In vivo anti-ageing activity of cream containing niosomes loaded with purple glutinous rice (Oryza sativa Linn.) extract.

OBJECTIVE: To evaluate the anti-ageing activity of cream containing the methanolic purple glutinous rice extract loaded in niosomes. METHODS: The in vitro biological activities of the purple glutinous rice extracted by methanol maceration were determined. The extract loaded in niosomes and the cream containing the niosomes were developed. The in vivo anti-ageing activity in 20 human volunteers including skin hydration, pigmentation, roughness and elasticity after daily application for 28 days compared to at initial was evaluated by Corneometer, Mexameter, Visiometer and Cutometer, respectively. RESULTS: The purple glutinous rice extract showed free radical scavenging (SC50 ), lipid peroxidation inhibition (IPC50 ), metal ion chelating (CC50 ) and tyrosinase inhibition (IC50 ) values at 32.31 ± 1.28, 57.40 ± 2.12, 85.05 ± 5.43 and 43.89 ± 2.14 mg/mL which were 0.00031, 0.011, 0.0078 and 0.0016 times of the standards (0.01 ± 0.00, 0.62 ± 0.14, 0.66 ± 0.05 and 0.07 ± 0.01), respectively. The purple glutinous rice extract contained 0.35 µg of anthocyanin/1 mg of the extract determined by HPLC. After loaded in niosomes, the solubility of the extract was not only increased in various solvents, but also the chemical stability in different environments (weak base, reducing agent and acid salt) was improved. The cream formulation containing niosomes loaded with 1%w/v of the purple glutinous rice extract indicated the anthocyanin remaining percentages after 6 cycles of heating and cooling test at 52.28% of the initial. For in vivo anti-ageing activities, cream containing niosomes loaded with the extract gave significant decreased melanin index and skin roughness reduction of -14.05 and -9.95% of the initial, respectively. The % changes of the increased skin hydration, skin elastic extension and skin elastic recovery when applied on human volunteers' skin with this formulation were +48.73, -24.51 and +35.98%, respectively. CONCLUSION: The cream containing niosomes loaded with the 1%w/v methanolic purple glutinous rice extract gave not only the suitable in vitro antioxidant activity and physical stability of the active anthocyanin, but also the superior in vivo anti-ageing activity on human skin compared to the cream base and before application which can be further developed as a novel anti-ageing cosmeceutical product.

OBJECTIF: Evaluer l'activité anti-âge d'une crème contenant de l'extrait de riz gluant violet méthanolique chargé en niosomes. MÉTHODES: Les activités biologiques in vitro de l'extrait du riz gluant violet par macération au méthanol ont été déterminées. L'extrait chargé en niosomes et la crème contenant les niosomes ont été développés. L'activité anti-âge in vivo sur 20 volontaires humains, y compris de l'hydratation de la peau, la pigmentation, la rugosité et l'élasticité après une application quotidienne pendant 28 jours a été évaluée par comparaison avec T0 par cornéomètre, mexamètre, visiomètre et cutomètre, respectivement. RÉSULTATS: L'extrait de riz gluant violet a montré des valeurs de piégeage des radicaux libres (SC50), d'inhibition de la peroxydation lipidique (IPC50), de chélation des ions métalliques (CC50) et d'inhibition de la tyrosinase (IC50) à 32,31 ± 1,28, 57,40 ± 2,12, 85,05 ± 5,43 et 43,89 ± 2,14 mg / ml qui étaient 0,00031, 0,011, 0,0078 et 0,0016 fois des standards respectivement (0,01 ± 0,00, 0,62 ± 0,14, 0,66 ± 0,05 et 0,07 ± 0,01). L'extrait de riz gluant violet contenait 0.35 pg d'anthocyanine / 1 mg de l'extrait déterminé par HPLC. Après avoir été chargé dans les niosomes, la solubilité de l'extrait a non seulement été augmentée dans divers solvants, mais aussi la stabilité chimique dans différents environnements (base faible, agent réducteur et sel d'acide) a été améliorée. La formulation de crème contenant des niosomes chargés avec 1% p / v de l'extrait de riz gluant violet a indiqué les pourcentages d'anthocyanine restants après 6 cycles de test de chauffage et de refroidissement à 52,28% de la valeur initiale. Pour les activités anti-âge in vivo, la crème contenant des niosomes chargés de l'extrait a donné une réduction significative de l'indice de mélanine et de la rugosité cutanée de -14,05 et -9,95% de la valeur initiale, respectivement. Les pourcentages de variation de l'hydratation accrue de la peau, de l'extension élastique de la peau et de la récupération de l'élasticité de la peau lors de l'application sur la peau de volontaires humains avec cette formulation étaient respectivement de +48,73, -24,51 et + 35,98%. CONCLUSION: La crème contenant des niosomes chargée avec l'extrait de riz gluant violet méthanolique à 1% p / v a donné non seulement une activité antioxydante in vitro appropriée et une stabilité physique de l'anthocyanine active, mais également une activité anti-vieillissement in vivo supérieure sur la peau humaine par rapport à la base de la crème et avant application, qui peut être développée en tant que nouveau produit cosméceutique anti-âge. Mots clés: riz gluant violet, niosomes, antioxydant, inhibition de la tyrosinase, efficacité anti-âge in vivo, produit cosméceutique.

