEG/CKC recovered tear production and goblet cell density more efficiently than the commercial cationic nanoemulsion on a dry eye disease rat model. All results indicated that a combination of PEGylation and cationization might provide a promising strategy to coordinate mucus penetration and cellular uptake for enhanced drug delivery to the ocular epithelium for nanomedicine-based eye drops.

Bio-synthesis, purification and structural analysis of Cyclosporine-A produced by Tolypocladium inflatum with valorization of agro-industrial wastes.

Cyclosporine A (CyA) holds significant importance as a strategic immunosuppressive drug for organ transplant patients. In this study, we aimed to produce pure and cost-effective Cyclosporine A (CyA) by fermenting a culture medium containing dairy sludge, using Tolypocladium inflatum PTCC 5253. Following the fermentation stage, ethyl acetate extraction and fast protein liquid chromatography were employed for sample purification. The initial evaluation of the effectiveness of CyA obtained from these processes was performed through bioassay, wherein the antimicrobial clear zone diameter was found to be larger compared to the sample obtained from the fermentation culture. The concentration of CyA was determined using high-performance liquid chromatography, yielding values of 334 mg/L, 456 mg/L, and 578 mg/L for the fermented, extracted, and purified samples, respectively. Further analysis utilizing liquid chromatography tandem mass spectrometry (LC/MS/MS) confirmed a purity of 91.9% and proper agreement with the standard sample based on the ion intensity of Z/m 1205. To validate the structure of CyA, nuclear magnetic resonance spectroscopy, Fourier-transform infrared (FT-IR), and Raman spectroscopy were employed. X-ray diffraction and differential scanning calorimetry analyses demonstrated that the purified CyA exhibited a crystal structure similar to the standard sample, characterized by two broad peaks at 2θ = 9° and 20°, and comparable glass transition temperatures (57-68 °C for the purified sample; 53-64 °C for the standard sample). Dynamic light scattering analysis confirmed a uniform particle size distribution in both the purified and standard samples. The zeta potentials of the purified and standard samples were determined to be - 25.8 ± 0.16 and - 23.63 ± 0.12 mV, respectively. Our results demonstrate that dairy sludge can serve as a suitable culture medium for the production of (CyA).

A sterilizable platform based on crosslinked xanthan gum for controlled-release of polymeric micelles: Ocular application for the delivery of neuroprotective compounds to the posterior eye segment.

TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate) polymeric micelles show interesting properties for ocular administration thanks to their solubilization capability, nanometric size and tissue penetration ability. However, micelles formulations are generally characterized by low viscosity, poor adhesion and very short retention time at the administration site. Therefore, the idea behind this work is the preparation and characterization of a crosslinked film based on xanthan gum that contains TPGS micelles and is capable of controlling their release. The system was loaded with melatonin and cyclosporin A, neuroprotective compounds to be delivered to the posterior eye segment. Citric acid and heating at different times and temperatures were exploited as crosslinking approach, giving the possibility to tune swelling, micelles release and drug release. The biocompatibility of the platform was confirmed by HET-CAM assay. Ex vivo studies on isolated porcine ocular tissues, conducted using Franz cells and two-photon microscopy, demonstrated the potential of the xanthan gum-based platform and enlightened micelles penetration mechanism. Finally, the sterilization step was approached, and a process to simultaneously crosslink and sterilize the platform was developed.

Silk Fibroin Formed Bioadhesive Ophthalmic Gel for Dry Eye Syndrome Treatment.

PURPOSE: Dry eye syndrome (DES), arising from various etiologic factors, leads to tear film instability and ocular surface damage. Given its anti-inflammatory effects, cyclosporine A (CsA) has been widely used as a short-term treatment option for DES. However, poor bioavailability and solubility of CsA in aqueous phase make the development of a cyclosporine A-based eye drop for ocular topical application a huge challenge. METHODS: In this study, a novel strategy for preparing cyclosporine A-loaded silk fibroin nanoemulsion gel (CsA NBGs) was proposed to address these barriers. Additionally, the rheological properties, ocular irritation potential, tear elimination kinetics, and pharmacodynamics based on a rabbit dry eye model were investigated for the prepared CsA NBGs. Furthermore, the transcorneal mechanism across the ocular barrier was also investigated. RESULTS: The pharmacodynamics and pharmacokinetics of CsA NBGs exhibited superior performance compared to cyclosporine eye drops, leading to a significant enhancement in the bioavailability of CsA NBGs. Furthermore, our investigation into the transcorneal mechanism of CsA NBGs revealed their ability to be absorbed by corneal epithelial cells via the paracellular pathway. CONCLUSION: The CsA NBG formulation exhibits promising potential for intraocular drug delivery, enabling safe, effective, and controlled administration of hydrophobic drugs into the eye. Moreover, it enhances drug retention within the ocular tissues and improves systemic bioavailability, thereby demonstrating significant clinical translational prospects.

