Cyanobacteria Secondary Metabolites as Biotechnological Ingredients in Natural Anti-Aging Cosmetics: Potential to Overcome Hyperpigmentation, Loss of Skin Density and UV Radiation-Deleterious Effects.

The loss of density and elasticity, the appearance of wrinkles and hyperpigmentation are among the first noticeable signs of skin aging. Beyond UV radiation and oxidative stress, matrix metalloproteinases (MMPs) assume a preponderant role in the process, since their deregulation results in the degradation of most extracellular matrix components. In this survey, four cyanobacteria strains were explored for their capacity to produce secondary metabolites with biotechnological potential for use in anti-aging formulations. Leptolyngbya boryana LEGE 15486 and Cephalothrix lacustris LEGE 15493 from freshwater ecosystems, and Leptolyngbya cf. ectocarpi LEGE 11479 and Nodosilinea nodulosa LEGE 06104 from marine habitats were sequentially extracted with acetone and water, and extracts were analyzed for their toxicity in cell lines with key roles in the skin context (HaCAT, 3T3L1, and hCMEC). The non-toxic extracts were chemically characterized in terms of proteins, carotenoids, phenols, and chlorophyll a, and their anti-aging potential was explored through their ability to scavenge the physiological free radical superoxide anion radical (O2•−), to reduce the activity of the MMPs elastase and hyaluronidase, to inhibit tyrosinase and thus avoid melanin production, and to block UV-B radiation (sun protection factor, SPF). Leptolyngbya species stood out for anti-aging purposes: L. boryana LEGE 15486 presented a remarkable SPF of 19 (at 200 µg/mL), being among the best species regarding O2•− scavenging, (IC50 = 99.50 µg/mL) and also being able to inhibit tyrosinase (IC25 = 784 µg/mL), proving to be promising against UV-induced skin-aging; L. ectocarpi LEGE 11479 was more efficient in inhibiting MMPs (hyaluronidase, IC50 = 863 µg/mL; elastase, IC50 = 391 µg/mL), thus being the choice to retard dermal density loss. Principal component analysis (PCA) of the data allowed the grouping of extracts into three groups, according to their chemical composition; the correlation of carotenoids and chlorophyll a with MMPs activity (p < 0.01), O2•− scavenging with phenolic compounds (p < 0.01), and phycocyanin and allophycocyanin with SPF, pointing to these compounds in particular as responsible for UV-B blockage. This original survey explores, for the first time, the biotechnological potential of these cyanobacteria strains in the field of skin aging, demonstrating the promising, innovative, and multifactorial nature of these microorganisms.

The SOS Chromotest applied for screening plant antigenotoxic agents against ultraviolet radiation.

In this work, we investigated the usefulness of the SOS Chromotest for screening plant antigenotoxic agents against ultraviolet radiation (UV). Fifty Colombian plant extracts obtained by supercritical fluid (CO2) extraction, twelve plant extract constituents (apigenin, carvacrol, ß-caryophyllene, 1,8-cineole, citral, p-cymene, geraniol, naringenin, pinocembrin, quercetin, squalene, and thymol) and five standard antioxidant and/or photoprotective agents (curcumin, epigallocatechin gallate, resveratrol, α-tocopherol, and Trolox®) were evaluated for their genotoxicity and antigenotoxicity against UV using the SOS Chromotest. None of the plant extracts, constituents or agents were genotoxic in the SOS Chromotest at tested concentrations. Based on the minimal extract concentration that significantly inhibited UV-genotoxicity (CIG), five plant extracts were antigenotoxic against UV as follows: Baccharis nítida (16 µg mL-1) = Solanum crotonifolium (16 µg mL-1) > Hyptis suaveolens (31 µg mL-1) = Persea caerulea (31 µg mL-1) > Lippia origanoides (62 µg mL-1). Based on CIG values, the flavonoid compounds showed the highest antigenotoxic potential as follows: apigenin (7 µM) > pinocembrin (15 µM) > quercetin (26 µM) > naringenin (38 µM) > epigallocatechin gallate (108 µM) > resveratrol (642 µM). UV-genotoxicity inhibition with epigallocatechin gallate, naringenin and resveratrol was related to its capability for inhibiting protein synthesis. A correlation analysis between compound antigenotoxicity estimates and antioxidant activity evaluated by the oxygen radical absorbance capacity (ORAC) assay showed that these activities were not related. The usefulness of the SOS Chromotest for bioprospecting of plant antigenotoxic agents against UV was discussed.

