Photodegradation and Photobinding of Tiaprofenic Acid: in vitro Versus in vivo

Abstract— The photoreactivity of the photosensitizing nonsteroidal anti-inflammatory drug tiaprofenic acid (TA) and its photoproduct decarboxytiaprofenic acid (DTA) was studied both in the presence and in the absence of bovine serum albumin (BSA). The photoproduct DTA was found to be photostable in buffered aqueous solution at pH 7.4, but photodecomposed when BSA was present in the reaction medium. Both TA and DTA underwent irreversible photobinding to BSA in an almost quantitative way, as evidenced by radioactivity measurements using labeled (3H) compounds. In the case of TA, it has been proven that photobinding is mainly attributable to the phototoreactivity of in situ -generated DTA. The photo-degradation and photobinding of TA were also investigated in the epidermis in vivo. Rats were exposed to UVA after application of TA to their shaven dorsal skin. During the initial periods of irradiation, the amount of TA decreased sharply, and the yield of the corresponding photoproduct (DTA) reached a maximum. Prolonged irradiation led to photodegradation of DTA. In vivo photobinding was studied using 3H-TA. Photobinding took place slowly at the beginning, but its rate increased sharply after complete photoconversion of TA, when the photoproduct DTA reached the maximum concentration. Thereafter, the decrease of DTA was more pronounced than that of TA. This indicates thatalso in vivoDTA rather than TA is responsible for the photobinding to biomacromolecules in the viable layer of the epidermis. Overall, the above results suggest that irradiation of TA in buffered aqueous solution, in the presence of proteins, is a reasonable model system to study the photodegradation and photobinding behavior of this drug in vivo. From the qualitative point of view, the main conclusion is that DTA plays a key role both in vivo and in vitro: it is the major photoproduct, it undergoes further photodegradation upon prolonged irradiation, and it appears to be responsible for the photobinding process.     

Peptides that anneal to natural collagen in vitro and ex vivo

Collagen comprises ? of the protein in humans and ? of the dry weight of human skin. Here, we implement recent discoveries about the structure and stability of the collagen triple helix to design new chemical modalities that anchor to natural collagen. The key components are collagen mimetic peptides (CMPs) that are incapable of self-assembly into homotrimeric triple helices, but are able to anneal spontaneously to natural collagen. We show that such CMPs containing 4-fluoroproline residues, in particular, bind tightly to mammalian collagen in vitro and to a mouse wound ex vivo. These synthetic peptides, coupled to dyes or growth factors, could herald a new era in assessing or treating wounds.     

Plasma protein adsorption: In vitro and ex vivo observations

The in vitro and ex vivo adsorption of blood proteins is studied in order to elucidate the protein-surface interactions which determine the thrombogenicity and thus the applicability of various polymers in blood contacting devices. The in vitro adsorption of albumin and fibrinogen to four polymers shows that at low solution concentrations, more fibrinogen is adsorbed than albumin. At higher solution concentrations, albumin adsorbs in multilayers while fibrinogen adsorbs, and then partially desorbs spontaneously from the surface. Sequential adsorption studies show that fibrinogen and albumin can partially replace each other. Fibrinogen is preferentially adsorbed over albumin in competitive adsorption studies. In ex vivo experiments, more albumin than fibrinogen is adsorbed from blood during the first 120 minutes of whole blood contact. When exposed to flowing whole blood, pre-adsorbed fibrinogen desorbs more rapidly than albumin.     

Photobinding of 8-methoxypsoralen, 4,6,4'-trimethylangelicin and chlorpromazine to Wistar rat epidermal biomacromolecules in vivo.

Photoinduced binding of drugs to endogenous biomacromolecules may cause both toxic and therapeutic effects. For example, photobinding of certain phenothiazines to biomolecules possibly underlies their phototoxic and photoallergic potential, whereas photobinding of furocoumarins to epidermal DNA is held responsible for their advantageous effects in the photochemotherapy of psoriasis. Usually, the in vitro photobinding of drugs is investigated. However, under in vivo conditions, the metabolism and distribution of the drug and the light absorption by endogenous compounds will significantly affect the photobinding of drugs to biomolecules. Therefore, in the present study, the photobinding of 8-methoxypsoralen (8-MOP), 4,6,4'-trimethylangelicin (TMA) (two therapeutically used furocoumarins) and chlorpromazine (CPZ) (a member of the phenothiazines) was investigated in vivo. The compounds were applied topically on the shaven skin of Wistar rats; one group was exposed to UVA and the other was kept in a dimly lit environment. Immediately, and at certain time intervals after UVA exposure, members of the two groups were sacrificed. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP, TMA or CPZ to each of these biomacromolecules was determined. In contrast with in vitro experiments, photobinding of CPZ to epidermal DNA/RNA was not found in vivo; apparently the bioavailability in the nucleus is very low. Compared with TMA, 8-MOP was observed to bind more extensively to epidermal DNA/RNA (again in contrast with findings from in vitro experiments) and proteins, but less extensively to lipids. The rates of removal of photobound 8-MOP and TMA were comparable. Photobound CPZ was more slowly removed from epidermal proteins and lipids than the furocoumarins. The observed in vivo photobinding is discussed with respect to the UVA-induced (side) effects of these drugs.     

