Effects of prostaglandin F2α on angiogenic and steroidogenic pathways in the bovine corpus luteum may depend on its route of administration.

Prostaglandin (PG) F2α and its analogs (aPGF2α) are used to induce regression of the corpus luteum (CL); their administration during the middle stage of the estrous cycle causes luteolysis in cattle. However, the bovine CL is resistant to the luteolytic actions of aPGF2α in the early stage of the estrous cycle. The mechanisms underlying this differential luteal sensitivity, as well as acquisition of luteolytic sensitivity by the CL, are still not fully understood. Therefore, to characterize possible differences in response to aPGF2α administration, we aimed to determine changes in expression of genes related to (1) angiogenesis-fibroblast growth factor 2 (FGF2), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2); and (2) steroidogenesis-steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (P450scc), and hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 1 (HSD3B) in early- and middle-stage CL that accompany local (intra-CL) versus systemic (i.m.) aPGF2α injection. Cows at d 4 (early stage) or d 10 (middle stage) of the estrous cycle were treated as follows: (1) systemic saline injection, (2) systemic aPGF2α injection (25 mg), (3) local saline injection, and (4) local aPGF2α injection (2.5 mg). Progesterone (P4) concentration was measured in jugular vein blood samples during the entire set of experiments. After 4 h of treatment, CL were collected by ovariectomy, and mRNA and protein expression levels were determined by reverse transcription quantitative-PCR and Western blotting, respectively. Local and systemic aPGF2α injections upregulated FGF2 expression but decreased expression of VEGFA in both CL stages. Both aPGF2α injections increased the expression of STAR in early-stage CL, but downregulated it in middle-stage CL. In the early-stage CL, local administration of aPGF2α upregulated HSD3B, whereas systemic injection decreased its mRNA expression in early- and middle-stage CL. Moreover, we observed a decrease in the P4 level earlier after local aPGF2α injection than after systemic administration. These results indicate that aPGF2α acting locally may play a luteotrophic role in early-stage CL. The systemic effect of aPGF2α on the mRNA expression of genes participating in steroidogenesis seems to be more substantial than its local effect in middle-stage CL.

Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization.

Retinal neovascularization is a major cause of blindness in such disorders as retinopathy of prematurity, proliferative diabetic retinopathy and senile macular degeneration. Because ligation of vitronectin receptor-type integrins appears to be required for the survival and maturation of newly formed but not quiescent blood vessels in several vascular beds including the retina, blockade of this downstream adhesion receptor system was investigated. In a mouse model of hypoxia-induced retinal neovascularization twice daily administration of 1 to 20 mg cyclic alpha v-integrin antagonist peptide per kilogram of body weight reduced capillary proliferation in a dose-dependent fashion--maximum 76%--without obvious side effects. A cyclic control peptide displayed no inhibitory effect on neovascularization. These findings indicate that systemic application of vitronectin receptor antagonists appears to be clinically feasible and is efficient in preventing retinal neovascularization and superior to cytokine-blocking strategies.

The effect of basic fibroblast growth factor 2 on the bovine corpus luteum depends on the stage of the estrous cycle and modulates prostaglandin F2α action.

The roles of fibroblast growth factor 2 (FGF2) in the corpus luteum (CL) function and its modulatory effect on prostaglandin (PG) F2α during the bovine estrous cycle were studied using the following design of in vivo and in vitro experiments: (1) effects of FGF2 and FGF receptor 1 inhibitor (PD173074) on bovine CL function in the early (PGF2α-resistant) and mid (PGF2α-responsive) luteal stage in vivo, (2) the modulatory effect of FGF2 on PGF2α action during the luteal phase in vivo and (3) effects of FGF2 and PD173074 on bovine CL secretory function in vitro. Cows were treated by injection into the CL with: (1) saline (control), (2) FGF2, (3) PD173074, (4) FGF2 followed by intramuscular (i.m.) PGF2α, (5) PD173074 followed by i.m. PGF2α and (6) i.m. PGF2α as a positive control. For in vitro experiments, CL explants were treated with the aforementioned factors. Progesterone (P4) concentrations of blood samples or culture media were determined by radioimmunoassay. Relative mRNA expressions of the genes involved in angiogenesis and steroidogenesis were determined by quantitative real-time PCR. Although FGF2 treatment on day 4 of the estrous cycle did not change the cycle length, FGF2 with PGF2α decreased the P4 concentrations observed during the estrous cycle compared to the control group (P < 0.001). Moreover, FGF2 treatment on day 10 prolonged CL function as indicated by a significantly greater concentration of P4 on day 21 compared to the control group. In the in vitro study, FGF2 decreased cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) mRNA expression (P < 0.01) and decreased P4 production in the early-stage CL (P < 0.001). However, FGF2 + PGF2α or PGF2α alone resulted in an elevation of steroidogenic acute regulatory protein and CYP11A1 mRNA expression and P4 secretion in the early-stage CL (P < 0.01). In the mid-luteal phase, FGF2 upregulated CYP11A1 and HSD3B1 mRNA expression (P < 0.01), while FGF2 + PGF2α increased only HSD3B1 mRNA expression (P < 0.001). In conclusion, FGF2 seems to play a modulatory role in CL development or luteolysis, differentially regulating steroidogenesis and angiogenic factors as well as PGF2α actions.

