scholarly journals Biopharmaceutical Assessment of Dexamethasone Acetate-Based Hydrogels Combining Hydroxypropyl Cyclodextrins and Polysaccharides for Ocular Delivery

Pharmaceutics ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 717 ◽  
Author(s):  
Roseline Mazet ◽  
Xurxo García-Otero ◽  
Luc Choisnard ◽  
Denis Wouessidjewe ◽  
Vincent Verdoot ◽  
...  
Keyword(s):  
Water Solubility ◽  
Ex Vivo ◽  
Cytotoxic Effects ◽  
Corneal Epithelial ◽  
Pet Ct ◽  
Dexamethasone Acetate ◽  
Mixture Designs ◽  
Ocular Bioavailability

We previously developed two optimized formulations of dexamethasone acetate (DXMa) hydrogels by means of special cubic mixture designs for topical ocular administration. These gels were elaborated with hydroxypropyl-β-CD (HPβCD) and hydroxypropyl-γ-CD (HPγCD) and commercial hydrogels in order to enhance DXMa water solubility and finally DXMa’s ocular bioavailability and transcorneal penetration. The main objective of this study was to characterize them and to evaluate in vitro, ex vivo, and in vivo their safety, biopermanence, and transcorneal permeation. Gels A and B are Newtonian fluids and display a viscosity of 13.2 mPa.s and 18.6 mPa.s, respectively, which increases their ocular retention, according to the in vivo biopermanence study by PET/CT. These hydrogels could act as corneal absorption promoters as they allow a higher transcorneal permeation of DXMa through porcine excised cornea, compared to DEXAFREE® and MAXIDEX®. Cytotoxicity assays showed no cytotoxic effects on human primary corneal epithelial cells (HCE). Furthermore, Gel B is clearly safe for the eye, but the effect of Gel A on the human eye cannot be predicted. Both gels were also stable 12 months at 25 °C after sterilization by filtration. These results demonstrate that the developed formulations present a high potential for the topical ocular administration of dexamethasone acetate.

scholarly journals Amyloid-Targeting PET Tracer [18F]Flutemetamol Accumulates in Atherosclerotic Plaques

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1072 ◽  
Author(s):  
Sanna Hellberg ◽  
Johanna Silvola ◽  
Heidi Liljenbäck ◽  
Max Kiugel ◽  
Olli Eskola ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Mouse Strains ◽  
Aortic Tissue ◽  
Atherosclerotic Plaques ◽  
Carotid Artery Plaque ◽  
Pet Tracer ◽  
Pet Ct ◽  
Human Carotid

Atherosclerosis is characterized by the accumulation of oxidized lipids in the artery wall, which triggers an inflammatory response. Oxidized low-density lipoprotein (ox-LDL) presents amyloid-like structural properties, and different amyloid species have recently been recognized in atherosclerotic plaques. Therefore, we studied the uptake of the amyloid imaging agent [18F]Flutemetamol in atherosclerotic plaques. The binding of [18F]Flutemetamol to human carotid artery plaque was studied in vitro. In vivo uptake of the tracer was studied in hypercholesterolemic IGF-II/LDLR−/−ApoB100/100 mice and C57BL/6N controls. Tracer biodistribution was studied in vivo with PET/CT, and ex vivo by gamma counter and digital ex vivo autoradiography. The presence of amyloid, ox-LDL, and macrophages in the plaques was examined by immunohistochemistry. [18F]Flutemetamol showed specific accumulation in human carotid plaque, especially in areas positive for amyloid beta. The aortas of IGF-II/LDLR−/−ApoB100/100 mice showed large thioflavin-S-positive atherosclerotic plaques containing ox-LDL and macrophages. Autoradiography revealed 1.7-fold higher uptake in the plaques than in a lesion-free vessel wall, but no difference in aortic tissue uptake between mouse strains were observed in the in vivo PET/CT. In conclusion, [18F]Flutemetamol binds to amyloid-positive areas in human atherosclerotic plaques. Further studies are warranted to clarify the uptake mechanisms, and the potential of the tracer for in vivo imaging of atherosclerosis in patients.