Quantitative analysis of hindered amine light stabilizers by CZE with UV detection and quadrupole TOF mass spectrometric detection.

The current work describes the development of a CZE method with quadrupole QTOF-MS detection and UV detection for the quantitation of Cyasorb 3529, a common hindered amine light stabilizer (HALS), in polymer materials. Analysis of real polymer samples revealed that the oligomer composition of Cyasorb 3529 changes during processing, a fact hampering the development of a straightforward method for quantitation based on calibration with a Cyasorb 3529 standard. To overcome this obstacle in-depth investigations of the oligomer composition of this HALS using QTOF-MS and QTOF-MS/MS had to be performed whereby 22 new oligomer structures, in addition to the ten structures already described, were identified. Finally, a CZE method for quantitative analysis of this HALS was developed starting with a comprehensive characterization of a Cyasorb 3529 standard using CZE-QTOF-MS, subsequently allowing the correct assignment of most Cyasorb 3529 oligomers in an electropherogram with UV detection. Employing the latter detection technique and hexamethyl-melamine as internal standard, peak areas obtained for the melamine could be correlated with those from the triazine ring, the UV-absorbing unit present in the HALS. This approach finally allowed proper quantitation of the single oligomers of Cyasorb 3529, an imperative for the quantitative assessment of this HALS in real polymer samples.

Analysis of UV-absorbing photoprotectant mycosporine-like amino acid (MAA) in the cyanobacterium Arthrospira sp. CU2556.

Mycosporine-like amino acids (MAAs) are ecologically important biomolecules with great photoprotective potential. The present study aimed to investigate the biosynthesis of MAAs in the cyanobacterium Arthrospira sp. CU2556. High-performance liquid chromatography (HPLC) with photodiode-array detection studies revealed the presence of a UV-absorbing compound with an absorption maximum at 310 nm. Based on its UV absorption spectrum and ion trap liquid chromatography/mass spectrometry (LC/MS) analysis, the compound was identified as a primary MAA mycosporine-glycine (m/z: 246). To the best of our knowledge this is the first report on the occurrence of MAA mycosporine-glycine (M-Gly) in Arthrospira strains studied so far. In contrast to photosynthetic activity under UV-A radiation, the induction of the biosynthesis of M-Gly was significantly more prominent under UV-B radiation. The content of M-Gly was found to increase with the increase in exposure time under UV-B radiation. The MAA M-Gly was highly stable under UV radiation, heat, strongly acidic and alkaline conditions. It also exhibited good antioxidant activity and photoprotective ability by detoxifying the in vivo reactive oxygen species (ROS) generated by UV radiation. Our results indicate that the studied cyanobacterium may protect itself by synthesizing the UV-absorbing/screening compounds as important defense mechanisms, in their natural brightly-lit habitat with high solar UV-B fluxes.

Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria.

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

A collagen extraction and deuterium oxide stable isotope tracer method for the quantification of bone collagen synthesis rates in vivo.

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short-term (days-weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D2 O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope-ratio mass spectrometry (GC-pyrolysis-IRMS). Forty-six male and female rats from a selectively bred model ingested D2 O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T-PRO), and distal (T-DIS) epiphyses-metaphyses and tibia mid-shaft diaphyses (T-MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106-0.156]) was greater than the FSR at T-MID (0.055 ± 0.049%/day; 95% CI [0.040-0.070]; p < 0.001). The T-PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166-0.241]) and T-DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022-0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site-specific rates of synthesis, which could be applicable to studies of human bone collagen turnover.