Exploring the Effects of Cyclosporin A to Isocyclosporin A Rearrangement on Ion Mobility Separation.

Cyclosporin A (CycA) is a peptide secondary metabolite derived from fungi that plays a crucial role in transplantation surgery. Cyclic traveling wave ion mobility mass spectrometry (IM-MS) revealed an N → O peptidyl shift in singly protonated CycA to isocyclosporin A (isoA), whereas no such isomerization was observed for doubly protonated and sodiated molecules. CycA and isoA were able to be separated by considering doubly protonated precursors using a specific ion fragment. In parallel, sodium ion stabilization facilitated the simultaneous separation and quantitation of singly charged cyclosporin isomers with the limit of detection and coefficient of determination of 1.3% and 0.9908 for CycA in isoA and 1.0% and 0.9830 for isoA in CycA, respectively. Finally, 1H-13C gHSQC NMR experiments permitted parallel recording of up to 11 cyclosporin conformers. The ratios were determined by integrating the volume of cross-peaks of the upfield resonating hydrogen in the diastereotopic methylene group of sarcosine-3.

Targeting Cyclophilin A and CD147 to Inhibit Replication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and SARS-CoV-2-Induced Inflammation.

Identification and development of effective therapeutics for coronavirus disease 2019 (COVID-19) are still urgently needed. The CD147-spike interaction is involved in the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 invasion process in addition to angiotensin-converting enzyme 2 (ACE2). Cyclophilin A (CyPA), the extracellular ligand of CD147, has been found to play a role in the infection and replication of coronaviruses. In this study, our results show that CyPA inhibitors such as cyclosporine A (CsA) and STG-175 can suppress the intracellular replication of SARS-CoV-2 by inhibiting the binding of CyPA to the SARS-CoV-2 nucleocapsid C-terminal domain (N-CTD), and the IC50 is 0.23 µM and 0.17 µM, respectively. Due to high homology, CsA also had inhibitory effects on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), and the IC50 is 3.2 µM and 2.8 µM, respectively. Finally, we generated a formulation of phosphatidylserine (PS)-liposome-CsA for pulmonary drug delivery. These findings provide a scientific basis for identifying CyPA as a potential drug target for the treatment of COVID-19 as well as for the development of broad-spectrum inhibitors for coronavirus via targeting CyPA. Highlights: 1) SARS-CoV-2 infects cells via the binding of its S protein and CD147; 2) binding of SARS-CoV-2 N protein and CyPA is essential for viral replication; 3) CD147 and CyPA are potential therapeutic targets for SARS-CoV-2; and 4) CsA is a potential therapeutic strategy by interrupting CD147/CyPA interactions. SIGNIFICANCE STATEMENT: New severe acute respiratory syndrome coronavirus (SARS-CoV)-2 variants and other pathogenic coronaviruses (CoVs) are continually emerging, and new broad-spectrum anti-CoV therapy is urgently needed. We found that binding sites of cyclophilin A/cyclosporin A (CyPA/CsA) overlap with CyPA/N-CTD (nucleocapsid C-terminal domain), which shows the potential to target CyPA during SARS-CoV-2 infection. Here, we provide new evidence for targeting CyPA in the treatment of coronavirus disease 2019 (COVID-19) as well as the potential of developing CyPA inhibitors for broad-spectrum inhibition of CoVs.

Exploring the Therapeutic Potential of Cyclosporine for Ophthalmic Indications by Novel Carrier Systems.