Luteinizing hormone and ovarian steroids affect in vitro prostaglandin production in the equine myometrium and endometrium.

Prostaglandins (PGs) play crucial roles in the regulation of the oestrus cycle and establishment of pregnancy in animals. Luteinizing hormone (LH) and ovarian steroids are involved in regulating endometrial PG production in many species. Their effects on PG production and associated pathways in the mare myometrium and endometrium are the subjects of our interest. This study aimed to evaluate the specific effects of LH and ovarian steroids on equine myometrial and endometrial tissues on (i) PGE2 and PGF2α secretion and (ii) transcription of genes encoding specific enzymes responsible for PG synthesis, such as prostaglandin-endoperoxide synthase (PTGS2), PGE2 synthases (PGES), PGF2α synthases (PGFS), and PGI2 synthases (PGIS), using equine myometrial and endometrial explants. Equine myometrial and endometrial tissues were collected at the mid-luteal (n = 6) and follicular (n = 6) phases of the oestrus cycle and were exposed to: (1) vehicle (control), (2) arachidonic acid (AA, 50 ng/mL, positive control), (3) LH (10 ng/mL), (4) progesterone (P4, 10-7M) and (5) 17-ß oestradiol (E2, 10-9M) for 24 h. After exposure, PGF2α and PGE2 concentrations were determined using direct enzyme immunoassays. Alterations in PG synthase mRNA expression were determined using RT-qPCR. After 24 h, LH and P4 increased PGE2 and PGF2α secretion by myometrial tissues at the mid-luteal phase (P < 0.05), whereas PG secretion was augmented by LH and E2 during the follicular phase (P < 0.01). In contrast, LH and E2 increased PGE2 and PGF2α secretion by endometrial tissues during the mid-luteal phase (P < 0.05), while E2 enhanced PGE2 secretion during the follicular phase of the oestrus cycle (P < 0.01). These results indicate that LH and ovarian steroids modulate PG production in equine myometrial and endometrial tissues and affect PG synthase expression at the mRNA level. We conclude that the equine myometrium is an alternative source of PG production and participates in the regulation of uterus function during the oestrus cycle.

Decreased angiogenesis and arthritic disease in rabbits treated with an alphavbeta3 antagonist.

Rheumatoid arthritis (RA) is an inflammatory disease associated with intense angiogenesis and vascular expression of integrin alphavbeta3. Intra-articular administration of a cyclic peptide antagonist of integrin alphavbeta3 to rabbits with antigen-induced arthritis early in disease resulted in inhibition of synovial angiogenesis and reduced synovial cell infiltrate, pannus formation, and cartilage erosions. These effects were not associated with lymphopenia or impairment of leukocyte function. Furthermore, when administered in chronic, preexisting disease, the alphavbeta3 antagonist effectively diminished arthritis severity and was associated with a quantitative increase in apoptosis of the angiogenic blood vessels. Therefore, angiogenesis appears to be a central factor in the initiation and persistence of arthritic disease, and antagonists of integrin alphavbeta3 may represent a novel therapeutic strategy for RA.

Investigating hair properties relevant for hair 'handle'. Part I: hair diameter, bending and frictional properties.