Antioxidant Activity of Encapsulated Extracts and Bioactives from Natural Sources

2020 ◽  
Vol 26 (31) ◽  
pp. 3847-3861
Author(s):  
Odinei H. Gonçalves ◽  
Thaysa F.M. Moreira ◽  
Anielle de Oliveira ◽  
Lívia Bracht ◽  
Rafael P. Ineu ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Water Solubility ◽  
Ex Vivo ◽  
Bioactive Substances ◽  
Natural Sources ◽  
In Vitro Techniques ◽  
Materials Used ◽  
Endogenous Antioxidant

The low water solubility and low bioavailability of natural bioactive substances such as polyphenols and flavonoids, either in pure form or extracts, are a major concern in the pharmaceutical field and even on the food development sector. Although encapsulation has demonstrated success in addressing these drawbacks, it is important to evaluate the antioxidant activity of the encapsulated compounds. This article reviews the encapsulation of bioactive compounds from natural sources focusing their antioxidant activity after encapsulation. Attention is given to the methods and wall materials used, and the antioxidant activity methodologies (classical in vitro techniques such as DPPH, ORAC, FRAP and others, as well as in vivo/ex vivo tests to evaluate endogenous antioxidant enzymes or oxidative stress) applied to assess the antioxidant capacity are also comprehensively summarized.

scholarly journals Design, Synthesis, In Vitro and In Vivo Evaluation of Heterobivalent SiFAlin-Modified Peptidic Radioligands Targeting Both Integrin αvβ3 and the MC1 Receptor—Suitable for the Specific Visualization of Melanomas?

2021 ◽  
Vol 14 (6) ◽  
pp. 547
Author(s):  
Xia Cheng ◽  
Ralph Hübner ◽  
Valeska von von Kiedrowski ◽  
Gert Fricker ◽  
Ralf Schirrmacher ◽  
...  
Keyword(s):  
Malignant Melanoma ◽  
Ex Vivo ◽  
Integrin Αvβ3 ◽  
In Vivo Evaluation ◽  
Design Synthesis ◽  
Pet Ct ◽  
In Vivo Testing ◽  
Radiochemical Yields

Combining two peptides addressing two different receptors to a heterobivalent peptidic ligand (HBPL) is thought to enable an improved tumor-targeting sensitivity and thus tumor visualization, compared to monovalent peptide ligands. In the case of melanoma, the Melanocortin-1 receptor (MC1R), which is stably overexpressed in the majority of primary malignant melanomas, and integrin αvβ3, which is involved in lymph node metastasis and therefore has an important role in the transition from local to metastatic disease, are important target receptors. Thus, if a radiolabeled HBPL could be developed that was able to bind to both receptor types, the early diagnosis and correct staging of the disease would be significantly increased. Here, we report on the design, synthesis, radiolabeling and in vitro and in vivo testing of different SiFAlin-modified HBPLs (SiFA = silicon fluoride acceptor), consisting of an MC1R-targeting (GG-Nle-c(DHfRWK)) and an integrin αvβ3-affine peptide (c(RGDfK)), being connected by a symmetrically branching framework including linkers of differing length and composition. Kit-like 18F-radiolabeling of the HBPLs 1–6 provided the labeled products [18F]1–[18F]6 in radiochemical yields of 27–50%, radiochemical purities of ≥95% and non-optimized molar activities of 17–51 GBq/µmol within short preparation times of 25 min. Besides the evaluation of radiotracers regarding logD(7.4) and stability in human serum, the receptor affinities of the HBPLs were investigated in vitro on cell lines overexpressing integrin αvβ3 (U87MG cells) or the MC1R (B16F10). Based on these results, the most promising compounds [18F]2, showing the highest affinity to both target receptors (IC50 (B16F10) = 0.99 ± 0.11 nM, IC50 (U87MG) = 1300 ± 288 nM), and [18F]4, exhibiting the highest hydrophilicity (logD(7.4) = −1.39 ± 0.03), were further investigated in vivo and ex vivo in a xenograft mouse model bearing both tumors. For both HBPLs, clear visualization of B16F10, as well as U87MG tumors, was feasible. Blocking studies using the respective monospecific peptides demonstrated both peptide binders of the HBPLs contributing to tumor uptake. Despite the somewhat lower target receptor affinities (IC50 (B16F10) = 6.00 ± 0.47 nM and IC50 (U87MG) = 2034 ± 323 nM) of [18F]4, the tracer showed higher absolute tumor uptakes ([18F]4: 2.58 ± 0.86% ID/g in B16F10 tumors and 3.92 ± 1.31% ID/g in U87MG tumors; [18F]2: 2.32 ± 0.49% ID/g in B16F10 tumors and 2.33 ± 0.46% ID/g in U87MG tumors) as well as higher tumor-to-background ratios than [18F]2. Thus, [18F]4 demonstrates to be a highly potent radiotracer for the sensitive and bispecific imaging of malignant melanoma by PET/CT imaging and impressively illustrates the suitability of the underlying concept to develop heterobivalent integrin αvβ3- and MC1R-bispecific radioligands for the sensitive and specific imaging of malignant melanoma by PET/CT.