Cyclosporine (CsA) stays the most intangible molecule holding a good history for treating several ophthalmic conditions and it even attributes to multiple off-label uses. Topical delivery of CsA is the most preferred route but owing to the molecule's physicochemical properties such as poor aqueous solubility and high molecular weight as well as its encounter with multiple barriers of eye causes hindrance for proper delivery of the molecule to the site of action. However, Restasis®, Cequa®, and Verkazia® are the marketed formulations that have been approved by U.S. Food and Drug Administration, whereas Cyclokat® and Ikervis® by the European Medicines Agency. Although these medications are in use, they are associated with severe discomfort and poor patient compliance. This review gives an overview regarding current formulations available in the market, the products in pipeline and the recent advances undertaken for improving ocular delivery of CsA for various ophthalmic indications.

Preclinical characterization of water-free cyclosporine eye drops - Factors impacting ocular penetration ex vivo and in vivo.

Although the efficacy of Cyclosporine A (CsA) in the management of ocular inflammation is well-demonstrated, ocular delivery remains challenging due to its hydrophobic nature. The semifluorinated alkane, perfluorobutylpentane (F4H5) has previously been suggested as an efficient vehicle for preparation of CsA eyedrops. Here we evaluated the influence of drop volume and the formulation aid, ethanol (EtOH), on ocular penetration of CsA and compared it to that of the commercial eyedrop, Ikervis, ex vivo and in vivo. Moreover, conjunctival and corneal tolerability after EtOH addition were evaluated ex vivo. The F4H5/EtOH vehicle was well tolerated and resulted in better corneal CsA penetration (AUC(0-4h): 63,008 ± 3,946 ng.h.g-1) than Ikervis (AUC(0-4h): 10,328 ± 1,462 ng.h.g-1) or F4H5 alone (AUC(0-4h): 50,734 ± 3,472 ng.h.g-1) ex vivo. Interestingly, in vivo the CsA concentration in cornea, conjunctiva and lacrimal glands observed after administration of the F4H5 formulation (AUC(0.133-24h): 7,741 ± 1,334 ng.h.g-1, 1,313 ± 291 ng.h.g-1, 48.2 ± 26.3 ng.h.g-1) and F4H5/EtOH, both at a reduced dose of 11 µl (AUC(0.133-24h): 9,552 ± 1,738 ng.h.g-1, 1,679 ± 285 ng.h.g-1, 50.3 ± 21.1 ng.h.g-1) was similar or even greater than that observed on administration of 50 µl Ikervis (AUC(0.133-24h): 9,943 ± 1,413 ng.h.g-1, 2,069 ± 263 ng.h.g-1, 30.6 ± 18.4 ng.h.g-1). Thus, F4H5-based eyedrops were shown to deliver CsA more efficiently to anterior ocular tissues at a reduced dose in comparison to Ikervis, reducing dose wastage and minimizing any potential systemic side effects.

Comparative study of cyclosporine A liposomes and emulsions for ophthalmic drug delivery: Process optimization through response surface methodology (RSM) and biocompatibility evaluation.

Herein, cyclosporine A loaded liposomes (CsA-Lips) were fabricated aimed at improving the biocompatibility of the ophthalmic formulation and getting rid of the direct contact of ocular tissues with irritant excipients. Response surface methodology was exploited in order to investigate the influence of miscellaneous factors on the key characteristics of CsA-Lips. Ratio of EPC:CsA, ratio of EPC:Chol, and stirring speed were selected as the independent variables, while size, drug-loading content (DL), and drug-loading content (DL) loss rate were applied as the response variables. In case of the maximal lack-of-fit p-value and minimum sequential p-value, quadratic model was regarded as the fittest model to analyze the data. The correlation of independent variables with response variables was described by three-dimension surface figures. Optimized formulation for CsA-Lips was obtained with ratio of EPC:CsA set as 15, ratio of EPC:Chol set as 2, and stirring speed set as 800 rpm. The particle size of CsA-Lips was 129.2 nm after optimalization while their TEM images exhibited spherical unilamellar vesicles with clearly shell-core structure. CsA released more rapidly from CsA-Lips in comparison with self-made emulsion and Restasis®. Besides, minimum cytotoxicity of CsA-Lips was perceived via both MTT method and LDH method, indicating the excellent compatibility of the ophthalmic formulation. Simultaneously, CsA-Lips showed enhanced nonspecific internalization in the cytoplasm with a time-dose-dependent manner. In conclusion, CsA-Lips could be adhibited as the hopeful ophthalmic drug delivery system clinically for dry eye syndrome (DES).

