Preparation of Rubra (Paeonia lactiflora Pall.) extract and studies in activity and skin penetration.

The inhibition rate of tyrosinase activity was used to determine extraction solvent of Paeoniae Radix Rubra extract (PRRE), which was established quality control standards by HPLC and verified the antioxidant activity. Ternary phase diagram was used to screen the best formulation of PRRE nanoemulsion, the skin permeability of PRRE and nanoemulsion were compared. The results show that 70% ethanol as the extraction solvent were highest (88.89%) and the contents of catechin (CC) and paeoniflorin (PF) in PRRE were 0.145 ± 0.0006 µg/mg and 21.783 ± 0.0247 µg/mg, respectively. The inhibition rate of PRRE on pyrogallol autoxidation was 6.94% ± 0.53%. The optimal formulation is Isopropyl myristate (IPM) as oil phase, Ethoxylated hydrogenated castor oil (RH40) as emulsifier, glycerine as coemulsifier, Km 3:1. The skin penetration of CC in PRRE nanoemulsion (0.79 ± 0.04 µg·cm-2) was significantly higher than that PRRE (0.17 ± 0.09 µg·cm-2) after 12 h.

A Non-Lipolysis Nanoemulsion Improved Oral Bioavailability by Reducing the First-Pass Metabolism of Raloxifene, and Related Absorption Mechanisms Being Studied.

OBJECTIVE: A non-lipolysis nanoemulsion (NNE) was designed to reduce the first-pass metabolism of raloxifene (RAL) by intestinal UDP-glucuronosyltransferases (UGTs) for increasing the oral absorption of RAL, coupled with in vitro and in vivo studies. METHODS: In vitro stability of NNE was evaluated by lipolysis and the UGT metabolism system. The oral bioavailability of NNE was studied in rats and pigs. Finally, the absorption mechanisms of NNE were investigated by in situ single-pass intestinal perfusion (SPIP) in rats, Madin-Darby canine kidney (MDCK) cells model, and lymphatic blocking model. RESULTS: The pre-NNE consisted of isopropyl palmitate, linoleic acid, Cremophor RH40, and ethanol in a weight ratio of 3.33:1.67:3:2. Compared to lipolysis nanoemulsion of RAL (RAL-LNE), the RAL-NNE was more stable in in vitro gastrointestinal buffers, lipolysis, and UGT metabolism system (p < 0.05). The oral bioavailability was significantly improved by the NNE (203.30%) and the LNE (205.89%) relative to the suspension group in rats. However, 541.28% relative bioavailability was achieved in pigs after oral NNE intake compared to the suspension and had two-fold greater bioavailability than the LNE (p < 0.05). The RAL-NNE was mainly absorbed in the jejunum and had high permeability at the intestine of rats. The results of both SPIP and MDCK cell models demonstrated that the RAL-NNE was absorbed via endocytosis mediated by caveolin and clathrin. The other absorption route, the lymphatic transport (cycloheximide as blocking agent), was significantly improved by the NNE compared with the LNE (p < 0.05). CONCLUSION: A NNE was successfully developed to reduce the first-pass metabolism of RAL in the intestine and enhance its lymphatic transport, thereby improving the oral bioavailability. Altogether, NNE is a promising carrier for the oral delivery of drugs with significant first-pass metabolism.

Comparisons of in vitro Fick's first law, lipolysis, and in vivo rat models for oral absorption on BCS II drugs in SNEDDS.