The expert working group 'Hair Care Products' of the DGK currently conducts a wide study to contribute to the understanding of how single hair fibre and hair collective properties contribute towards hair 'handle' and 'feel'. During the first stage of this study four hair types were selected from a large group of individual European hair braids, according to either similar or widely different panel ratings for handle. Against the background of the panel test and the state of the literature the working group readily identified the bending properties of single fibres interacting in the tress as a fibre collective and fibre friction as being of central relevance for hair 'handle' and 'feel'. Fibre diameters of the hair types were determined by Optical Fibre Diameter Analyzer and by weighing. From these data mean ellipticity and bending stiffness distributions were calculated. Single fibre friction was determined by the capstan method in the root, middle and tip regions. Significant differences were determined between the hair types in diameters, ellipticity, bending stiffness and friction. The results lead to conclude that 'handle' is perceived as inferior when the hair is thick and bending stiffness thus high. For such hair differences in handle rating are related to differences in friction, namely in the tip region. For thin and thus 'soft' hair fibre friction seems to play only a minor role.

Cilengitide--exceptional pseudopolymorphism of a cyclic pentapeptide.

Cilengitide (Cil) represents a cyclic pentapeptide, cyclo-(Arg-Gly-Asp-D-Phe-N-MeVal). Existence of an anhydrate form (A1) and a tetrahydrate form Cil1(H2O)4 has been observed. Surprisingly the anhydrate form proved to be more stable in aqueous environment compared to the tetrahydrate form. Assessment of thermodynamic stability has been carried out by competitive slurry experiments as well as by investigation of thermodynamic solubility. The lower solubility of the anhydrate form A1 can be explained by the hydrogen bonding motifs within the crystal structures. The tetrahydrate form Cil1(H2O)4 represents a special manifestation of a class of non-stoichiometric water-alcohol solvates Cil1(H2O)x(alcohol)y where methanol and ethanol can substitute water molecules in the crystal lattice of the tetrahydrate form leading to the hydrate-solvate systems Cil1(H2O)x(methanol)y named S1 and Cil1(H2O)x(ethanol)y named S2 with x ⩽ 4, y ⩽ 1 and y ⩽ 2-0.5x. The non-stoichiometric water alcohol solvates exhibit a higher solubility compared to the anhydrate form but convert rapidly to the anhydrate form in aqueous environments. Accordingly, the better soluble non-stoichiometric water alcohol solvates cannot be obtained by crystallization from aqueous media. However slurries or crystallization from solvent mixtures containing methanol and ethanol represent a means to obtain the highly soluble pseudo-polymorphs S1 and S2 and to circumvent formation of the low soluble anhydrate form A1.

Substrate analogue renin inhibitors containing replacements of histidine in P2 or isosteres of the amide bond between P3 and P2 sites.

Incorporation of beta-alanine or gamma-aminobutyric acid in position P2 of ACHPA or Leu psi [CHOHCH2]Val-based tetrapeptides gave highly active renin inhibitors (compounds V, VI, and XVII) with high specificity for renin and a remarkable stability against chymotrypsin. Replacement of the amide bond between P2 and P3 by isosteres (ketomethylenes, hydroxyethylenes, and the corresponding thio-insertion analogues) led to compounds (VIII-XIII, XVIII, and XIX) with renin inhibitory activity in the nanomolar range. Oral activity was achieved by incorporation of polar functionalities at the N-terminus of beta-alanine-containing tetrapeptides. One of these compounds (XXVIII) was chosen for further studies. This inhibitor demonstrated excellent efficacy and a long duration of action after intravenous and oral administration to cynomolgus monkeys.

Renin inhibitors containing new P1-P1' dipeptide mimetics with heterocycles in P1'.