Combined effects of UVR and temperature on the survival of crab larvae (Zoea I) from Patagonia: the role of uv-absorbing compounds.

The aim of our study was to assess the combined impact of UVR (280-400 nm) and temperature on the first larval stage (Zoea I) of three crab species from the Patagonian coast: Cyrtograpsus altimanus, C. angulatus, and Leucippa pentagona. We determined the survival response of newly hatched Zoea I after being exposed for 8-10 h under a solar simulator (Hönle SOL 1200) at 15 and 20 degrees C. There was no mortality due to Photosynthetic Active Radiation (PAR, 400-700 nm) or ultraviolet-A radiation (UV-A, 315-400 nm), and all the observed mortality was due to ultraviolet-B radiation (UV-B, 280-315 nm). The data of larval mortality relative to exposure time was best fit using a sigmoid curve. Based on this curve, a threshold (Th) and the lethal dose for 50% mortality (LD(50)) were determined for each species. Based on the Th and LD(50), C. altimanus was found to be the most resistant species, while L. pentagona was found to be the most sensitive to UV-B. For both species of Cyrtograpsus, mortality was significantly lower at 20 degrees C than at 15 degrees C; however, no significant differences between the two temperature treatments were found in L. pentagona. Bioaccumulation of UV-absorbing compounds in the gonads and larvae of C. altimanus, and to a lesser extent in C. angulatus, might have contributed for counteracting the impact of UV-B. However, most of the resilience to UV-B observed with the increase in temperature might be due to an increase in metabolic activity caused by a repair mechanism mediated by enzymes.

Evaluation of a method for measuring the radioprotective metabolite WR-1065 in plasma using chemical derivatization combined with UHPLC-MS/MS.

Hypotension is the dose-limiting side effect of the radio-protective drug Amifostine and results from relaxation of the vascular smooth muscle, which is directly mediated by the active metabolite, WR-1065, of Amifostine. The route of administration (currently FDA-approved only for intravenous administration) and the rapid metabolic conversion of Amifostine combine to yield high systemic levels of WR-1065 and facilitate the onset of hypotension. Research efforts aiming to optimize the delivery of WR-1065 to maintain efficacy while reducing its peak, systemic concentration below levels that induce hypotension are underway. To fully characterize the effect of reduced dose levels and alternative routes of administration of Amifostine on systemic WR-1065 concentrations, improved analytical techniques are needed. We have developed and evaluated a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The method exhibits a limit of quantification (LOQ) of 7.4 nM in plasma, which is a significant improvement over conventional approaches that utilize LC-electrochemical detection (ECD) (LOQ 150 nM or higher). The method was assessed in a pharmacokinetics study in rats administered Amifostine intravenously and via direct jejunal injection (10 mg/kg each route). The bioavailability of WR-1065 was 61.5% after direct jejunal injection indicating rapid conversion and absorption of the metabolite in the intestinal tract. This demonstrates that an oral formulation of Amifostine designed for site-specific release of the drug in the upper GI tract can deliver systemic absorption/conversion to WR-1065, provided that the formulation protects the therapeutic from gastric decomposition in the stomach.

Development and validation of a sensitive liquid chromatographic method for the analysis of a novel radioprotectant: ON 01210.Na.