[Rapid and simultaneous determination of two immunosuppressants in whole blood by high performance liquid chromatography].

Cyclosporine A and sirolimus are immunosuppressants that are widely used in many organ transplantation procedures. They exhibit some complementary mechanisms of action and interact synergistically when used together. However， they are critical-dose drugs and have a narrow therapeutic index. They provide the desired therapeutic effect with acceptable tolerability only within a specific range of blood concentrations. Therefore， the rapid and simultaneous detection of the concentrations of cyclosporine A and sirolimus in whole blood could provide valuable information on planning medicine administration after organ transplantations. In this study， firstly， the chromatographic behaviors of cyclosporine A and sirolimus on a biological liquid chromatography （BioLC） column and traditional liquid chromatography （TraLC） columns were investigated systematically under the same chromatographic conditions. The results suggested that the peak height and peak width of cyclosporine A and sirolimus on the BioLC column， ZORBAX 300SB C8 （250 mm×4.6 mm， 5.0 µm）， were the highest and narrowest， respectively. The number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column increased significantly when the volume ratio of acetonitrile in the mobile phases was greater than 70%. Their retention time on the BioLC and TraLC columns was negligibly affected by the use of formic acid and trifluoroacetic acid as the mobile phases. In the range of the experimental column temperature， the number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column was significantly higher than that on the two TraLC columns. Furthermore， the relationship between the retention factor and column temperature of cyclosporine A on the ZORBAX 300SB C8 column was different from that on the two TraLC columns. Subsequently， a high performance liquid chromatography method based on the ZORBAX 300SB C8 column was established for the rapid separation and determination of cyclosporin A and sirolimus in whole blood. A sample of whole blood with a volume of 50 µL was prepared by protein precipitation with 1 mol/L sodium hydroxide and then extracted into 500 µL of ether-methanol （95∶5， v/v）. After centrifugation at 14000 r/min for 10 min， the organic layer was removed and evaporated under a stream of nitrogen at 50 ℃. The residue was then reconstituted in 200 µL of methanol for use. Cyclosporin A and sirolimus were separated through isocratic elution on the ZORBAX 300SB C8 column. The column temperature was set at 60 ℃. The mobile phase was acetonitrile-water （70∶30， v/v） and the flow rate was 1.0 mL/min. The detection wavelengths were 205 nm for cyclosporine A and 278 nm for sirolimus. The injection volume was 20 µL. The external standard method was used to quantify cyclosporine A and sirolimus. Under the optimized conditions， cyclosporine A and sirolimus were well-separated within 6 min with a resolution of 3.7 at 205 nm. In addition， the endogenous substances in whole blood negligibly interfered in the detection of sirolimus， while two endogenous substances slightly affected the detection of cyclosporine A. Cyclosporine A and sirolimus both showed good linear relationships in their respective concentration （r>0.997）. The limits of detection （LODs） of cyclosporine A and sirolimus were respectively calculated to be 10 ng/mL and 1 ng/mL based on a signal-to-noise ratio of 3， and the limits of quantification （LOQs） were 30 ng/mL and 2 ng/mL based on a signal-to-noise ratio of 10. In the whole blood samples， the recoveries of cyclosporine A and sirolimus at three spiked levels were in the ranges of 83.5%-89.7% and 95.8%-97.8% with relative standard deviations （RSDs） of 3.2%-9.0% and 3.4%-6.7% （n=5）， respectively. The established method is simple in operation， can be performed with a simple mobile phase， has a short analysis time， and provides a wide linear range and high sensitivity； hence， it can be applied to the determination of cyclosporine A and sirolimus in whole blood.

Cyclosporine A-loaded apoferritin alleviates myocardial ischemia-reperfusion injury by simultaneously blocking ferroptosis and apoptosis of cardiomyocytes.