Potent and prolonged hypoglycemic activity of an oral insulin--Tat mixture in diabetic mice.

Trans-activator of transcription (Tat) is a cell penetrating peptide which can translocate and carry macromolecular cargoes through cell membranes. This study investigated the hypoglycemic activity of orally delivered insulin - Tat mixture in alloxan-induced diabetic mice. The mixtures of insulin and Tat at 1:1, 1:3 and 1:6 molar ratios were given orally at the insulin doses ranging from 1-200 IU/kg. The fasting blood glucose (FBG) levels were measured at initial, 1, 2, 4, 6, and 12 h after administration. At 1:3 molar ratio of the mixture and after 12 h of administration, insulin at 200 IU/kg showed the highest with prolonged hypoglycemic activity of 74.0±10.3% FBG reduction (2.18 folds of subcutaneously injected (SC) insulin). Free insulin administered orally did not show any hypoglycemic activity. The mixtures at the insulin doses of 100 and 50 IU/kg also showed potent FBG reduction of 73.8±8.2 and 71.3±16.9% at 12 h after administration (2.18 and 2.10 folds of SC insulin, respectively). After incubation with Mono-Mac-6 cells, only the -mixtures but not the free insulin showed intra-cellular insulin uptake, indicating the insulin penetration through the cell membranes via Tat. In simulated gastric fluid, the insulin content in the mixture was not found, demonstrating the degradation of insulin in the gastric environments. Insulin may be absorbed at upper gastrointestinal tract facilitated by Tat. The potent and prolonged hypoglycemic activity of insulin co-administered orally with Tat can be further developed as an effective oral insulin delivery system.

Safety assessment of azelaic acid and its derivatives entrapped in nanovesicles.

The aim of this study was to determine the safety of azelaic acid (AA) and its derivatives in nanovesicles for pharmaceutical and cosmetic uses. The hydrophilic property of AA was modified by complexing AA with hydroxypropyl-beta-cyclodextrin (AACD). The lipophilic property of AA was improved to diethyl azelate (DA) by esterification with Fischer reaction. AA, AACD and DA were entrapped in liposomes and niosomes with the compositions of L-alpha-dipalmitoyl phosphatidylcholine/cholesterol = 7:3 and Tween 61/cholesterol = 1:1, respectively, by chloroform film method with sonication. The size of the vesicles ranged from 50 to 200 nm, indicating nanosize characteristics. The cytotoxicity of AA, AACD and DA entrapped nanovesicular formulations on mouse epidermal cell lines (JB6, normal cell lines) by the sulforhodamine B assay was modest when compared with cisplatin. Blank liposomes and niosomes gave no growth inhibitory effect. The irritation of AA, AACD and DA entrapped and not entrapped in nanovesicles on rabbit skin was examined according to the Environmental Protection Agency health effect test guidelines. The results showed no signs of erythema or edema within 72 h. AA and its derivatives were safe for topical use when entrapped in nanovesicles because of no toxicity to normal cell lines and no allergy on rabbit skin.

Anti-inflammatory activity of gel containing novel elastic niosomes entrapped with diclofenac diethylammonium.