Photobinding of carprofen to protein

Carprofen is a non-steroidal antiinflammatory drug with marked photosensitising properties. In order to elucidate the mechanisms underlying the phenomenon of drug-protein photobinding, mixtures of the drug and human serum albumin were irradiated under different experimental conditions. After irradiation and subsequent gel-filtration chromatography of the photomixture, the eluting protein fraction was analysed by means of fluorescence spectroscopy. The formation of drug-protein photoadducts could be evidenced by the characteristic emission properties of the carbazole chromophore. The photobinding of the drug to human serum albumin appears to involve the formation of aryl radicals resulting from carbon-halogen photocleavage. This mechanistic interpretation is supported by the observed variations in the intensity of the fluorescence spectra, which can be correlated with the lower quantum yield emission of chlorocarbazoles as compared to non-halogenated analogues. The results from laser flash photolysis studies are also in agreement with this proposal.     

In vitro and ex vivo antitubercular activity of diarylheptanoids from the rhizomes of Alpinia officinarum Hance

Phytochemical investigation of methanol extract of the rhizomes of Alpinia officinarum Hance afforded four known diarylheptanoids 1,7-diphenylhept-4-en-3-one (1), 5-hydroxy-1,7-diphenyl-3-heptanone (2), 5-hydroxy-7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3-heptanone (3), and 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl heptan-3-one (4).The acetate derivative of (4), 7-(4″-actetate-3″-methoxy phenyl)-1-phenyl heptan-3-one (5), was prepared. These diarylheptanoids exhibited promising in vitro and ex vivo antitubercular activity for the first time against dormant Mycobacterium tuberculosis H37Ra with the IC₅₀ values between 0.34–47.69 and 0.13–22.91 μM, respectively. All compounds showed comparable activity against Mycobacterium bovis BCG (dormant phage) and did not show any activity against two gram + ve and two gram –ve bacterial strains. These compounds were also weakly cytotoxic up to 300 μM against three human cancer cell lines THP-1, Panc-1 and A549.     

Indirect detection of photosensitizer ex vivo

Photodynamic therapy induces the production of reactive oxygen species (ROS) within tissues exposed to laser light after administration of a sensitizer. In the context of continuing clinical and commercial development of chemicals with sensitizing properties, a minimally invasive assay is needed to determine the tissue kinetics of fluorescent or non-fluorescent photoreactive drugs. The level of ROS was determined ex vivo from 1 mm3 biopsy samples using 2'-7' dichlorofluorescin diacetate (DCFH-DA), a fluorescent probe which was converted into highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This assay was tested on meta(tetrahydroxyphenyl)chlorin (m-THPC, FOSCAN), a powerful and fluorescent sensitizer, and bacteriochlorophyll derivative WST09 (TOOKAD), a near-infrared absorbing sensitizer that is only slightly fluorescent. In conjunction with the ROS assay, the tissue accumulation of m-THPC was determined on biopsy samples using an optic fibre spectrofluorometer (OFS). DCF fluorescence was proportional to the level of oxidation induced by horseradish peroxidase used as a control and to the concentration (range: 0-5 microg x ml(-1)) of both selected photosensitizers irradiated in a tube together with DCFH. Regardless of the organ studied, an excellent correlation was found between fluorescence measurement by OFS and ROS determination for m-THPC. m-THPC (2 mg x kg(-1) iv) accumulation in tumour tissues was best after 48 h, and the best signal was obtained in liver. With non-fluorescent WST09 (2 mg x kg(-1)), ROS determination showed the best tumour uptake 48 h after injection, with a tumour/muscle ratio of 5.4. The ROS assay appears to be feasible for determining sensitizer concentration in regular grip biopsy tissue samples.     