scholarly journals Cell-based approaches in drug development – a concise review

2020 ◽  
Vol 8 (1) ◽  
pp. 44-49
Author(s):  
Ievgeniia Kocherova ◽  
Bartosz Kempisty ◽  
Greg Hutchings ◽  
Lisa Moncrieff ◽  
Claudia Dompe ◽  
...  
Keyword(s):  
Drug Development ◽  
Development Process ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Cytotoxic Effects ◽  
Safety Issues ◽  
Third Phase ◽  
Concise Review

AbstractIn vitro models represent an alternative technique to in vivo or ex vivo studies in the drug development process. Cell-based assays are used to measure the level of proliferation and toxicity, as well as activation of signalling pathways and changes in morphology in cultivated cells. The studies conducted in vitro are aimed to estimate the newly synthesised drugs’ ability to permeate biological barriers and exert their therapeutic or cytotoxic effects. However, more than half of all studied drugs fail in the second or third phase of clinical trials due to a lack of confirmed efficacy. About a third of drugs fail because of safety issues, such as unacceptable levels of toxicity. To reduce attrition level in drug development, it is crucial to consider the implementation of translational phenotypic assays as well as to decipher various molecular mechanisms of action for new molecular entities. In this review, we summarise the existing cell-based methods most frequently used in the studies on drugs, taking into account their advantages and drawbacks.Running title: Cell-based approaches in drug development

scholarly journals Role of Defensins in Corneal Epithelial Barrier Function againstPseudomonas aeruginosaTraversal

2010 ◽  
Vol 79 (2) ◽  
pp. 595-605 ◽  
Author(s):  
Danielle K. Augustin ◽  
Susan R. Heimer ◽  
Connie Tam ◽  
Wing Y. Li ◽  
Jeff M. Le Due ◽  
...  
Keyword(s):  
Barrier Function ◽  
Ex Vivo ◽  
Bacterial Colonization ◽  
Corneal Epithelial Cell ◽  
Epithelial Barrier ◽  
Epithelial Barrier Function ◽  
Corneal Epithelial

ABSTRACTStudies have shown that epithelium-expressed antimicrobial peptides (AMPs), e.g., β-defensins, play a role in clearing bacteria from mouse corneas already infected withPseudomonas aeruginosa. Less is known about the role of AMPs in allowing the cornea to resist infection when healthy. We previously reported that contact lens exposure, a major cause ofP. aeruginosakeratitis, can inhibit the upregulation of human β-defensin 2 (hBD-2) by corneal epithelial cells in response toP. aeruginosaantigensin vitro. Here, we studied the role of AMPs in maintaining the corneal epithelial barrier toP. aeruginosapenetration using bothin vitro(human) andin vivo(mouse) experiments. Results showed that preexposing human corneal epithelial multilayers to bacterial antigens in a culture supernatant (known to upregulate AMP expression) reduced epithelial susceptibility toP. aeruginosatraversal up to 6-fold (P< 0.001). Accordingly, small interfering RNA (siRNA) knockdown of any one of four AMPs expressed by human epithelia promotedP. aeruginosatraversal by more than 3-fold (P< 0.001). The combination knockdown of AMPs further enhanced susceptibility to bacterial traversal by ∼8-fold (P< 0.001).In vivoexperiments showed that the loss of murine β-defensin 3 (mBD-3), a murine ortholog of hBD-2, enhanced corneal susceptibility toP. aeruginosa. The uninjured ocular surface of mBD-3−/−mice showed a reduced capacity to clearP. aeruginosa, and their corneal epithelia were more susceptible to bacterial colonization, even when inoculatedex vivoto exclude tear fluid effects. Together, thesein vitroandin vivodata show functional roles for AMPs in normal corneal epithelial cell barrier function againstP. aeruginosa.