The objective of this study was to develop a novel elastic bilayer vesicle entrapped with the non-steroidal anti-inflammatory drug (NSAID), diclofenac diethylammonium (DCFD) for topical use. Eighteen bilayer vesicular formulations composing of DPPC or Tween 61 or Span 60 mixed with cholesterol (at 1:1, 3:7 and 1:1 molar ratios, respectively) and ethanol at 0-25% (v/v), by chloroform film method with sonication were developed. The elastic Tween 61 niosomes which gave no sedimentation, no layer separation, unchanged particle sizes (about 200 nm) were selected to entrap DCFD. The entrapment efficiency of the drug in the conventional and elastic Tween 61 niosomes was 65 and 93%, respectively. At least 87% of DCFD determined by HPLC remained in elastic Tween 61 niosomes when kept at 4, 27 and 45 degrees C for 3 months. The deformability index values of the elastic niosomes were 13.76 and 3.44 times higher than the conventional niosomes entrapped and not entrapped with the drug, respectively, indicating the higher flexibility of the elastic vesicle especially, when entrapped with the drug. Transdermal absorption through excised rat skin was performed by vertical Franz diffusion cell at 32+/-2 degrees C for 6h. Gel containing elastic niosomes exhibited fluxes of DCFD in the stratum corneum (SC), deeper skin layer (viable epidermis and dermis, VED) and receiver chamber at 191.27+/-9.52, 16.97+/-2.77 and 3.76+/-0.54 microg/(cm2 h), whereas the commercial emulgel, containing an equivalent DCFD, gave 60.84+/-13.63, 7.33+/-1.70 and 0.14+/-0.01 microg/(cm2 h), respectively. The in vivo anti-inflammatory activity was evaluated by ethyl phenylpropiolate (EPP)-induced rat ear edema (n=3). DCFD entrapped in the developed elastic niosomes and incorporated in gel gave the same ear edema inhibition percentages of 23.81% at 30 min, but 2 and 9 times more inhibition percentages at 45 and 60 min than the commercial emulgel, respectively. This result has not only demonstrated the enhancement of transdermal absorption through rat skin, but also the in vivo anti-inflammatory effect of DCFD when entrapped in the developed novel elastic Tween 61 niosomes, as well.

Stability of luciferase plasmid entrapped in cationic bilayer vesicles.

Characteristics and physical stability of luciferase plasmid (pLuc) entrapped in cationic bilayer vesicles prepared from various molar ratios of amphiphiles (DPPC, Tween61 or Span60), cholesterol (Chol) and cationic charge lipid (DDAB) were investigated. The cationic liposomes were composed of DPPC/Chol/DDAB in the molar ratio of 7:2:1. The cationic (Tween61 or Span60) niosomes were composed of Tween61/Chol/DDAB or Span60/Chol/DDAB in the molar ratio of 1:1:0.05. The maximum loading of pLuc was 15.29, 22.70, and 18.92 microg/mg of the total lipids or surfactants of liposomes, Tween61 and Span60 niosomes, respectively. The morphology of the vesicles showing multilamellar structure was characterized by transmission electron microscope (TEM). The particle sizes of the vesicles in nanosize range (160-850 nm) were determined by Photon Correlation Spectroscopy (PCS). Gel electrophoresis and gel documentation were modified to determine the entrapment efficiency of pLuc in cationic bilayer vesicles. The cationic bilayer vesicles gave the pLuc entrapment efficiency of 100%. The pLuc entrapped in cationic liposomes exhibited higher stability than pLuc in solution and pLuc entrapped in cationic Tween61 or Span60 niosomes, when stored at 4, 30 and 50 degrees C for 8 weeks. After 8 weeks at 4 degrees C, pLuc contents remained in cationic liposomes was 2 and 3 times higher than cationic Span60 and Tween61 niosomes, respectively. After 3 weeks, 50 and 2% of pLuc was remained in cationic liposomes at 30 and 50 degrees C respectively, whereas all pLuc in cationic Span 60 and Tween61 niosomes were degraded within 2 and 1 week, respectively. At 30 and 50 degrees C, pLuc in an aqueous solution or in bilayer vesicular formulations were not in a stable supercoil form. This study has indicated that the stability of pLuc can be enhanced by entrapping in cationic liposomes more than in niosomes. Higher temperature with increase storage time can affect the stability of pLuc even entrapped in bilayer vesicles.