PURPOSE: The objective of this study was to compare the in vitro Fick's first law, in vitro lipolysis, and in vivo rat assays for oral absorption of Biopharmaceutical Classification Systems Class II (BCS II) drugs in self-nanoemulsifying drug delivery system (SNEDDS), and studied drugs and oils properties effects on the absorption. METHODS: The transport abilities of griseofulvin (GRI), phenytoin (PHE), indomethacin (IND), and ketoprofen (KET) in saturated water solutions and SNEDDS were investigated using the in vitro Madin-Darby canine kidney cell model. GRI and cinnarizine (CIN) in medium-chain triglycerides (MCT)-SNEDDS and long-chain triglycerides (LCT)-SNEDDS were administered in the in vivo SD rat and in vitro lipolysis models to compare the oral absorption and the distribution behaviors in GIT and build an in vitro-in vivo correlation (IVIVC). RESULTS: In the cell model, the solubility of GRI, PHE, IND, and KET increased 6-8 fold by SNEDDS, but their permeability were only 18%, 4%, 8%, and 33% of those of their saturated water solutions, respectively. However, in vivo absorption of GRI-SNEDDS was twice that of the GRI suspension and those of CIN-SNEDDS were 15-21 fold those of the CIN suspension. In the lipolysis model, the GRI% in aqueous and pellet phases of MCT were similar to that in LCT. In contrast, the CIN% in the aqueous and pellet phases were decreased but that of the lipid phase increased. In addition, an IVIVC was found between the CIN% in the lipid phase and in vivo relative oral bioavailability (F r). CONCLUSION: The in vitro cell model was still a suitable tool to study drug properties effects on biofilm transport and SNEDDS absorption mechanisms. The in vitro lipolysis model provided superior oral absorption simulation of SNEDDS and helped to build correlation with in vivo rats. The oral drug absorption was affected by drug and oil properties in SNEDDS.

Nanoemulsion improves hypoglycemic efficacy of berberine by overcoming its gastrointestinal challenge.

Berberine (BBR) is an important natural product with poor gastrointestinal behavior includes low permeability, P-glycoprotein efflux, and mass elimination in the intestine. The aim of this study was to develop a novel nanoemulsion (NE) to improve the hypoglycemic efficacy of BBR. NE was prepared and characterized by morphology and droplet size detection, stored stability, in vitro intestinal lipolysis and metabolism, Caco-2 cells transport, in situ single-pass intestinal perfusion, oral bioavailability in rats, and hypoglycemic efficacy in high-fat diet and streptozocin-induced mice. BBR-loaded NE exhibits small droplet size (30.56 ±â€¯0.35 nm) and good stability. NE could remain intact after lipolysis and protect BBR against the intestinal metabolism mediated by CYP2D6 and CYP3A4. Cells transport and intestinal perfusion studies revealed that NE decreases the P-glycoprotein efflux of BBR by 2-fold and enhances its permeability by 5.5-fold. Consequently, NE increased the oral bioavailability of BBR in rats by 212.02%. Compared to BBR control, blood glucose level of diabetic mice by NE was decreased by 3-fold. This novel NE provides a promising carrier to improve the hypoglycemic efficacy of BBR by overcoming its gastrointestinal deficiency, which may offer a product for the therapy of diabetes.

Niosomal Nanocarriers for Enhanced Skin Delivery of Quercetin with Functions of Anti-Tyrosinase and Antioxidant.

This study aimed to screen an effective flavonoid with promising whitening and antioxidant capacities, and design flavonoid-loaded niosomes to improve its solubility, stability, and penetration. In vitro anti-tyrosinase and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging experiments were conducted to investigate the whitening and antioxidant capacities of several flavonoids, including quercetin, morin, festin, myricetin, rutin, and breviscapine. The conductivity, viscosity, and particle size of Span60-RH40-based formulation of nonionic surfactant vesicles (niosomes) with different mass ratios were studied to determine the most appropriate formulation. Drug-loaded niosomes were characterized for size, zeta potential, morphology, and entrapment efficiency. The photostability, solubility, release behavior, ex vivo drug penetration, and skin retention were also studied. The results showed that quercetin has considerable whitening and antioxidant capacities and Span60-RH40 at a mass ratio of 9:11 forms spherical or oval niosomes of 97.6 ± 3.1 nm with a zeta potential range of 31.1 ± 0.9 mV, and drug entrapment efficiency as high as 87.3 ± 1.6%. Niosomes remarkably improved the solubility and photostability of quercetin. Furthermore, compared to quercetin solution, quercetin-niosomes had the advantages of sustained release and improved transdermal penetration, with skin retention 2.95 times higher than quercetin solution.

Research progress on berberine with a special focus on its oral bioavailability.

The natural product berberine (BBR) has become a potential drug in the treatment of diabetes, hyperlipidemia, and cancer. However, the oral delivery of BBR is challenged by its poor bioavailability. It is necessary to improve the oral bioavailability of BBR before it can be used in many clinical applications. Understanding the pharmacokinetic characteristics of BBR will enable the development of suitable formulas that have improved oral bioavailability. The key considerations for BBR are how to enhance the drug absorption and to avoid the intestinal first-pass effect. This review summarizes the pharmacological activities of BBR and analyzes the factors that lead to its poor oral bioavailability. In particular, the therapeutic potential of BBR in new indications from the aspect of oral bioavailability is discussed. In conclusion, BBR is a promising drug candidate for metabolic disorders and cancer but faces considerable challenges due to its poor oral bioavailability.

