The effect of cryoprotectants on the physical properties of large liposomes containing sodium diclofenac.
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Large liposomes (1-10 microm) containing sodium diclofenac were prepared and lyophilized using lactose or mannitol (7.5% in respect to the lipid content) as cryoprotectants. The physical studies of liposomes were performed during 30 days of storage in a dry or resuspended form. Lyophilization of large liposomes and storage in the dry form at 5 degrees C increases their physical stability. Lactose is a cryoprotectant which does not influence changes of properties of liposomes regarding their size, encapsulation efficacy and release rate. Large liposomes lyophilized in the presence of mannitol tend to increase in size and encapsulation efficacy, but the lipid bilayers are stabilized and less permeable to the drug. (+info)
Lyophilisates for drug delivery in ophthalmology: pharmacokinetics of fluorescein in the human anterior segment.
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AIMS: To assess the ocular bioavailability of fluorescein from a novel water free, freeze dried ophthalmic drug delivery system compared to conventional preservative-free fluorescein eye drops. METHODS: Sodium fluorescein 0.17% was dissolved in an aqueous solution of hydroxypropylmethyl cellulose 1.0% (HPMC), deposited on sterilised flexible hydrophobic poly(tetrafluoroethylene) (PTFE) carrier strips and freeze dried under aseptic conditions. The fluorescein dose of the lyophilisate was 68 micro g, corresponding to a single conventional drop of 40 micro l fluorescein 0.17% solution. In a randomised, open label study 12 healthy volunteers applied the lyophilised fluorescein to one eye and one drop of conventional fluorescein ophthalmic solution to the fellow eye. Fluorophotometry measurements of fluorescein concentrations in the anterior segment were performed with the Fluorotron Master II (Ocumetrics, USA) before and +15, 30, 45, 60, 120, and 180 minutes after application. RESULTS: At all times anterior chamber fluorescein concentration was greater in the lyophysilate treated eye than the solution treated eye. The magnitude of this difference ranged from 2-5.3 times and was statistically significant. CONCLUSION: The greater intraocular bioavailability of fluorescein from the lyophilisate relative to the solution suggests that it may be a useful method for delivering substances to the eye. (+info)
Stabilization of S-adenosyl-L-methionine promoted by trehalose.
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S-adenosyl-L-methionine (SAM), an important metabolic intermediate of mammals, is a well-known therapeutic agent. The molecule is chemically unstable, both in solution and in dry state, and forms different degradation products. Because the chemical instability represents a real problem during the preparation of therapeutic formulations, we investigated the capacity of some sugars to improve the SAM stability over time. In the present work, we demonstrated that the disaccharide trehalose exercises a protective effect towards the lyophilized SAM slackening its degradation (65% of SAM was detected after 50 days at 37 degrees C). A parallel study, performed to stabilize the SAM into lyophilized yeast cells enriched in the sulfonium compound, assessed the positive effect of trehalose also in whole cells, but in lesser measure. (+info)
Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis.
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The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4oC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions. (+info)
Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa.
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The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution. (+info)
Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis.
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In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential. (+info)
Thermal aggregation of ribonuclease A. A contribution to the understanding of the role of 3D domain swapping in protein aggregation.
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By lyophilizing RNase A from 40% acetic acid solutions, two dimeric aggregates, the "minor" and "major" dimers (named here N-dimer and C-dimer, respectively), form by 3D domain swapping at a ratio of 1:4. Trimeric and tetrameric aggregates are also obtained. The two dimers and the higher oligomers also form without a lyophilization step. By keeping RNase A dissolved at a high concentration (generally 200 mg/ml) in various media at temperatures ranging from 23 to 70 degrees C for times varying from a few minutes to 2 h, various oligomers, in particular the two dimeric conformers, formed in quite different amounts, often inverting their relative quantities depending on the more or less severe unfolding conditions. When unfolding mainly concerned the N terminus of the protein, richer in hydrophilic residues, the N-dimer, formed by 3D domain swapping of the N-terminal alpha-helix of each monomer, prevailed over the C-dimer. Under more vigorous denaturing conditions, where also the C terminus of RNase A, richer in hydrophobic amino acids, unfolded, the C-dimer, formed by 3D domain swapping of the C-terminal beta-strand, prevailed over the other, possibly because of the induction to aggregation promoted by the hydrophobic residues present in the C termini of the two monomers. (+info)
European Concerted Action on Anticoagulation (ECAA). An assessment of lyophilised plasmas for ISI calibration of CoaguChek and TAS whole blood prothrombin time monitors.
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AIMS: The recommended method for the international sensitivity index (ISI) calibration of whole blood point of care testing (POCT) prothrombin time (PT) systems was originally described by Tripodi et al in 1993 but is too complex and demanding. The present European Concerted Action on Anticoagulation (ECAA) study aimed to assess the reliability of simpler ISI calibration using lyophilised plasma samples. METHODS: ISI calibrations using three different types of ECAA lyophilised plasma samples (artificially depleted, individual, and pooled coumarin) were compared with whole blood calibrations on CoaguChek Mini and TAS PT-NC POCT monitors at 10 centres. RESULTS: With CoaguChek Mini systems, lyophilised coumarin plasma samples (both single donation and pooled) gave ISI and international normalised ratio (INR) values comparable to whole blood. With artificially depleted plasma, ISI and INR values were too high. With TAS PT-NC systems, all three types of lyophilised plasma samples gave inaccurate ISI and unreliable INR results, similar to previous ECAA findings with fresh plasma calibrations. CONCLUSIONS: With CoaguChek Mini systems, ISI calibration can be simplified by the use of ECAA lyophilised plasma samples from coumarin treated patients. Further study is needed to devise a simpler calibration method for the TAS PT-NC system. (+info)