ON 01210.Na is a chlorobenzylsulfone derivative with potential property to mitigate the effects of accidental or intentional exposure to life threatening levels of radiation. A simple and sensitive HPLC method was developed and validated for the assay of ON 01210.Na. The isocratic system used a mobile phase consisting of acetonitrile:0.1% trifluroacetic acid in water (60:40, v/v) at a flow rate of 1 ml/min. The method used a C-18 Gemini column (250 mm x 4.6 mm) with column effluents monitored at 254 nm. Forced degradation of the drug was achieved by autoclaving ON 01210.Na with 0.05 N HCl, 0.05 N NaOH or 1.5% (v/v) hydrogen peroxide. The assay validation parameters evaluated include specificity, linearity, precision, accuracy and sensitivity. The retention time of the drug and the other effluents were well within 7 min. Standard curves were linear over the concentration range of 10-500 microg/ml. The R.S.D. values for the within-day and day-to-day precision ranged from 0.4 to 2.5 and 2.2 to 4.4%, respectively. The R.S.D. for accuracy measurement ranged from 0.85 to 1.7%. The critical level, the detection level and the determination level for this assay were 2.86+/-0.67, 5.69+/-0.67 and 15.6+/-1.8 microg/ml, respectively. A simple, sensitive and stability indicating HPLC assay was developed and validated for the analysis of a novel radioprotectant. This method was used to evaluate the aqueous as well as solid-state stability of this drug during autoclaving.

Radioprotective Effect of Nigella Sativa Oil on Heart Tissues of Rats Exposed to Irradition

Abstract Background Various studies are ongoing related to the radioprotective agents. Herbal preparations are currently becoming popular because of their beneficial effects with fewer side effects compared to the synthetic/semi-synthetic medicines, and Nigella sativa oil (NSO) is only one of them. Objective To investigate NSO for its antioxidant effects on the heart tissue of rats exposed to ionizing radiation (IR). Methods Thirty six male albino Wistar rats, divided into four groups, were designated to group I (IR plus NSO group) that received both 5 Gray of gamma IR to total cranium and NSO; group II (IR alone group) that received IR plus saline, group III (control group of NSO) that received saline and did not receive NSO or IR; group IV (control group) that received only sham IR. Alterations in Total antioxidant status (TAS) and Total oxidant status (TOS), Oxidative stres index (OSI), Sulhydryl group (SH), Lipid hydroperoxide (LOOH), Paraoxonase (PON) levels, Arylesterase (ARE) and Ceruloplasmin (CER) activities in homogenized heart tissue of rats were measured by biochemical methods. Results In heart tissue of the rats in the IR alone group (group II) LOOH, TOS and OSI levels were found to be higher, ARE activity and TAS level were found to be lower than all of the other groups (p < 0.01). These results also support that IR increases oxidative stress and NSO's protective effect. Conclusion NSO would reduce the oxidative damage in the irradiated heart tissue in the experimental rat model.

In vitro screening of radioprotective properties in the novel glucosylated flavonoids.

Novel glucosyl flavonoids are developed by the addition of glucose to naturally occurring flavonoids. Flavonoids are known antioxidants that possess radioprotective properties. In order to investigate the radioprotective properties of novel glucosyl flavonoids, in vitro DNA double-strand breaks (DSBs) analysis was carried out. In the present study, Quercetin, Naringenin, and Hesperetin groups of flavonoids included in the natural and novel glucosyl 13 flavonoids were investigated. Flavonoids were mixed with Lambda DNA, and subsequently exposed to gamma­rays. Furthermore, DNA DSB yields were visualized by gel electrophoresis. Quercetin derivatives displayed reduced DNA DSB formation at 10 µM. At a high concentration, the majority of flavonoids displayed radioprotective properties as a reduction of DSB yields. Suppression of DSB formation was confirmed via the molecular combing assay for Quercetin, and three monoglucosyl flavonoids. Glucosylation showed positive effects for radioprotection and monoglucosyl-Rutin showed superior radioprotective properties when compared to monoglucosyl-Naringin and Hesperidin. In addition, Quercetin derivatives had greater total antioxidant capacities and DPPH radical scavenging ability than other flavonoid groups. Since Quercetin, Isoquercetin, and Rutin display poor water solubility, monoglucosyl-Rutin, maltooligosyl-Isoquercetin, and maltooligosyl-Rutin may be better radioprotective agents and easily bioavailable with increased water solubility.

Radioprotective properties of sodium humate in radiation-induced mutagenesis in cultured lymphocytes of thyroid cancer patients.