[Preparation and Dissolution Evaluation of Breviscapine Self-microemulsion].

OBJECTIVE: To study the prescription and preparation technology of breviscapine self-microemulsion for oral administration, and to evaluate the quality, stability and in vitro dissolution. METHODS: The prescription and preparation technology were selected and optimized through the solubility experiment, compatibility test, and pseudo-ternary phase diagram method, using the self-emulsifying time, appearance, particle diameter and stability as indexes. The droplet morphous, drug content, stability and dissolution were evaluated. Results:The prescription composition of breviscapine self-microemulsion was caprylic/capric triglyceride(GTCC,40%), Cremophor RH-40(50%), and PEG-400 (10%), with the drug loading of 7. 0 mg/g. The breviscapine self-microemulsion exhibited uniform and transparent,with the particle size of 38. 57 nm,Zeta potential of - 8. 80 mV. The results of dissolution indicated that the accumulative dissolution in 0. 1 mol/L hydrochloric acid was able to reach 90. 30% after 90 min, being 5. 9 times to that of the raw material medicine. The stability result showed that the content of breviscapine self-microemulsion was affected by high temperature, indicating it should be stored at low temperature. CONCLUSION: The preparation of breviscapine self-microemulsion is simple, which can increase the solubility of breviscapine in water and the absorption of breviscapine in the stomach and intestine, and conform to the main indexes of oral drug delivery system. It offers the basis for further research of breviscapine.

Evaluation of the anaphylactoid potential of Andrographis paniculata extracts using the popliteal lymph node assay and P815 cell degranulation in vitro.

BACKGROUND: The anaphylactoid reactions induced by andrographis injection have repeatedly been reported. The aim of our study was to evaluate the immuno-sensitizing potential of extracts from Andrographis paniculata Nees and to screen for the constituent that is responsible for inducing the anaphylactoid reaction. METHODS: In the direct popliteal lymph node assay (D-PLNA), female BALB/c mice were randomly divided into several groups with ten mice per group according to the experiment design, the right hind footpads of mice received a single subcutaneous injection of Andrographis paniculata (50 µl), and the left hind footpads received the same volume of vehicle. Seven days later, the mice were sacrificed by cervical dislocation, and the popliteal lymph nodes from both the left and right sides were removed. The weight (WI) and cellularity indices (CI) of the popliteal lymph nodes (PLNs) were then calculated, and the pathological changes of the PLNs were measured. In addition, P815 mast cells were collected for the in vitro cell degranulation experiment. The level of histamine, the percentage of cell degranulation and the ratio of ammonia glycosidase released were measured to further evaluate the potential allergenicity. RESULTS: Alcohol extract (AEE), ethyl acetate extract (EAE) and n-butanol extract (NBE) significantly increased the weight (WI > 2) and cell number (CI > 5) of PLNs (P < 0.05, P < 0.01). Additionally, all the three monomers of andrographis, namely NAD, AND, and DDA, significantly increased the weight (WI > 2) and cell number (CI > 5) of the PLNs (P < 0.05, P < 0.01). In the cell model, all of the different extract fractions (AEE, EAE and NBE) and the three monomers of andrographis markedly elevated the level of histamine, the percentage of cell degranulation and the ratio of ammonia glycosidase released. CONCLUSION: The diterpene lactone compounds of Andrographis paniculata Nees (total lactones of andrographolide) may have a potential sensitizing capacity in andrographis injection.

Lobelia chinensis: chemical constituents and anticancer activity perspective.

Research has demonstrated that many chemical constituents dominated by piperidine alkaloids and flavonoids, such as lobelanidine, lobeline, and lobelanine, have been obtained from Lobelia chinensis Lour. Experimental studies and clinical applications have also indicated that L. chinensis possesses a number of pharmacological activities (e.g., diuretic, choleretic, breathing excitement, anti-venom, anti-bacterial, and anticancer). This paper focuses on the properties, chemical constituents, and anticancer activity of L. chinensis to clarify the connection among them, and identify the active anticancer compounds. This work serves as the foundation for further research and development of L. chinensis.