High-wavenumber FT-Raman spectroscopy for in vivo and ex vivo measurements of breast cancer

The identification of normal and cancer breast tissue of rats was investigated using high-frequency (HF) FT-Raman spectroscopy with a near-infrared excitation source on in vivo and ex vivo measurements. Significant differences in the Raman intensities of prominent Raman bands of lipids and proteins structures (2,800?C3,100?cm^?1) as well as in the broad band of water (3,100?C3,550?cm^?1) were observed in mean normal and cancer tissue spectra. The multivariate statistical analysis methods of principal components analysis (PCA) and linear discriminant analysis (LDA) were performed on all high-frequency Raman spectra of normal and cancer tissues. LDA results with the leave-one-out cross-validation option yielded a discrimination accuracy of 77.2, 83.3, and 100% for in vivo transcutaneous, in vivo skin-removed, and ex vivo biopsy HF Raman spectra. Despite the lower discrimination value for the in vivo transcutaneous measurements, which could be explained by the breathing movement and skin influences, our results showed good accuracy in discriminating between normal and cancer breast tissue samples. To support this, the calculated integration areas from the receiver-operating characteristic (ROC) curve yielded 0.86, 0.94, and 1.0 for in vivo transcutaneous, in vivo skin-removed, and ex vivo biopsy measurements, respectively. The feasibility of using HF Raman spectroscopy as a clinical diagnostic tool for breast cancer detection and monitoring is due to no interfering contribution from the optical fiber in the HF Raman region, the shorter acquisition time due to a more intense signal in the HF Raman region, and the ability to distinguish between normal and cancerous tissues.     

HPLC–UV method for temozolomide determination in complex biological matrices: Application for in vitro,ex vivo and in vivo studies

A high‐performance liquid chromatography method for temozolomide (TMZ) determination in complex biological matrices was developed and validated for application in in vitro, ex vivo and in vivo studies of new nanotechnology‐based systems for TMZ nasal delivery. The method was able to quantify TMZ in nanoemulsions, following cellular uptake, in the porcine nasal mucosa and in mouse plasma and brain. Analyses were performed on a C₁₈ column at 35°C, under UV detection at 330 nm. The mobile phase was methanol–acetic acid 0.5% (30:70, v/v), eluted at an isocratic flow rate of 1.1 mL/min. The method was found to be specific, precise, accurate, robust and linear (0.05 to 5 μg/mL) for TMZ determination in all matrices. No interference of TMZ degradation products was found under various stress conditions such as acidic, alkaline, oxidative, light and thermal exposure, demonstrating stability. The method was applied for the quantification of TMZ in different matrices, i.e. the efficiency of nanoemulsions in vitro in increasing TMZ cellular uptake, ex vivo TMZ permeation and retention in the porcine nasal mucosa tissue, and for in vivo TMZ quantification in mouse brain following intranasal nanoemulsion administration compared with free TMZ.     

MALDI-MS imaging of lipids in ex vivo human skin

Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 μm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i ^TM (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMS^TM system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.     

Comparison of protein adsorption patterns onto differently charged hydrophilic superparamagnetic iron oxide particles obtained in vitro and ex vivo.

Protein adsorption patterns of superparamagnetic iron oxides (SPIO) were evaluated by two-dimensional electrophoresis (2-DE) after in vitro incubation of the particles in plasma or serum. SPIO particles having positive (MKK 1211), negative (MKA 1211), or neutral (MKG 1411) charge were used. Protein adsorption patterns of different charged SPIO particles acquired in vitro and recollected 5 min after intravenous injection into rats (ex vivo) were compared. For the uncharged MKG 1411 particles, the differences of protein adsorption patterns were negligible and only minor differences were found for the negatively charged MKA 1211 and positively charged MKK 1211 particles. A good correlation between in vitro and ex vivo data could be shown. For the evaluation of protein adsorption patterns of SPIO particles determining organ distribution and allowing estimation of site-specific delivery (drug targeting), the currently used protocol for 2-DE analysis could be confirmed.     