UNLABELLED: To investigate the effect of sodium humate on the level of cytogenetic damage in culture of lymphocytes of patients with thyroid cancer after γ-irradiation. MATERIALS AND METHODS: Metaphase analysis of chromosome aberrations in cultured peripheral blood lymphocytes of 10 individuals with thyroid cancer was performed after irradiation of lymphocytes in vitro at a dose of 1 Gy from (137)Cs source at the early G0 phase of cell cycle. Sodium humate was added to cell culture for 30 ± 15 min after phytohemagglutinin stimulation at concentrations of 10 and 100 µg/ml. RESULTS: Sodium humate exhibited antimutagenic properties. The preparation at a concentration of 10 µg/ml was more effective than at a concentration of 100 µg/ml, reducing the average incidence of radiation-induced chromosome aberrations by 51.88 and 38.77%, respectively. The most pronounced antimutagenic effect of sodium humate was the reduction of the frequency of chromosomal type aberrations, however, such efficiency varied between individual patients with thyroid cancer. CONCLUSIONS: Sodium humate could be considered as a potential therapeutic modifier of radiation damage.

Radioprotection by oral administration of Aegle marmelos (L.) Correa in vivo.

The radioprotective effect of an extract of Aegle marmelos (L.) Correa (AME), family Rutaceae, was investigated in mice exposed to different doses of gamma-radiation. Mice were administered orally AME 250 mg/kg b.wt. orally daily for 5 consecutive days before exposure to 6, 7, 8, 9, 10, or 11 Gy of gamma-radiation. The animals were monitored daily up to 30 days after irradiation for the development of symptoms of radiation sickness or death. Treatment of mice with AME before irradiation reduced the symptoms of radiation sickness and delayed death compared to the irradiated controls given sterile physiological saline (SPS). AME provided protection against both gastrointestinal and hematopoietic toxicities. Reducing the administration schedule of AME to 1 or 3 consecutive days or increasing the schedule to 7 consecutive days was not as effective as 5 consecutive days of preradiation schedule. The administration of AME after irradiation was not effective, and no survivors could be reported 30 days after irradiation. The LD50/30 was found to be 8.1 Gy for the SPS + irradiation group and 9.7 Gy for the AME + irradiation group. The oral administration of AME resulted in an increase in radiation tolerance by 1.6 Gy, and the dose reduction factor was found to be 1.2. Preradiation treatment of mice with AME caused a significant depletion in lipid peroxidation followed by a significant elevation in glutathione concentration in the liver of mice 31 days after irradiation. The drug was nontoxic up to a dose of 6000 mg/kg b.wt., the highest drug dose that could be tested for acute toxicity.

[The estimation of the perspectives of the 1,3,4-thiadiazine derivatives as the protectors means for the health tissues under radiation therapy of malignant tumour patients].

We generalized the results of our own researches of the mechanisms, determined the high (90% BALB-line mice were survived) radioprotection activity by 1,3,4-thiadiazine derivatives. It was determined that this preparat achieves the highest concentrations in the critical for the acute radiation influence tissues. The preparate bind with the cell's membranes, nucleus and mitochondries, blockade the development of the radial reactions on the tissues level. Small quantity passes to the brain marrow, takes part in the regulative processes, which central nervous system is produced, reduces the metabolitical processes in the organism. It doesn't possess the election accumulation in the tumour and it is perspective for the prevention of damage health tissues under irradiation cancroid's therapy.

Natural UV-protective organic matter in thermal water.

Medical significance of the organic fractions of natural waters is still poorly understood. Nevertheless, there are putative biologically active organic compounds found in natural medical waters and related clay or mud samples. Organic fractions of five thermal (spa) water samples of different geochemical origin were tested for photo-biological effects. To study possible effects on the UV sensitivity of Salmonella typhimurium TA strains, the organic isolates were applied in the "plate incorporation" Ames test combined with UV-irradiation. Four samples showed measurable survival of TA100 his+ revertants following exposure to a normally lethal UV dose. Metabolic activation with a mammalian microsomal fraction (S9) elevated the effect detected (up to 61% survival). This is the first study to demonstrate the UV-protective property of organic matter in natural thermal water samples used in balneotherapy.