Myocardial ischemia-reperfusion injury (MI/RI) seriously restricts the therapeutic effect of reperfusion. It is demonstrated that ferroptosis and apoptosis of cardiomyocytes are widely involved in MI/RI. Therefore, simultaneous inhibition of ferroptosis and apoptosis of cardiomyocytes can be a promising strategy to treat MI/RI. Besides, transferrin receptor 1 (TfR1) is highly expressed in ischemic myocardium, and apoferritin (ApoFn) is a ligand of the transferrin receptor. In this study, CsA@ApoFn was prepared by wrapping cyclosporin A (CsA) with ApoFn and actively accumulated in ischemic cardiomyocytes through TfR1 mediated endoctosis in MI/RI mice. After entering cardiomyocytes, ApoFn in CsA@ApoFn inhibited ferroptosis of ischemic cardiomyocytes by increasing the protein expression of GPX4 and reducing the content of labile iron pool and lipid peroxides. At the same time, CsA in CsA@ApoFn attenuated the apoptosis of ischemic cardiomyocytes through recovering mitochondrial membrane potential and reducing the level of reactive oxygen species, which played a synergistic role with ApoFn in the treatment of MI/RI. In conclusion, CsA@ApoFn restored cardiac function of MI/RI mice by simultaneously blocking ferroptosis and apoptosis of cardiomyocytes. ApoFn itself not only served as a safe carrier to specifically deliver CsA to ischemic cardiomyocytes but also played a therapeutic role on MI/RI. CsA@ApoFn is proved as an effective drug delivery platform for the treatment of MI/RI. STATEMENT OF SIGNIFICANCE: Recent studies have shown that ferroptosis is an important mechanism of myocardial ischemia-reperfusion injury (MI/RI). Therefore, simultaneous inhibition of ferroptosis and apoptosis of cardiomyocytes can be a promising strategy to treat MI/RI. Apoferritin, as a delivery carrier, can actively target to ischemic myocardium through binding with highly expressed transferrin receptor on ischemic cardiomyocytes. At the same time, apoferritin plays a protective role on ischemic cardiomyocytes by inhibiting ferroptosis. This strategy of killing two birds with one stone significantly improves the therapeutic effect on MI/RI while does not need more pharmaceutical excipients, which has the prospect of clinical transformation.

Product Quality Research for Developing and Assessing Regulatory Submissions for Generic Cyclosporine Ophthalmic Emulsions.

Approval of the first generic 0.05% cyclosporine ophthalmic emulsion (COE) in the U.S. represents a milestone achievement of the science and research program in the U.S. Food and Drug Administration's Center for Drug Evaluation and Research (CDER). COE is a locally acting complex drug product indicated to increase tear production in patients whose production is presumed to be suppressed due to ocular inflammation associated with keratoconjunctivitis sicca. The path to approval required overcoming numerous scientific challenges to determining therapeutic equivalence to the reference listed drug. Researchers in CDER's Office of Pharmaceutical Quality and Office of Generic Drugs developed a quality by design approach to understand the effects of process and formulation variables on the product's critical quality attributes, including globule size distribution (GSD), turbidity, viscosity, zeta potential, surface tension, and osmolality. CDER researchers explored multiple techniques to perform physicochemical characterization and analyze the GSD including laser diffraction, nanoparticle tracking analysis, cryogenic transmission electron microscopy, dynamic light scattering, asymmetric field flow fractionation, and two-dimensional diffusion ordered spectroscopy nuclear magnetic resonance. Biphasic models to study drug transfer kinetics demonstrated that COEs with qualitative and quantitative sameness and comparable GSDs, analyzed using earth mover's distance, can be therapeutic equivalents. This body of research facilitated the review and approval of the first U.S. generic COE. In addition, the methods and fundamental understanding developed from this research may support the development and assessment of other complex generics. The approval of a generic COE should improve the availability of this complex drug product to U.S. patients.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

Rationale: Human coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively. Methods: Primary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication. Results: Both CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model. Conclusion: The immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.

Oil-free eye drops containing Cyclosporine A/cyclodextrin/PVA supramolecular complex as a treatment modality for dry eye disease.