Biotransformation of cortexolone to hydrocortisone by molds using a rapid color development assay.

Capabilities of 22 molds were assessed for 11beta-hydroxylation of cortexolone (Reichstein's compound S) to hydrocortisone. The biotransformation capability was compared for solid-state and submerged monocultures of the molds under otherwise identical conditions. A novel rapid color development assay and thin layer chromatography were used to qualitatively establish the ability of the fungi to convert cortexolone to hydrocortisone. These assays were validated and supplemented with data from high performance liquid chromatography to obtain quantitative information on the biotransformation. Nearly all the fungi consumed a significant fraction of the cortexolone fed, but only four (i.e. two isolates of Cunninghamella blakesleeana, C. echinulata and Curvularia lunata) yielded measurable quantities of hydrocortisone. Submerged cultures generally gave significantly greater yield of hydrocortisone compared to equivalent solid-state cultures.

In vivo immunomodulating activity of wood extracts from Clausena excavata Burm. f.

Immunomodulating effects in Balb/C mice of aqueous extract (CEHW) and the Thai folklore preparation (CEHF) of Clausena excavata Burm. f. were investigated. Haemagglutinating antibody (HA) titers at day 0, 7, 14, 21, 28 and 35 from the serum of animals fed or injected intraperitoneally with the extracts for 5 days were compared and evaluated for humoral mediated immunity (HMI). Footpad swelling test was used to determine delayed type hypersensitivity for cell mediated immunity (CMI). CEHW and CEHF injected intraperitoneally and administered orally exhibited the same maximum antibody production of 1/800. Both extracts given orally reached the maximum antibody titer at day 7, which was 2 weeks faster than by intraperitoneal administration. However, antibody titers from CEHW injected intraperitoneally diminished without retaining whereas the CEHF retained for 1 week. Moreover, CEHW gave CMI response more than the CEHF. This study suggested the potential in vivo immunomodulating activities of extracts from Clausena excavata supporting our previous in vitro studies.

In vitro immunomodulatory effect of Pouteria cambodiana (Pierre ex Dubard) Baehni extract.

The methanolic stem bark extract from Pouteria cambodiana (Pierre ex Dubard) Baehni was evaluated for immunomodulating activity on BALB/c mice. The antioxidant effect was also assessed. The extract presented a good dose-response effect in the peritoneal macrophage phagocytosis assay with higher activity at 1mg/ml and an EC50 of 0.02 mg/ml and also activated lysosomal enzyme activity with an EC50 of 0.16 mg/ml. In the splenocyte proliferation assay, the extract without mitogen was active (EC50, 0.01 mg/ml) while the EC50 of the extract with lipopolysaccharide (LPS) and pokeweed mitogen (PWM) were 0.02 and 0.41 mg/ml, respectively. The extract showed low free radical scavenging activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay with an IC50 of 0.24 mg/ml, being less active than ascorbic acid, butylated hydroxytoluene (BHT) and alpha-tocopherol which showed IC50 of 0.08, 0.10 and 0.11 mg/ml, respectively. The extract at doses up to 0.073 mg/ml had no effect on lipid peroxidation. The potent immunological but no antioxidant activity of the extract presented in this study can explain, at least in part, the Thai folklore application of this plant in the treatment of fever and skin eruption.

The entrapment of kojic oleate in bilayer vesicles.