Combined use of phospholipid complexes and self-emulsifying microemulsions for improving the oral absorption of a BCS class IV compound, baicalin.

The aim of this study was to develop a formulation to improve the oral absorption of baicalin (BA) by combining a phospholipid complex (PC) and self-emulsifying microemulsion drug delivery system (SMEDDS), termed BA-PC-SMEDDS. BA-PC was prepared by a solvent evaporation method and evaluated by complexation percentage (CP). The physicochemical properties of BA-PC were determined. The synergistic effect of PC and SMEDDS on permeation of BA was studied in vitro with Caco-2 cells and in situ with a single pass intestinal perfusion model. The improved bioavailability of BA in BA-PC-SMEDDS was confirmed in an in vivo rat model. The CP of BA-PC reached 100% when the molar ratio of drug to phospholipid (PP) was ≥1:1. The solubility of BA-PC increased in both water and octanol, and the log P o/w of BA-PC was increased significantly. BA-PC-SMEDDS could be dispersed more evenly in water, compared to BA and BA-PC. Both the Caco-2 cell uptake and single-pass intestinal perfusion models illustrated that transport of BA in BA-PC was lower than that of free BA, while improved significantly in BA-PC-SMEDDS. The relative bioavailability of BA-PC(1:2)-SMEDDS was 220.37%. The combination system of PC and SMEDDS had a synergistic effect on improving the oral absorption of BA.

Formulation and characterization of Brucea javanica oil microemulsion for improving safety.

OBJECTIVE: This study engaged in investigation of optimal formulation, characteristics analysis of Brucea javanica oil microemulsion (BJOM) in order to address safety concerns and make recommendations for improvements in BJOM safety during clinical use in vivo. METHODS: Pseudo-ternary phase diagram techniques were used to determine the appropriate ratio of surfactant, cosurfactant and oil phases. Subsequent stability testing of BJOM was performed by dilution, centrifugation and accelerated stability testing. The results were expounded through additional assessment utilizing the classical thermostat method to establish the shelf life of the material. These results were utilized to evaluate the safety of BJOM by haemolytic, irritative and allergic testing in vitro. In addition, the cytotoxicity of BJOM was examined using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), with particular emphasis given to potential uses in cancer treatment. RESULTS: The most suitable method of preparation for BJOM was found to be a one to one ratio (Km 1:1) of Solutol HS15 surfactant matched with sorbitol cosurfactant in the ratio. The microemulsion droplets of BJOM possessed a spherical shape, uniform size and average diameter of 23.8 nm. The expiration date of BJOM was found to be 568 d. The safety study demonstrated no hemolysis activity at the experimental BJOM concentrations; however, mild hemolysis was observed at higher concentrations of Brucea javanica oil emulsion (BJOE), a common commercially available product. Irritation observed upon BJOM treatment can be primarily attributed to Brucea javanica oil (BJO) with little influence of BJOM excipients. In addition, BJOM caused no observed hypersensitivity or other visible allergic reactions in guinea pigs. The anticancer activity curves of BJOM and BJOE demonstrate that both BJOM and BJOE inhibit Hela cells, with BJOM demonstrating significantly more dramatic anticancer activity. CONCLUSION: An optimal formulation of BJOM superior to commercially available products and safe for medical application such as intravenous injection has been outlined along with its anticancer activity rating.

[Preparation and quality evaluation of tanshinone IIA microemulsion for parenteral injection].

OBJECTIVE: To study the prescription and preparation technology of tanshinone IIA microemulsion for parenteral injection, and to evaluate its quality. METHODS: The prescription was selected and optimized through single-factor test, compatibility experiment and the pseudo-ternary phase diagram method. The preparation technology was investigated, and the droplet morphous, particle diameter, zeta potential, stability and haemolyticus were evaluated. RESULTS: The prescription composition of tanshinone IIA microemulsion was MCT:Solutol HS-15: fabaceous lecithin: absolute alcohol = 9:10:5:6(m/m), oil phase: aqueous phase = 1:10, with the drug-loaded of 1. 0 mg/g. The acquired microemulsion exhibited salmon pink,uniform and transparent, with the average particle diameter of 16. 04 nm, Zeta potential of -11. 57 mV, and the encapsulation efficiency of 98. 53%. The stability result showed that tanshinone IIA content in microemulsion was influenced by high temperature and illumination, indicating tanshinone IIA microemulsion should to be stored at low temperature and protected from light. The preparation was without hemolytic crisis. CONCLUSION: The preparation of tanshinone IIA micro- emulsion is simple,corresponding to the main index of parenteral injection and offering the basis for new dosage form development of tanshinone IIA.