According to the global mapping of dry eye disease (DED), nearly 5 to 50 % of people suffer from DED, and this number is on the rise. The drug of choice Cyclosporine A (CsA) exhibits poor ocular bioavailability due to high molecular weight and lipophilicity. Moreover, formulations of CsA currently available are in the form of oil-based emulsions that are known to cause ocular irritation and pain. In this study, sulfobutylether-ß-cyclodextrin (SBE-ß-CD) based binary and ternary supramolecular complexes of CsA were developed as completely oil-free, and particle-free eye drops to treat DED. The physicochemical characterizations were supplemented with relevant in silico studies, to ascertain the findings. Further, the efficacy of the complexes was evaluated in the scopolamine-induced mouse model of DED. The complexation improved the CsA solubility by ~21-fold, with ~4-fold improvement in dissolution and transcorneal permeation. The non-irritancy and non-toxicity were confirmed by hen's egg chorioallantoic membrane assay and cytotoxicity assay using human corneal epithelial cells, respectively. The in vivo treatment with the ternary CD complex demonstrated better management of the dry eye supported by the tear volume assessment, corneal fluorescein staining, and histopathological studies of the cornea, lacrimal gland, and harderian gland. The study demonstrates the potential of the supramolecular complex as an alternative to the oil-based formulation of eye drops for drugs that show low solubility and poor corneal permeation.

Non-Immunosuppressive Cyclophilin Inhibitors.

Cyclophilins, enzymes with peptidyl-prolyl cis/trans isomerase activity, are relevant to a large variety of biological processes. The most abundant member of this enzyme family, cyclophilin A, is the cellular receptor of the immunosuppressive drug cyclosporine A (CsA). As a consequence of the pathophysiological role of cyclophilins, particularly in viral infections, there is a broad interest in cyclophilin inhibition devoid of immunosuppressive activity. This Review first gives an introduction into the physiological and pathophysiological roles of cyclophilins. The presentation of non-immunosuppressive cyclophilin inhibitors will commence with drugs based on chemical modifications of CsA. The naturally occurring macrocyclic sanglifehrins have become other lead structures for cyclophilin-inhibiting drugs. Finally, de novo designed compounds, whose structures are not derived from or inspired by natural products, will be presented. Relevant synthetic concepts will be discussed, but the focus will also be on biochemical studies, structure-activity relationships, and clinical studies.

Transition from Amorphous Cyclosporin A Nanoparticles to Size-Reduced Stable Nanocrystals in a Poloxamer 407 Solution.

Amorphous drug nanoparticles usually exhibit low storage stability. A comprehensive understanding of the molecular states and physicochemical properties of the product is indispensable for designing stable formulations. In the present study, an amorphous cyclosporin A (CyA) nanosuspension with a mean particle size of approximately 370 nm was prepared by wet bead milling with poloxamer 407 (P407). Interestingly, the prepared amorphous CyA nanoparticles were transformed into uniform CyA nanocrystals with a reduced mean particle size of approximately 200 nm during storage at 25 °C. The CyA nanocrystals were stably maintained for at least 1 month. The particle morphologies and molecular structures of the CyA nanosuspensions before and after storage were thoroughly characterized by cryogenic transmission electron microscopy and magic-angle spinning nuclear magnetic resonance spectroscopy, respectively. They revealed that the freshly prepared amorphous CyA nanoparticles (∼370 nm) were secondary particles composed of aggregated primary particles with an estimated size of 50 nm. A portion of P407 was found to be entrapped at the gaps between the primary particles due to aggregation, while most of P407 was dissolved in the solution either adsorbing at the solid/liquid interface or forming polymeric micelles. The entrapped P407 is considered to play an important role in the destabilization of the amorphous CyA nanoparticles. The resultant CyA nanocrystals (∼200 nm) were uniform single crystals of Form 2 hydrate and showed corner-truncated bipyramidal features. Owing to the narrow particle size distribution of the CyA nanocrystals, the rate of Ostwald ripening was slow, giving long-term stability to the CyA nanocrystals. This study provides new insights into the destabilization mechanism of amorphous drug nanoparticles.

Cyclosporine A Nanosuspensions for Ophthalmic Delivery: A Comparative Study between Cationic Nanoparticles and Drug-Core Mucus Penetrating Nanoparticles.