The entrapment of kojic acid and its newly synthesized ester (kojic oleate) has been evaluated. Kojic oleate was synthesized by DCC (N,N'-dicyclohexylcarbodiimide, DCC)/(4-(N,N-dimethylamino)pyridine, DMAP) esterification method and identified by FAB-MS and 1H NMR. The synthesized product was mainly 7-O-kojic oleate with more than 80% yield. It was entrapped in vesicular membrane prepared from 9.5:9.5:1.0 molar ratio of amphiphiles (Span 60, Tween 61 or DPPC), cholesterol and dicetyl phosphate. Kojic acid was encapsulated in the water compartment of these vesicles in order to confirm the vesicle formation. The morphology and particle size of the vesicles were characterized by an optical microscope and transmission electron microscope (TEM). The entrapment efficiencies of kojic acid and kojic oleate in the vesicles were investigated by dialysis and column chromatography, respectively. The contents of the entrapped kojic acid and kojic oleate were assayed by HPLC. The entrapment efficiency of kojic acid was 0.01-0.04 mol, whereas kojic oleate gave higher entrapment efficiency of 0.25-0.35 mol/mol of the total compositions of amphiphile/cholesterol/dicetyl phosphate. Structural modification of kojic acid improved its entrapment in the vesicles. Tween 61 vesicles could entrap kojic oleate more than did Span 60 vesicles. The pi-A isotherms revealed the lower area per molecule of Span 60, which formed a more rigid pack of its molecule on air/water interface than that of Tween 61. This implied the high rigidity of vesicular membrane prepared with Span 60 led to the lower amount of kojic oleate entrapped in the vesicles. From the release study of kojic acid through the dialysis membrane, it indicated that the intercalation of kojic oleate in the vesicular membranes did not significantly affect the release of kojic acid from the vesicles.

Free Radical Scavenging Activity of Extracts from Thai Plants in Guttiferae and Schisandraceae Families.

Five Thai plants from the Guttiferae (Hypericum hookerianum. Wight & Arn, Garcinia speciosa. Wall, Garcinia xanthochymus. Hook f. ex. T. Anderson, Cratoxylum formosum. ssp. pruniflorum. (Kurz) Gogel, and Calophyllum polyanthum. Wall ex Choisy) and one from the Schisandraceae (Schisandra verruculosa.) were extracted by methanol and chloroform. The extracts were screened for free radical scavenging activity using the DPPH assay. All extracts showed a dose-dependent antioxidant activity. The most potent with the lowest IC50 values were observed in the methanol extracts from the wood of G. speciosa., which were 2.5- and 5.3-fold more potent than the two standard antioxidants, ascorbic acid and α.-tocopherol, respectively. Free radical scavenging activities ranging from moderate to high were observed in both methanol and chloroform extracts from H. hookerianum., C. formosum. ssp. pruniflorum., G. xanthochymus., S. verruculosa. and C. polyanthum.. The information from this study can explain the traditional use and the further development of these extracts into new pharmaceuticals.

Characterization of amphotericin B liposome formulations.

Liposomes composed of hydrogenated soya phosphatidylcholine (Emulmetik 950)/cholesterol/charged lipids [dicetyl phosphate (-) or stearylamine (+)] were developed. The hydrogenated soya phosphatidylcholine/cholesterol/charged lipid liposomes at molar ratios of 1:1:0, 7:2:0, 7:2:1 (-), and 7:2:1 (+), with and without the entrapped amphotericin B (0.05 mg AmB/mg lipid), were prepared by a chloroform-film method with sonication. The charges of liposomes were characterized by a Zeta-Meter. The negative liposomes with and without the entrapped AmB showed higher surface charge density than other formulations. The size distribution of liposomes determined by standard error of the mean (SEM) was in the range of 0.115 to 0.364 microm. The smallest size was observed in the negative liposomes with the entrapped drug [7:2:1 (-) AmB]. The lamellarity of more than 15 layers was observed by transmission electron microscope (TEM) in the neutral liposomes with the entrapped drug [7:2 AmB]. The transition temperature and enthalpy of transition (deltaH) were determined by differential scanning calorimetry (DSC). Positive liposomes with the entrapped and unentrapped AmB demonstrated higher deltaH of the first peak than other formulations, indicating higher rigidity of liposomal membrane. The AmB contents in liposomes were determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection at 382 nm. The percentages of entrapment of AmB in all formulations were above 85%. The positive liposome [7:2:1 (+) AmB] formulation, which gave the highest thermal stability, was selected for further skin absorption evaluation.

Immunomodulatory activities of fractions from hot aqueous extract of wood from Clausena excavata.

The effects of fractions from hot aqueous extract, acetone extract and the folklore preparation of Clausena excavata were studied on mouse splenocyte proliferation. The fractions of hot aqueous and acetone extracts were found to be the most active. On the contrary, the fractions from the crude folklore preparation resulted less active. This result could partly explain the popularity of this plant in folk medicine as a remedy for cancer and HIV patients in the eastern part of Thailand.

Stability and transdermal absorption of topical amphotericin B liposome formulations.