[Drug delivery systems of baicalin, baicalin-phospholipid complex and self-microemulsifying drug across Caco-2 cell model].

OBJECTIVE: To study the absorption of baicalin (BA), baicalin-phospholipid complex (BA-PC), and two kinds of self-microemulsifying drug delivery system (SMEDDS) of BA-PC (BA-PC-NE-SMEDDS with natural emulsifier and BA-PC-NS-SMEDDS with nonionic surfactants) and predict the ability of improving bioavailability through changing the formulation of BA. METHODS: Transmembrane transports of each formulation were studied by Caco-2 cell model and the concentration of BA was determined by HPLC. RESULTS: With the increasing concentration of BA, the transport rate and apparent permeability coefficient (Papp) of BA was increased,indicating the passive absorption mechanism of BA. While with the increase of transport time, the transport rate and Papp of BA was decreased slowly, most likely due to the biological transformation of BA during the permeation process as reported in other people's paper. When coupled with P-gp inhibitor (Verapamil), the efflux rate (ER) of BA decreased from 2.07 to 0.48, indicating it was the substrate of P-gp. Compared with BA,the cumulative permeate quantity of BA-PC and BA-PC-SMEDDS were with no significant increase before 90 min (P > 0.05), but increased obviously after 90 min (P < 0.05). Three hours later, the cumulative permeate quantity and Papp showed significant differences (P < 0.05) among each formulation and were arranged in the following order: BA-PC-NS-SMEDDS > BA-PC-NE-SMEDDS > BA-PC > BA. Furthermore, the Papp of BA-PC and BA-PC-SMEDDS was significantly greater than that of BA coupled with Verapamil (P < 0.05). CONCLUSION: PC promotes the permeation of BA; PC-SMEDDS further accelerates its permeation bases on BA-PC; And BA-PC-NS-SMEDDS shows the better effect than BA-PC-NE-SMEDDS to promote the permeation of BA.

The pharmacokinetics of recombinant human interferon-alpha-2b poly(lactic-co-glycolic acid) microspheres in rats.

Interferon-alpha2b (IFN α-2b) microspheres were prepared at various concentrations (5%, 10%, 15%, 20% and 25%) and viscosities (0.39, 0.6, 0.89 and 1.13 dL/g) of poly(lactic-co-glycolic acid) (PLGA) using double emulsion solvent evaporation. The optimal formulation of IFN α-2b microspheres was determined to be 0.89 dL/g PLGA, as assessed by the in vitro release test. The pharmacokinetics of IFN α-2b microspheres was investigated. Nine groups of rats were injected intramuscularly with three doses (0.5, 1 and 2 MIU) of commercial lyophilized IFNα-2b injection or IFN α-2b microspheres. At a dose of 0.5 MIU, the IFN α-2b microsphere released significantly longer than that of the IFN α-2b injection. At a dose of 2 MIU, each pharmacokinetics parameter of microspheres prepared with the IFNa-2b stock solution was manifestly greater than those of the injection. Our study indicated that the IFN α-2b microspheres prepared in 15% of 0.89 dL/g PLGA provided a sustained drug effect for up to 21 days in rats.

[Preparation and in vitro evaluations of topically applied capsaicin transfersomes].