The effect of mucin on ocular bioavailability depends on the extent to which it acts as a barrier or retention site. Mucus penetrating particles (MPPs) can evade the mucus entrapment and associated rapid clearance, but cationic nanoparticles have high adhesion to the mucosa. Both formulations can prolong the drug residence time on the surface of the eyes. The purpose of this work is to compare the effects of mucoadhesion of cationic nanoparticles and mucous permeability of MPPs on ocular bioavailability. Cationic nanosuspensions and drug-core MPP nanosuspensions were developed using the anti-solvent precipitation method. The results of X-ray diffraction revealed that CsA was amorphous. In vitro mucoadhesion evaluation demonstrated that cationic nanosuspensions enhanced the interaction with pig mucin about 5.0-6.0 fold compared to drug-core MPP nanosuspensions. A mucus permeation study by the transwell diffusion system showed that the Papp values of drug-core MPP nanosuspensions were 5.0-10.0 times higher than those of cationic nanosuspensions. In vivo ocular bioavailability evaluation of those CsA formulations was conducted in rabbits using a conventional nanosuspension as a comparison. The CsA concentrations in the cornea following the administration of a cationic nanosuspension and a drug-core MPP nanosuspension were 13,641.10 ng/g and 11,436.07 ng/g, respectively, significantly higher than that of the conventional nanosuspension (8310.762 ng/g). The results showed that both the cationic and MPP nanosuspensions were able to deliver CsA to anterior ocular tissues in effective therapeutic concentrations (10-20 µg/g) with topical drop instillation. The cationic nanosuspension could achieve relatively higher bioavailability than the MPP nanosuspension. The cationic nanosuspension would be a promising ocular drug delivery system.

Molecular Dynamics Simulations and Experimental Results Provide Insight into Clinical Performance Differences between Sandimmune® and Neoral® Lipid-Based Formulations.

OBJECTIVE: Molecular dynamics (MD) simulations provide an in silico method to study the structure of lipid-based formulations (LBFs) and the incorporation of poorly water-soluble drugs within such formulations. In order to validate the ability of MD to effectively model the properties of LBFs, this work investigates the well-known cyclosporine A formulations, Sandimmune® and Neoral®. Sandimmune® exhibits poor dispersibility and its absorption from the gastrointestinal tract is enhanced when administered after food, whereas Neoral® disperses comparatively well and shows no food effect. METHODS: MD simulations were performed of both LBFs to investigate the differences observed in fasted and fed conditions. These conditions were also tested using an in vitro experimental model of dispersion and digestion. RESULTS: These MD simulations were able to show that the food effect observed for Sandimmune® can be explained by large changes in drug solubilization on addition of bile. In contrast, Neoral® is well dispersed in water or in simulated fasted conditions, and this dispersion is relatively unchanged on moving to fed conditions. These differences were confirmed using dispersion and digestion in vitro experimental model. CONCLUSIONS: The current data suggests that MD simulations are a potential method to model the fate of LBFs in the gastrointestinal tract, predict their dispersion and digestion, investigate behaviour of APIs within the formulations, and provide insights into the clinical performance of LBFs.

Nanoparticle-Mediated Simultaneous Targeting of Mitochondrial Injury and Inflammation Attenuates Myocardial Ischemia-Reperfusion Injury.

Background The opening of mitochondrial permeability transition pore and inflammation cooperatively progress myocardial ischemia-reperfusion (IR) injury, which hampers therapeutic effects of primary reperfusion therapy for acute myocardial infarction. We examined the therapeutic effects of nanoparticle-mediated medicine that simultaneously targets mitochondrial permeability transition pore and inflammation during IR injury. Methods and Results We used mice lacking cyclophilin D (CypD, a key molecule for mitochondrial permeability transition pore opening) and C-C chemokine receptor 2 and found that CypD contributes to the progression of myocardial IR injury at early time point (30-45 minutes) after reperfusion, whereas C-C chemokine receptor 2 contributes to IR injury at later time point (45-60 minutes) after reperfusion. Double deficiency of CypD and C-C chemokine receptor 2 enhanced cardioprotection compared with single deficiency regardless of the durations of ischemia. Deletion of C-C chemokine receptor 2, but not deletion of CypD, decreased the recruitment of Ly-6Chigh monocytes after myocardial IR injury. In CypD-knockout mice, administration of interleukin-1ß blocking antibody reduced the recruitment of these monocytes. Combined administration of polymeric nanoparticles composed of poly-lactic/glycolic acid and encapsulating nanoparticles containing cyclosporine A or pitavastatin, which inhibit mitochondrial permeability transition pore opening and monocyte-mediated inflammation, respectively, augmented the cardioprotection as compared with single administration of nanoparticles containing cyclosporine A or pitavastatin after myocardial IR injury. Conclusions Nanoparticle-mediated simultaneous targeting of mitochondrial injury and inflammation could be a novel therapeutic strategy for the treatment of myocardial IR injury.