Are all granzymes cytotoxic in vivo?

2014 ◽  
Vol 395 (2) ◽  
pp. 181-202 ◽  
Author(s):  
Lars T. Joeckel ◽  
Phillip I. Bird
Keyword(s):  
Serine Proteases ◽  
Ex Vivo ◽  
Cytotoxic Lymphocytes ◽  
Cytotoxic Effects ◽  
Knock Out ◽  
Granzyme A ◽  
Knock Out Mice

Abstract Granzymes are serine proteases mainly found in cytotoxic lymphocytes. The most-studied member of this group is granzyme B, which is a potent cytotoxin that has set the paradigm that all granzymes are cyototoxic. In the last 5 years, this paradigm has become controversial. On one hand, there is a plethora of sometimes contradictory publications showing mainly caspase-independent cytotoxic effects of granzyme A and the so-called orphan granzymes in vitro. On the other hand, there are increasing numbers of reports of granzymes failing to induce cell death in vitro unless very high (potentially supra-physiological) concentrations are used. Furthermore, experiments with granzyme A or granzyme M knock-out mice reveal little or no deficit in their cytotoxic lymphocytes’ killing ability ex vivo, but indicate impairment in the inflammatory response. These findings of non-cytotoxic effects of granzymes challenge dogma, and thus require alternative or additional explanations to be developed of the role of granzymes in defeating pathogens. Here we review evidence for granzyme cytotoxicity, give an overview of their non-cytotoxic functions, and suggest technical improvements for future investigations.

scholarly journals BETULIN DERIVATIVES. BIOLOGICAL ACTIVITY AND SOLUBILITY IMPROVEMENT

2019 ◽  
pp. 407-430
Author(s):  
Ol'ga Aleksandrovna Vorobyeva ◽  
Darina Sergeyevna Malygina ◽  
Elizaveta Vladimirovna Grubova ◽  
Nina Borisovna Melnikova
Keyword(s):  
Betulinic Acid ◽  
Water Solubility ◽  
Ex Vivo ◽  
Mechanical Action ◽  
Biological Properties ◽  
Birch Bark ◽  
Solubility Improvement ◽  
Betulin Derivatives

In the review the biological properties (antitumor, antiviral, hypolipidemic, anti-inflammatory, etc.) and bioavailability of betulin and betulinic acid derivatives were discussed. These compounds are isolated from various natural sources, including birch bark (Betula, Betulaceae). The structure-activity correlation was considered for well-known betulinic acid derivatives. The perspectivity of this compounds as active pharmaceutical ingredients was demonstrated by in vitro, in vivo, and ex vivo experiments. The type of antitumor actions, generally, depends on substituents at the C-3 and C-28 carbon atoms of the lupane skeleton. It is very important that the carboxyl group of betulinic acid in the C-28 position was present. In this case, the cytotoxicity of C-3 modified derivatives is extremely high for all tested cell lines. The use of these compounds in the medical practice is complicated because they have low bioavailability and poor water solubility (from 1 to 100 µg*l-1). The main chemical syntheses for solubility improvement of betulin derivatives by grafting of hydrophilic groups were discussed. Moreover, the colloid-chemical approaches for the bioavailability improving of triterpenoids include: 1) including of these compounds in liposomes, vesicles and other nanoparticles; 2) obtaining of micelles with high-molecular compounds; 3) colloid-chemical dissolution due to physico-mechanical action; 4) inclusion complexes formation; 5) using of polymers for triterpenoids grafting. Chemical modification of betulin and betulinic acid by polar groups, such as phosphate/phosphonate, sulfate, amino acids, etc. has been shown for bioavailability improving.

scholarly journals Preclinical and Exploratory Clinical Studies of Novel 68Ga-labeled ᴅ-Peptide Antagonist for PET Imaging of TIGIT Expression in Cancers

2021 ◽  
Author(s):  
Xiaobo Wang ◽  
Ming Zhou ◽  
Bei Chen ◽  
Huanhuan Liu ◽  
Jianyang Fang ◽  
...  
Keyword(s):  
Pilot Study ◽  
Pet Imaging ◽  
Ex Vivo ◽  
Ct Imaging ◽  
Whole Body ◽  
Binding Assays ◽  
Pet Ct ◽  
Peptide Antagonist