The aim os this study was to characterize the stability and transdermal absorption of amphotericin B (AMB: 0.05 mg/mg lipid) in hydrogenated soya phosphatidylcholine/cholesterol/charged lipid [dicetyl phosphate (-) or stearylamine (+)] liposomes at molar ratios of 1:1:0, 7:2:0, 7:2:1(-) and 7:2:1(+). The AmB contents in liposomes were determined by HPLC with UV detection at 382 nm. Stabilities of AmB in liposome formulations were compared with those in solution and powder forms, during storage at 4, 30 and 45 degrees C for 90 days. Absorption studies of AmB across the rat skin were conducted, using vertical Franz diffusion cells at 37 degrees C for 24 h. The slowest degradation was observed in the positive liposome (7:2:1(+)AmB), with shelf life of approximately 1 year (30 degrees C). In comparison, the shelf lives of AmB in solution and powder were 4 and 14 days, respectively. AmB in positive liposomes seemed to demonstrate the highest flux in stratum corneum (58 ng/cm(2)/h), while the highest flux in viable epidermis (23 ng/cm(2)/h) was observed in negative liposomes. AmB entrapped in charged liposomes showed sustained skin absorption. The positively charged liposome might be the best formulation for AmB, due to its higher stability than other formulations.

Immunomodulatory activities of Clausena excavata Burm. f. wood extracts.

In vitro immunomodulatory activities of aqueous extract, acetone extract and the Thai folklore extract of Clausena excavata Burm. f. on mouse immune system were investigated. The phagocytic activity of macrophages and splenocyte proliferation in the absence and presence of mitogens (lipopolysaccharide, LPS) or pokeweed mitogen, PWM) were assayed. The aqueous extract exhibited the maximum effect on both respiratory burst response and lysosomal enzyme activity more than the acetone extract and the Thai folklore extract indicating effective phagocytic activation. For splenocyte proliferation assay, the Thai folklore extract with LPS gave the maximum activity higher than that with PWM, suggesting specificity towards B cell proliferation through T cell independent pathway the same as LPS. The present study revealed the immunomodulating activity, which could be explained the traditional use of this plant in Thailand.

Development of novel topical tranexamic acid liposome formulations.

The aims of this study were to develop novel liposome formulations for tranexamic acid (TA) from various lipid compositions [neutral (hydrogenated soya phosphatidylcholine and cholesterol), positive (stearylamine) or negative (dicetyl phosphate) charged lipid], and to investigate the effects of concentrations of TA (5 and 10% in DI water) and charges on the physicochemical properties of liposomes. Liposomes were prepared by chloroform film method with sonication. The physical (appearance, pH, size, morphology) and chemical (drug encapsulation efficiency, transition temperature, enthalpy of transition) properties of liposomes were characterized. The TA contents were determined spectrophotometrically at 415 nm, following derivatization with 2,4,6-trinitrobenzosulfonic acid. The charged liposomes demonstrated better physical stability than the neutral liposomes. The percentages of TA entrapped in all liposome formulations varied between 13.2 and 15.6%, and were independent of TA concentrations and charges of liposomes. Charges affected the physical stability, pH and size of liposomes. The particle sizes of negative blank and positive liposomes (with and without the entrapped drug) were approximately 10 times larger than the negative liposome with the entrapped TA. The multilamellar 7:2:1 molar ratio of hydrogenated soy phosphatidylcholine/cholesterol/dicetyl phosphate entrapped with 10% TA liposome (10%TA,-) was selected for further release study, due to its high physical stability, small particle size and relatively high drug encapsulation efficiency.

Stability and release of topical tranexamic acid liposome formulations.