In situ growth and phenyl functionalization of titania nanoparticles coating for solid-phase microextraction of ultraviolet filters in environmental water samples followed by high performance liquid chromatography-UV detection.

Based on TiO2-nanoparticles coating fabricated by a one-step anodization method on titanium wire substrate, a novel phenyl functionalized solid-phase microextraction (SPME) fiber coating was prepared by simple and rapid in situ chemical assembling technique between the fiber surface titanol groups and trichlorophenylsilane reaction. The as-fabricated fiber exhibited good extraction capability for some UV filters and was employed to determine the ultraviolet (UV) filters in combination with high performance liquid chromatography-UV detection (HPLC-UV). The main parameters affecting extraction performance were investigated and optimized. Under the optimized conditions, the developed method was applied to detect several UV filters at trace concentration levels with only 8 mL of sample volume. They were determined in the range from 0.005 to 25 µg L(-1) with detection limits (S/N=3) from 0.1 to 50 ng L(-1). The relative standard deviations (RSDs) for single fiber repeatability varied from 4.6 to 6.5% (n=5) and fiber-to-fiber reproducibility (n=5) ranged from 5.5 to 9.1%. The linear ranges spanned two-four magnitudes with correlation coefficients above 0.9990. Five real water samples including four Yellow River water samples and one rain water sample were determined sensitively with good recoveries ranging from 86.2 to 105.5%. The functionalized fiber coating performed good reproducible manner, high mechanical strength, good stability and long service life. Moreover, this study proposed an efficient sample pretreatment method for the determination of UV filters from environmental water samples.

Pharmacokinetic profile of amifostine.

This article reviews the chemistry, measurement, metabolism, and pharmacokinetics of the cytoprotective agent amifostine. Validated analytic methodology to measure parent drug and pharmacologically active metabolites and pharmacokinetic studies are essential to the efficient performance and analysis of clinical studies. Well-validated analytic methods developed in the authors' laboratory were used to characterize this agent. Amifostine [s-2-(3-aminopropylamino)ethyl-phosphorothioate] is the phosphorylated pro-drug form of the active free thiol drug WR-1065 [2-(3-aminopropylamino)ethanethiol]. Observations described here support the hypothesis that amifostine is dephosphorylated rapidly by alkaline phosphatase present on the plasma membranes of the arteriolar endothelium of various normal tissues and on the plasma membranes of the kidneys' proximal tubular epithelium to its active thiol metabolite WR-1065, which in turn immediately enters normal tissues. Other metabolites that have been identified are WR-33278, the symmetrical disulfide of WR-1065; the mixed disulfides WR-1065-cysteine and WR-1065-glutathione; and cysteamine. Amifostine was recently approved by the US Food and Drug Administration for use as a cytoprotector in cancer patients receiving chemotherapy. Current pharmacokinetic studies in cancer patients are focusing on establishing a population model as a basis for developing limited sampling strategies for future investigations of the pharmacokinetic-pharmacodynamic behavior of amifostine.

Analysis of S-35 labeled WR-2721 and its metabolites in biological fluids.