Stability indicating liquid chromatographic method for simultaneous quantification of betamethasone valerate and tazarotene in in vitro and ex vivo studies of complex nanoformulation

Low‐potency corticosteroid betamethasone valerate and vitamin‐A tazarotene are used in combination for effective treatment of psoriasis. There is no robust high‐performance liquid chromatography analytical technique available for simultaneous estimation of betamethasone valerate and tazarotene in conventional and nanocarriers based formulations. A simple, accurate, robust isocratic high‐performance liquid chromatography method was developed for simultaneous estimation of betamethasone valerate and tazarotene in topical pharmaceutical formulations. The developed method was validated as per the regulatory guidelines. The validated method was linear over the concentration range of 150–6000 ng/mL (r² > 0.999) at 239 nm wavelength. Limits of detection and quantification of two analytes were 50 and 150 ng/mL, respectively. The %relative standard deviation for intraday and interday precision was less than 2%. The method was also evaluated in the presence of forced degradation conditions. The developed method was successfully applied for in vitro and ex vivo drug release studies of in‐house designed nanoformulations.     

Phospholipid dynamics in ex vivo lung cancer and normal lung explants

In cancer cells, metabolic pathways are reprogrammed to promote cell proliferation and growth. While the rewiring of central biosynthetic pathways is being extensively studied, the dynamics of phospholipids in cancer cells are still poorly understood. In our study, we sought to evaluate de novo biosynthesis of glycerophospholipids (GPLs) in ex vivo lung cancer explants and corresponding normal lung tissue from six patients by utilizing a stable isotopic labeling approach. Incorporation of fully ¹³C-labeled glucose into the backbone of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) was analyzed by liquid chromatography/mass spectrometry. Lung cancer tissue showed significantly elevated isotopic enrichment within the glycerol backbone of PE, normalized to its incorporation into PI, compared to that in normal lung tissue; however, the size of the PE pool normalized to the size of the PI pool was smaller in tumor tissue. These findings indicate enhanced PE turnover in lung cancer tissue. Elevated biosynthesis of PE in lung cancer tissue was supported by enhanced expression of the PE biosynthesis genes ETNK2 and EPT1 and decreased expression of the PC and PI biosynthesis genes CHPT1 and CDS2, respectively, in different subtypes of lung cancer in publicly available datasets. Our study demonstrates that incorporation of glucose-derived carbons into the glycerol backbone of GPLs can be monitored to study phospholipid dynamics in tumor explants and shows that PE turnover is elevated in lung cancer tissue compared to normal lung tissue.Subject terms: Cancer metabolism, Lung cancer     

A green chemistry approach towards synthesizing hydrogel for sustained ocular delivery of brinzolamide: In vitro and ex vivo evaluation

Brinzolamide is a carbonic anhydrase inhibitor used in the eye drop form for the treatment of glaucoma. It requires frequent dosing to attain therapeutic concentration. Therefore, this study aimed to prepare sustained ocular drug delivery of brinzolamide. The objective of the study was to prepare a hydrogel loaded with a nanostructured lipid carrier (NLC) of brinzolamide. The hydrogel was prepared by a green synthesis approach using genipin as a natural crosslinking agent and polymers such as carboxymethyl chitosan and poloxamer 407. The melt emulsification-ultra sonication method was used to prepare a nanostructured lipid carrier of brinzolamide, which was loaded into a hydrogel using a swelling and loading method. The NLC formulation has shown small particle sizes of 111.20 ?± ?2.15 ?nm, polydispersity index of 0.280 ?± ?0.005 and % entrapment efficiency of 82.16% ?± ?0.14%. The NLC-loaded hydrogels of brinzolamide formulations were studied for swelling properties and showed temperature and pH-responsive swelling behavior. The optimized hydrogel formulation has been studied for in vitro drug release and showed drug release for a longer duration (24 ?h) than marketed eye drops (8 ?h). In an ex vivo study, hydrogel formulations showed transcorneal permeability 4.54 times greater than marketed eye drops. The hydrogel formulation of brinzolamide produced by the green synthesis method has shown sustained-release properties with no sign of ocular irritation. Hence, the hydrogel of brinzolamide-loaded NLC would be the potential drug delivery approach in the near future for sustained ocular drug delivery in glaucoma management.     