Tranexamic acid (TA) has been claimed to have whitening effects. The effects of TA contents (5% and 10%) and charges on the stability and release of TA entrapped in hydrogenated soya phosphatidylcholine/cholesterol/charged lipid [dicetyl phosphate (-) or stearylamine (+)] liposomes at molar ratios of 7:2:1(-) and 7:2:1 (+) were investigated. The TA contents were determined spectrophotometrically at 415 nm, following derivatization with 2,4,6-trinitrobenzosulfonic acid. Stability and leakage of TA from liposomes were characterized at 4 degrees, 30 degrees and 45 degrees C for 90 days. The leakage rates of TA in negative liposomes were lower than those in positive liposomes. The TA in all liposome formulations was relatively stable, as > 90% of total drug remained after up to two months. The release of TA from liposomes was examined using vertical Franz diffusion cells at 37 degrees C for 24 h. The release rates of TA from all liposome formulations were approximately 3 times lower than those from solutions. Charges appeared to affect the physical stability, leakage, and shelf life of TA in liposomes, whereas TA concentrations seemed to affect the release of TA. The 7:2:1 (10% TA,-) liposome was the best formulation, due to its small size, low leakage, high stability, and prolonged and sustained release profile.

Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli.

The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused to gpIII of PhiM13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII, a stop codon between K2S and the gpIII gene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-beta-D-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/microg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.

Dominant lethal test in rats treated with some plant extracts.

The present study was undertaken to investigate the toxic effect of aqueous extracts of Aegle marmelos (AM), Stevia rebaudiana (SR), Pouteria cambodiana (PC) and Clausena excavata (CE) on rats by dominant lethal test. The data of 8-week treatment suggested that none of the extracts adversely affected male body and testicular weights as well as cauda epididymal sperm counts. No notable changes in sperm morphology and motility were observed. On the other hand, sperm count in the CE group was significantly higher as compared to both control and other treatment groups. There were no abnormal changes in the number of implantation sites, number of viable fetuses and number of dead fetuses in females mated with plant extract-treated males relative to controls. Based on these results, it could be concluded that all the investigated plant extracts have no toxic effect on male rat reproduction and progeny outcome.

Microencapsulation of human insulin DEAE-dextran complex and the complex in liposomes by the emulsion non-solvent addition method.

Human insulin-DEAE (diethyl amino ethyl) dextran complex and human insulin DEAE-dextran complex in liposomes were encapsulated in cellulose acetate butyrate (CAB) microcapsules by the emulsion non-solvent addition method. The ratio of core-to-coat used was 1:1. The average diameters of the complex microcapsules and the complex liposome microcapsules were 239.5 +/- 77.5 and 182.9 +/- 52.2 microns respectively. In vitro dissolution studies of both types of microcapsules in simulated intestinal fluid at pH 7.2 showed a sustained release of the complex and the complex liposome microcapsules with t50 = 1.5 h and 4 h respectively. This study can be applied to the further development of oral formulations of human insulin liposomes for diabetic treatment.

Suppression of experimental systemic lupus erythematosus (SLE) with specific anti-idiotypic antibody-saporin conjugate.

The importance of the idiotypic network is represented in experimental SLE induced by active immunization of naive mice with an anti-DNA idiotype (Ab1) emulsified in adjuvant. The mice after 4 months of incubation generate Ab3 having anti-DNA activity. In addition, the mice develop other serological markers for SLE associated with clinical and histopathological manifestations characteristic of the disease. To confirm further the etiological role of the idiotype in this experimental model, the mice were treated with specific anti-idiotypic antibodies (anti-Id) which were also conjugated to a toxin-saporin (Immunotoxin (IT)). Pretreatment of hybridoma cell line producing the anti-anti-Id (anti-DNA = (Ab3)) for 48 h with the anti-Id MoAb (Ab2) reduced the production of anti-DNA by 58%, while pretreatment with the IT resulted in 86% decrease in anti-DNA secretion (saporin alone had only 12% effect). The anti-Id MoAb had no effect on the production of immunoglobulin by an unrelated cell line. In vivo treatment of mice with experimental SLE led to a significant decrease in titres of serum autoantibodies, with diminished clinical manifestations. The results were more remarkable when the IT was employed. These suppressive effects were specific, since an anti-Id treatment of experimental anti-phospholipid syndrome was of no avail. The anti-Id effect was mediated via a reduction in specific anti-DNA antibody-forming cells, and lasted only while anti-Id injections were given. Discontinuation of the anti-Id injection was followed by a rise in titres of anti-DNA antibodies. No immunological escape of new anti-DNA Ids was noted. Our results point to the importance of pathogenic idiotypes in SLE and to the specific potential of implementing anti-idiotypic therapy, enhanced by the conjugation of the anti-Id to an immunotoxin, in particular one with low spontaneous toxicity.