OBJECTIVE: Capsaicin transfersomes were prepared and its quality specifications were evaluated. METHOD: Capsaicin transfersomes were prepared by high shear dispersing machine and evaluated on the entrapment efficiency, drugs release rate and in vitro skin permeation. RESULT: Capsaicin transfersomes is composed of single unilamellar vesicles, with average size of 150.6 nm. Capsaicin entrapment efficiency achieved 96.7% while concentration of lecithin used was 8%. cumulative release amount of capsaicin was in direct proportion to the ethanol concentration in the medium. The in vitro rate cumulative penetration rate of capsaicin was higher in transfersomes than in cream and suspension in rats. Adomen skin cumulative penetration rate in vitro of capsaicin transfersomes in mouse was significantly higher than that from rat and men. In the same way,cumulative penetration rate in vitro of capsaicin transfersomes through abdomen skin epidermal membrance was significantly higher than that with derma and full skin in men. CONCLUSION: Entrapment efficiency of capsaicin transfersomes reached 96.7%, meeting the criterion of China pharmacopia( > 80%), skin penetration of capsaicin was enhanced by a capsaicin transfersomes preparation and was affected by diverse characters and levels of skin.

[Determination of capsaicin in Pheretima aspergillum from different habitats by RP-HPLC].

OBJECTIVE: To determine the content of hypoxanthine in Pheretima aspergillum from different habitats. METHOD: A RP-HPLC method was established. The chromatographic column was Inertsil ODS-EP. The mobile phase was H2O-CH3OH-C4H8O(93:: 7: 0.05). The flow rate was 1.0 ml/min, and the detection wavelength was 254 nm. RESULTS: The average recoveries for hypoxanthine was 98.6% , precision of the method was 0. 50% (RSD, n = 6). CONCLUSION: The method can be used to determine the content of hypoxanthine in Pheretima aspergillum from diffrent habitats.

Preparation and in vitro and in vivo evaluations of topically applied capsaicin transfersomes / 药学学报

<p><b>AIM</b>To prepare capsaicin transfersomes and evaluate them in vitro and in vivo.</p><p><b>METHODS</b>Capsaicin transfersomes were prepared by high shear dispersing machine and evaluated by entrapment efficiency, release rate, in vitro skin permeation and distribution in different tissues in vivo.</p><p><b>RESULTS</b>Capsaicin transfersomes were composed of single unilamellar vesicles with an average diameter of 150.6 nm. Capsaicin entrapment efficiency increased distinctly with increasing of concentration of lecithin and entrapment efficiency is 96.7% while concentration of lecithin to 8%. Cumulative release amount of capsaicin is in direct proportion to the ethanol concentration in the receptor medium. In vitro capsaicin cumulative penetration amount showed higher levels in transfersomes than cream and suspension in rat abdominal skin. Abdominal skin cumulative penetration amount in vitro of capsaicin transfersomes in mouse was significantly higher than that from rat and men. In the same way, abdominal skin epidermal membrane cumulative penetration amount in vitro of capsaicin transfersomes was significantly higher than that from derma and full skin in human abdominal skin. The capsaicin tissue distribution of capsaicin injection by multiple celiac injections in rats is different: bone > plasma > skin > muscle. There is a similar result by multiple thigh topical application of capsaicin transfersomes: bone > skin > plasma > muscle.</p><p><b>CONCLUSION</b>Entrapment efficiency of capsaicin transfersomes reached the criterion of China Pharmacopoeia (> 80%) and capsaicin skin penetration can be increased by capsaicin transfersomes. It should be noted that the diverse characters and levels of skin may probably affect the permeating capability of capsaicin. Capsaicin tissue distribution in bone and muscle is similar and is different in plasma and skin by multiple injections and topical skin apply.</p>

[The preparation and the in vitro release of OANO-1 microspheres].

OBJECTIVE: To prepare OANO-1 microspheres and test their release in vitro. METHOD: OANO-1 microspheres were made by W/O/W-liquid drying process. The surface morphology of the microspheres was observed by SEM. The mean diameter and the size distribution of microspheres, the drug loading and the incorporation efficiency were examined. The release of OANO-1 microspheres in vitro was examined by small cup method. The accumulated release percent of OANO-1 microspheres was examined. RESULT: The OANO-1 microspheres were regular in their morphology. The average particle size was 8.59 microm with over 90% of the microspheres being in the range of 1-12 microm. The drug loading and the incorporation efficiency were 48.39% and 19.32% respectively. The accumulated release percent of OANO-1 microspheres was 78.4% after 108 h. The release half-life t1/2 was 40.8 h and Higuchi equation was Y = 0.1326 X - 0.4782, r = 0.9951. CONCLUSION: The preparation of OANO-1 microspheres was well. The release in vitro of OANO-1 microspheres showed significant sustained release.