A series of renin inhibitors containing new P1-P1' dipeptide mimetics are presented. The P1-P1' mimetics were obtained from (4S,5S)-3-(tert-butoxycarbonyl)-4-(cyclohexylmethyl)-5-[(omega- mesyloxy)alkyl]-2,2-dimethyloxazolidines 5b, 9, and 11b by nucleophilic substitution of the mesylate groups with the sodium salts of mercapto- and hydroxyheterocycles. Removal of the protecting groups and stepwise acylations with amino acid derivatives provided renin inhibitors with a length of a tripeptide. Replacement of P2 histidine by other amino acids maintained or enhanced renin inhibitory potency. By alteration of P3 phenylalanine, compounds with IC50 values in the nanomolar range and stability against chymotrypsin were obtained. Finally, the effect of the C-terminal heterocycle on the renin inhibition was studied. Compound XVII was examined in vivo for its hypotensive effects. In salt-depleted cynomolgus monkeys, XVII inhibited plasma renin activity and lowered blood pressure after oral administration of a dose of 10 mg/kg.

N-Methylated cyclic RGD peptides as highly active and selective alpha(V)beta(3) integrin antagonists.

The alpha(V)beta(3) integrin receptor plays an important role in human tumor metastasis and tumor-induced angiogenesis. The in vivo inhibition of this receptor by antibodies or by cyclic peptides containing the RGD sequence may in the future be used to selectively suppress these diseases. Here we investigate the influence of N-methylation of the active and selective alpha(V)beta(3) antagonist cyclo(RGDfV) (L1) on biological activity. Cyclo(RGDf-N(Me)V-) (P5) was found to be even more active than L1 and is one of the most active and selective compounds in inhibiting vitronectin binding to the alpha(V)beta(3) integrin. Its high-resolution, three-dimensional structure in water was determined by NMR techniques, distance geometry calculations, and molecular dynamics calculations, providing more insight into the structure-activity relationship.

Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.

A cyclo peptide activates signaling events and promotes growth and the production of the bone matrix.

The interaction of bone cells and their underlying extracellular matrix impacts biological processes such as maintenance of tissue integrity. The biological recognition of the extracellular matrix by attached cells is mediated by the activity of integrins that recognize adhesive-specific domains. The most widely recognized adhesive motif is the RGD sequence, common to many of the adhesive matrix molecules. Here, we show that cyclo DFKRG which was previously selected to increase cell adhesion of human bone marrow stromal cells (HBMSC), increases both cell differentiation and mineralization through activation of tyrosine kinases, focal adhesion kinase (p(125)FAK) and Mitogen Activated Protein (MAP) kinases.

Multiple molecular forms of tachykinins in rat spinal cord: a study comparing different extraction methods.

Various procedures for extraction at acid, neutral and alkaline pH were compared with regard to the yield of different tachykinins and tachykinin-like substances from rat spinal cord. Reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay with various C-terminally directed tachykinin antisera and a newly developed N-terminally directed substance P (SP)-antiserum (SPN 1) were used. Antiserum SPN 1 fully reacts with SP-analogues modified at the C-terminal end (SP free acid and SP-Gly-Lys) and also (77%) with SP(1-9) but not with C-terminal SP-fragments lacking 2 or more N-terminal amino acids. The highest levels of SP-like immunoreactivity (LI) and neurokinin A (NKA)-LI were measured after combined water and acetic acid extraction procedures. Also when measuring cholecystokinin-like immunoreactivity the highest level was obtained following this extraction procedure. RP-HPLC revealed a major component of SP-LI at the position of synthetic SP irrespectively of the extraction method and if the C- or N-terminally directed antiserum was used. Neutral water extracts contained a late eluting component detected with the C-terminally, but not with the N-terminally, directed antiserum. Acid and alkaline extracts, in contrast, contained components which could be detected with the N-terminally, but not with the C-terminally, directed SP-antiserum. Immunoreactive components eluting at the position of NKA and NKB were found in all types of extracts with NKA-, kassinin- and eledoisin-antisera. The NKB- and neuropeptide K (NPK)-components were more prominent in acid than in neutral and alkaline extracts. In conclusion, the present results indicate that rat spinal cord may contain molecular forms of tachykinin-like immunoreactivity in addition to those previously described and illustrate the importance of the choice of extraction method in immunochemical studies. Combined extraction in water and acetic acid appears to be a suitable method when the content of peptides with different chemical properties are to be measured in a tissue sample.

Proton transport through a peptide-tethered bilayer lipid membrane by the H(+)-ATP synthase from chloroplasts measured by impedance spectroscopy.

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.

Incorporation of the acetylcholine receptor dimer from Torpedo californica in a peptide supported lipid membrane investigated by surface plasmon and fluorescence spectroscopy.