Studies with WR-2721 and related compounds have been hindered by the lack of a suitable assay for the drug and its major metabolites. We have developed a chromatographic method which requires no derivatization for the separation and detection of WR-2721, the free thiol, its symmetrical disulfide and other mixed disulfides. Our procedure involves ion-pairing for separation of ionizable compounds by causing polar molecules to become more lipophilic and hence separable using reverse phase HPLC. Detection is based upon liquid scintillation counting of S-35 incorporated during the synthesis of the parent compound. This method requires no pre-column preparation of samples and, by detecting the S-35 label, eliminates the chance that a coeluting species could interfere with detection, as might occur with post-column derivatization. Chromatography was done using a 10 micron C8RP column and 35% MeOH/65% 0.0113M NaH2PO4, 0.005 M hexanesulfonate, pH 5.9, flowing at 1 ml/min. Half-minute fractions were collected into scintillation vials for counting. Retention volumes for the various compounds were: column breakthrough (3.5 ml), WR-2721 (4.5 ml), WR-1065 (9 ml), and WR-33278 (24 ml). This analytical technique employing radiotracers can be used to study radioprotective mechanisms by time dependent measurements of the tissue distribution and chemical form of labeled drug. Such chemical information can then be correlated with biological measures of radiation protection.

Determination of the cytoprotective agent WR-2721 (Amifostine, Ethyol) and its metabolites in human blood using monobromobimane fluorescent labeling and high-performance liquid chromatography.

PURPOSE: WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] is a chemoprotective agent that is currently in pediatric clinical trials. It is a prodrug that is dephosphorylated by alkaline phosphatase to the active free thiol form, WR-1065 [S-2-(3-aminopropylamino)ethanethiol]. It is likely that adequate and sustained cellular levels of the drug are necessary for optimum cytoprotection. To date, a method to measure both plasma and cellular levels of WR-2721 and its metabolites in clinical samples has not been available. METHODS: In the study reported here the monobromobimane (mBBr) fluorescent labeling method was used to measure these levels when drug was added in vitro to blood samples from normal volunteers. In addition, we present pharmacokinetic data from a pediatric patient receiving WR-2721 (825 mg/m2 x 2). RESULTS: The results can be summarized as follows: (1) WR-2721 was detected in the patient's plasma with a half-life of about 10 min; (2) the WR-1065 concentration in the blood cellular fraction was similar to that of plasma; (3) both WR-1065 and WR-SS-low molecular weight (WR-SS-LMW) metabolites disappeared from plasma and the cellular fraction by 3.6 h after WR-2721 infusion; (4) a large proportion of WR-1065 was oxidized in plasma to WR-SS protein and WR-SS-LMW; (5) a large proportion of WR-1065 in the cellular fraction was oxidized to WR-SS-protein; (6) the WR-SS-LMW concentration in the cellular fraction was low; and (7) saturation of plasma and cellular protein binding sites was possible. CONCLUSIONS: The pharmacokinetic data that were generated with this technique could guide clinical trials using WR-2721.

Damage to liposomal lipids: protection by antioxidants and cholesterol-mediated dehydration.

Liposomes composed of egg phosphatidylcholine (EPC) (13.4%, of the acyl chains being polyunsaturated fatty acids (PUFA)) and EPC/cholesterol (10:1 mol/mol) were studied for factors that affect liposomal lipid oxidative damage and hydrolysis upon long-term (16 months) storage. Factors studied include: (1) levels of lipid/water interface hydration, related to the presence of cholesterol in the lipid bilayer; (2) the membrane-associated antioxidant vitamin E; (3) the water-soluble antioxidant Tempol; and (4) exposure to light. Liposomal dispersions were stored at room temperature, either exposed to or protected from daylight, for a period of 16 months. Chemical and physical changes were monitored at several time points to assess oxidative and hydrolytic degradation of liposomal lipids. The conclusions of the study are: (1) PUFA are the most sensitive component of the liposome bilayer to oxidative degradation damage during long-term storage; (2) EPC liposomes are more sensitive to degradation during storage than EPC cholesterol liposomes, the presence of cholesterol in the lipid bilayer having a protective effect, probably due to its effect in decreasing the lipid-bilayer hydration; (3) oxidative degradation is the major process during long-term storage, having an earlier onset than the hydrolytic degradation: and (4) Tempol provided significantly better protection than vitamin E to EPC liposomal PUFA against oxidative damage during long-term storage. The relevance of cholesterol's presence, as a 'drying agent', in membranes containing PUFA to resistance of biological membranes to oxidative damage is discussed.