In and ex vivo breast disease study by Raman spectroscopy

In this work, Raman spectra in the 900?C1,800?cm^?1 wavenumber region of in vivo and ex vivo breast tissues of both healthy mice (normal) and mice with induced mammary gland tumors (abnormal) were measured. In the case of the in vivo tissues, the Raman spectra were collected for both transcutaneous (with skin) and skin-removed tissues. To identify the spectral differences between normal and cancer breast tissue, the paired t-test was carried out for each wavenumber using the whole spectral range from both groups. Quadratic discriminate analysis based on principal component analysis (PCA) was also used to determine and evaluate differences in the Raman spectra for the various samples as a basis for diagnostic purposes. The differences in the Raman spectra of the samples were due to biochemical changes at the molecular, cellular and tissue levels. The sensitivity and specificity of the classification scheme based on the differences in the Raman spectra obtained by PCA were evaluated using the receiver operating characteristic (ROC) curve. The in vivo transcutaneous normal and abnormal tissues were correctly classified based on their measured Raman spectra with a discriminant proportion of 73%, while the in vivo skin-removed normal and abnormal tissues were correctly classified again based on their measured Raman spectra with a discriminant proportion of 86%. This result reveals a strong influence due to the skin of the breast, which decreased the specificity by 11%. Finally, the results from ex vivo measurements gave the highest specificity and sensitivity: 96 and 97%, respectively, as well as a largest percentage for correct discrimination: 94%. Now that the important bands have been experimentally determined in this and other works, what remains is for first principles molecular-level simulations to determine whether the changes are simply due to conformational changes, due to aggregation, due to changes in the environment, or complex interactions of all of the above.     

Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin

Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280-320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m(-2) UVB), the model finds that 14.70 x 10(-3) mol of ROS that react with DHR are generated in the stratum corneum of an average adult-size face (258 cm(-2)). Approximately 10(-4) mol are potentially generated in the lower epidermal strata. The data show that two-photon fluorescence imaging can be used to detect ROS in UV-irradiated skin.     

Photosensitization reactions in vitro and in vivo

This review of Photochemistry and Photobiology summarizes articles published in 2010, and highlights progress in the area of photosensitization. The synthesis of conjugated photosensitizers is an area of interest where increasing water solubility has been a goal. Targeting infrared sensitizer absorption has been another goal, and relates to the practical need of deep tissue absorption of light. Photodynamic techniques for inactivating microbes and destroying tumors have been particularly successful. Biologically, singlet oxygen [(1)O(2)((1)Δ(g))] is an integral species in many of these reactions, although photosensitized oxidations tuned to electron and hydrogen transfer (Type I) give rise to other reactive species, such as superoxide and hydrogen peroxide. How photoprotection against yellowing, oxygenation and degradation occurs was also an area of topical interest.     

DNA-lipid interactions in vitro and in vivo

The data on lipid-nucleic interactions and their role in vitro and in vivo are presented. The results of study of DNA-lipid complexes in absence and in presence of divalent metal cations (triple complexes) are discussed. The triple complexes represent the generation of cellular structures such as pore complexes of eucaryotes and "Bayer's junctions" of procaryotes. The participation of triple complexes in the formation of structure of bacterial and eucaryotic nucleoid and nuclear matrix is analysed. A model of formation of triple complexes and cellular structures and their role in DNA-lipid interactions are discussed.     

Design of meloxicam and lornoxicam transdermal patches: Preparation, physical characterization, ex vivo and in vivo studies

Transdermal patches of meloxicam (MX) and lornoxicam (LX) were aimed to be prepared in order to overcome their side effects by oral application. The strategy was formulation of optimized films to prepare transdermal patches by determination of physical properties and investigation of drug-excipient compatibility. As the next step, in vitro drug release, assesment of anti-inflammatory effect on Wistar Albino rats, ex vivo skin penetration and investigation of factors on drug release from transdermal patches were studied. Hydroxypropyl methylcellulose (HPMC) was concluded to be suitable polymer for formulation of MX and LX transdermal films indicating pharmaceutical quality required. MX and LX transdermal patches gave satisfactory results regarding to the edema inhibition in the assessment of anti-inflammatory effect. MX was found out to be more effective compared to LX on relieving of edema and swelling. These results were supported by data obtained from ex vivo penetration experiments of drug through rat skin. Indicative parameters like log P, molecular weight and solubility constraint on penetration rate of drugs also indicated good skin penetration. Transdermal patches of MX and LX can be suggested to be used especially for the immediate treatment of inflammated area since it displays anti-inflammatory effect, soon.     

Achieving natural product synthesis and diversity via catalytic networking ex vivo

Recent studies on ex vivo synthesis of natural products reveal that even complex multistep pathways can be successfully reconstructed. Genetic engineering of such reconstituted pathways has already been used to generate ‘unnatural’ natural products related to the original compound. In the future, it may be possible to use these approaches to make natural products that are currently inaccessible to conventional synthesis.