The dimer species (M(r) 580,000) of the nicotinic acetylcholine receptor, isolated from the electric organ of Torpedo californica, was incorporated into a thiopeptide supported lipid bilayer. The incorporation was achieved by fusion of liposomes with reconstituted receptor onto a gold-supported thiopeptide lipid monolayer. Surface plasmon resonance spectroscopy (SPS) was used to monitor in real time the fusion process as well as the specific binding of the antagonist alpha-bungarotoxin. A recently developed extension of SPS offering enhanced sensitivity and specificity, surface plasmon fluorescence spectroscopy (SPFS), was then used to monitor subsequent binding of the monoclonal WF6 and polyclonal antibody, respectively. The latter was fluorescence labeled with Cy5. The different binding assays indicate the successful incorporation of the receptor in the lipid bilayer.

Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy.

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.

Evaluation of absorption enhancement for a potent cyclopeptidic alpha(nu)beta(3)-antagonist in a human intestinal cell line (Caco-2).

Different absorption enhancing principles for a potent cyclopeptidic alpha(nu)beta(3)-antagonist (EMD 121974) were investigated in monolayers of a human intestinal cell line (Caco-2). Transepithelial transport was quantitated by reversed-phase high-performance liquid chromatography. Cytotoxic effects were characterized by determination of transepithelial electrical resistances (TEERs), propidium iodide (PI)-influx, FITC-phalloidin staining and the release of cytosolic lactate dehydrogenase (LDH). Medium chain fatty acids (MCFAs, NaC10, NaC12) and taurocholate (NaTC) were the most efficient enhancers of cyclopeptide and FITC-dextran 4400 permeability coefficients, displaying different time profiles of activity. Whereas NaTC (15 mM) showed almost a constant permeation enhancing effect from 20 min up to 120 min (ca. 12-fold), MCFA absorption enhancement was markedly dependent on incubation time (NaC10, 20 min: 1.2-fold, 120 min: 17-fold; NaC12, 20 min: 4.3-fold, 120 min: 13-fold). All cytotoxicity assays demonstrated that MCFAs were significantly more cytotoxic than NaTC. Ion pairing with hydrophobic amino acids and heptane sulfonate distinctly increased octanol-buffer partition coefficients of the cationic cyclopeptide but did not enhance its transepithelial permeability. Nanoparticles as well as beta-cyclodextrin neither affected integrity of the cells nor transport properties of the cyclopeptide. In summary, significant absorption enhancement was only observed with NaTC or MCFAs. Increase in permeability coefficients using NaTC occurred rapidly with acceptable cytotoxicities and merits further investigations.

Fusion of small unilamellar vesicles onto laterally mixed self-assembled monolayers of thiolipopeptides.

Monolayers of the thiolipopeptide NH(2)-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-(Myr)Lys(Myr)-OH (III) were formed on gold surfaces by self-assembly, mixed with a lateral spacer of the same peptide composition, NH(2)-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-Lys-OH (I). Different mixing ratios were employed ranging from 0.1 to 1, corresponding to 10-100% thiolipopeptide. These self-assembled monolayers (SAMs) were then exposed to a suspension of liposomes with the aim of forming lipid bilayers as a function of the mixing ratio. A clear optimum with respect to homogeneity and electrical properties of the membranes was obtained in the middle region (0.5) of mixing ratio, as revealed by surface plasmon resonance spectroscopy, impedance spectroscopy, and fluorescence microscopy. The combination of these methods was shown to be a powerful tool, although a true lipid bilayer was not obtained. Instead, vesicle adsorption was shown to be the predominant process, and FRAP (fluorescence recovery after photobleaching) measurements showed that the films were not fluid on the micrometer length scale.

Determination of hydrochlorothiazide, triamterene and propranolol hydrochloride by the spectrophotometric method and high-performance liquid chromatography (HPLC).

Spectrophotometric and chromatographic (HPLC) methods for determination of hydrochlorothiazide, triamterene and propranolol hydrochloride were elaborated. Both methods were appropriate for the determination of three compounds in pharmaceutical preparations containing their mixtures. Both the elaborated methods for the determination of the studied compounds give comparable results and can successfully be applied to the assay in their mixtures occurring in the composition of pharmaceutical preparations.