Abstract Purpose While TIGIT has been propelled under the spotlight as a next-generation target in cancer immunotherapy, anti-TIGIT therapy seems to be promising for a fraction of patients in clinical trials. Therefore, patient stratification is critical for this therapy, which could benefit from a whole-body, non-invasive and quantitative evaluation of TIGIT expression in cancers. In this study, a 68Ga-labeled ᴅ-peptide antagonist, 68Ga-GP12, was developed and validated for PET imaging of TIGIT expression in vitro, in vivo, and first-in-human pilot study. Methods The ᴅ-enantiomer peptide antagonists were modified and radiolabeled with 68Ga. In vitro binding assays were performed in human peripheral blood mononuclear cells (PBMCs) to assess their affinity and specificity. The imaging capacity, biodistribution, pharmacokinetics, and radiation dosimetry were investigated in vivo. Flow cytometry, autoradiography, and immunohistochemical staining were used to confirm the expression of TIGIT ex vivo. The safety and potential of 68Ga-GP12 for PET/CT imaging of TIGIT expression were further evaluated in a first-in-human pilot study with advanced NSCLC. Results 68Ga-labeled ᴅ-peptides were conveniently produced with high radiochemical yields,radiochemical purity and molar activities. In vitro binding assays demonstrated 68Ga-GP12 has favorable affinity and specificity for TIGIT with a KD of 37.28 nM. In vivo and ex vivo studies demonstrated the favorable pharmacokinetics of 68Ga-GP12 for PET imaging of TIGIT expression with high tumor uptake of 4.22 ± 0.68 %ID/g and the tumor-to-muscle ratio of 12.94 ± 2.64 at 60 min post-injection. The primary and metastatic lesions found in the first-in-human studies of 68Ga-GP12 PET/CT imaging were comparable to that in 18F-FDG PET/CT imaging. Moreover, the inhomogenous intra-and-inter-tumoral uptake of 68Ga-GP12 was presented, reflecting the heterogeneity of TIGIT expression levels. Conclusion 68Ga-GP12 is a promising radiotracer for PET imaging of TIGIT expression in cancers, indicating its potential as a potential companion diagnostic for anti-TIGIT therapies.

scholarly journals Investigation of Combined Cyclodextrin and Hydrogel Formulation for Ocular Delivery of Dexamethasone Acetate by Means of Experimental Designs

Pharmaceutics ◽  
2018 ◽  
Vol 10 (4) ◽  
pp. 249 ◽  
Author(s):  
Roseline Mazet ◽  
Luc Choisnard ◽  
Delphine Levilly ◽  
Denis Wouessidjewe ◽  
Annabelle Gèze
Keyword(s):  
Ex Vivo ◽  
Aqueous Solubility ◽  
Eye Drops ◽  
Ocular Delivery ◽  
Drug Bioavailability ◽  
Corneal Inflammation ◽  
Dexamethasone Acetate ◽  
Great Propensity

Dexamethasone acetate (DXMa) has proven its efficiency to treat corneal inflammation, without a great propensity to increase intraocular pressure. Unfortunately, its poor aqueous solubility, associated with a rapid precorneal elimination, results in a low drug bioavailability and a low penetration after topical ocular administration. The main objective of this study was to improve the apparent aqueous solubility of DXMa using cyclodextrins. First, hydroxypropyl-β-CD (HPβCD) and hydroxypropyl-γ-CD (HPγCD) were used to enhance DXMa concentration in aqueous solution. The β and γ HPCD derivatives allowed the increase of the DXMa amount in solution at 25 °C by a factor of 500 and 1500, respectively. Second, with the aim of improving the persistence of the complex solution after instillation in the eye, the formulations of DXMa-based CD solutions with marketed ophthalmic gels (CELLUVISC®, GEL-LARMES®, and VISMED®) were investigated and optimized by means of special cubic mixture designs, allowing the defining of mixed gels loaded with 0.7% (HPβCD) and 2% (HPγCD) DXMa with osmolality within acceptable physiological range. Finally, in vitro drug release assays from the mixed gels were performed and compared with reference eye drops. Similarly to MAXIDEX® and DEXAFREE®, in the case of mixed gel containing HPβCD, more than 90% of the drug was released within 2 h, while in mixed gel containing HPγCD, the release of DXMa was partial, reaching ≈60% in 2 h. This difference will have to be further addressed with ex vivo and in vivo ocular delivery experiments.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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