Evaluation of the degradation of clonidine-loaded PLGA microspheres

Abstract

Context: The release of an encapsulated drug is dependent on diffusion and/or degradation/erosion processes.

Objective: This work aimed to better understand the degradation mechanism of clonidine-loaded microparticles.

Methods: Gel permeation chromatography was used to evaluate the degradation of the polymer. The water-uptake and the weight loss were determined gravimetrically. The swelling behaviour and the morphological changes of the formulations were observed by microscopy. The glass transition temperature and the crystallinity were also determined by differential scanning calorimetry and X-ray diffraction, respectively. The pH of the medium and inside the microspheres was assessed.

Results: The microspheres captured a large amount of water, allowing a decrease in the molecular weight of the polymer. The pH of the medium decreased after release of the degradation products and the pH inside the microparticles remained constant due to the neutralization of these acidic products.

Conclusion: Clonidine and buffers both had an action on the degradation.     

Measurement of Protein Diffusion Through Poly(d,l-Lactide-Co-Glycolide)

A novel method was developed for studying the diffusion of proteins through poly(d,l-lactide-co-glycolide) (PLG), using a diffusion cell. To develop improved formulations for the controlled release of encapsulated drugs it is important to understand the underlying release mechanisms. When using low-molecular-weight PLG as the release-controlling polymer, diffusion through the pores is often proposed as the main release mechanism. The experimental set-up and method of determining the diffusion coefficient were thoroughly evaluated with regard to the reliability and the influence of the stirring rate. A procedure for spraying thin films of PLG onto a filter, which could be placed in the diffusion cell, was optimized. The method was then applied to the determination of the diffusion coefficient of human growth hormone (hGH) through a PLG film. The results show that the method enables measurements of the diffusion coefficient through the polymer film. Neither the stirring rate nor the concentration of hGH influenced the diffusion coefficient. The diffusion coefficient of hGH through degraded PLG films was 5.0 · 10^{? 13} m²/s, which is in the range that could be expected, i.e., several orders of magnitude smaller than its the diffusivity in pure water. The reproducibility was good, considering the dynamic properties of PLG, i.e., the difference in diffusion coefficients, at, for example, different stages of degradation and for different compositions of PLG, is expected to be much higher. The variation is probably also present in PLG films used for controlled-release formulations. Although the PLG film contains a large amount of water, a considerable time elapsed before pores of sufficient size formed and diffusion through the film started. In two-component diffusion experiments, the difference in diffusion rate did not correspond to the difference in molecular weight of the solutes, indicating a size exclusion effect. This method can be used to study the effect of changes in the formulation specification. By studying the change in the diffusion coefficient through the degradation process of PLG, or similar polymers, a better understanding of diffusion and, thus, also release mechanisms can be obtained.     

Encapsulated zinc salt increases the diffusion of protein through PLG films

The use of microspheres and nanospheres of poly(d,l-lactide-co-glycolide) (PLG) as a controlled-release drug delivery system has been the subject of great interest for at least two decades within the field of pharmaceuticals. Salts of zinc and other divalent cations are sometimes co-encapsulated in PLG particles to control the pH or to stabilize encapsulated proteins or peptides. Zinc salts are known to affect pore formation and other processes that may lead to the release of an encapsulated drug. In this study the effect of encapsulated zinc acetate on protein diffusion through PLG films was investigated. PLG films, with and without encapsulated zinc acetate, were degraded in Hepes buffer for different periods of time. The films were subsequently subjected to various kinds of analyses: diffusion properties (using a diffusion cell), porosity (using scanning electron microscopy) and thickness (using light microscopy and an image-analysis program). Encapsulated zinc acetate had a considerable effect and increased the diffusion coefficient of lysozyme through PLG films degraded for 18 days or longer. Films containing zinc acetate became porous, while those without zinc acetate only developed cavities on the surface. Zinc salts may thus be used as release-modifying agents. This effect should be considered when using zinc salts as protein stabilizers or pH neutralizers.     

Preparation and in vitro evaluation of thienorphine-loaded PLGA nanoparticles

Poly (d,l-lactic-co-glycolide) nanoparticles (PLGA-NPs) have attracted considerable interest as new delivery vehicles for small molecules, with the potential to overcome issue such as poor drug solubility and cell permeability. However, their negative surface charge decreases bioavailability under oral administration. Recently, cationically modified PLGA-NPs has been introduced as novel carriers for oral delivery. In this study, our aim was to introduce and evaluate the physiochemical characteristics and bioadhesion of positively charged chitosan-coated PLGA-NPs (CS-PLGA-NPs), using thienorphine as a model drug. These results indicated that both CS-PLGA-NPs and PLGA-NPs had a narrow size distribution, averaging less than 130?nm. CS-PLGA-NPs was positively charged (+42.1?±?0.4?mV), exhibiting the cationic nature of chitosan, whereas PLGA-NPs showed a negative surface charge (?2.01?±?0.3?mV). CS-PLGA-NPs exhibited stronger bioadhesive potency than PLGA-NPs. Furthermore, the transport of thienorphine-CS-PLGA-NPs by Caco-2 cells was higher than thienorphine-PLGA-NPs or thienorphine solution. CS-PLGA-NPs were also found to significantly enhance cellular uptake compared with PLGA-NPs on Caco-2 cells. An evaluation of cytotoxicity showed no increase in toxicity in either kind of nanoparticles during the formulation process. The study proves that CS-PLGA-NPs can be used as a vector in oral drug delivery systems for thienorphine due to its positive surface charge and bioadhesive properties.     

Drug release from core-shell type nanoparticles of poly(DL-lactide-co-glycolide)-grafted dextran

This study prepared core-shell type nanoparticles of a poly(DL-lactide-co-glycolide) (PLGA) grafted-dextran. The synthesis of the PLGA-dextran conjugate was confirmed by Fourier transform-infrared (FT-IR) spectroscopy. The PLGA grafted-dextran was able to form nanoparticles in water by self-assembly and their particle size was 245.3?±?95.1?nm. From fluorescence probe study using pyrene as a hydrophobic probe, critical association concentration (CAC) values were determined from the fluorescence excitation spectra and were found to be 0.006?g?l^?1. Morphological observations using a scanning electron microscope (SEM) showed that the polymeric nanoparticles of the PLGA-dextran conjugate have uniformly spherical shapes. Their size and morphology provide them with acceptable properties for use as a drug-targeting carriers. Drug release from core-shell type nanoparticles was faster in the presence of dextranase, indicating that core-shell type nanoparticles of PLGA grafted-dextran can be used as an oral drug carriers.     

Physico-chemical characterisation of PLGA nanoparticles after freeze-drying and storage

Nanoparticles represent promising carriers for controlled drug delivery. Particle size and size distribution of the particles are important parameters for the in vivo behaviour after intravenous injection and have to be characterised precisely. In the present study, the influence of lyophilisation on the storage stability of poly(d,l lactic-co-glycolic acid) (PLGA) nanoparticles, formulated with several cryoprotective agents, was evaluated. Nanoparticles were prepared by a high pressure solvent evaporation method and freeze-dried in the presence of 1%, 2%, and 3% (m/v) sucrose, trehalose, and mannitol, respectively. Additionally, to all samples containing 3% of the excipients, l-arginine hydrochloride was added in concentrations of 2.1% or 8.4% (m/V). Dynamic light scattering (DLS), analytical ultracentrifugation and transmission electron microscopy (TEM) were used for particle characterisation before and after freeze-drying and subsequent reconstitution. In addition, glass transition temperatures were determined by differential scanning calorimetry (DSC), and the residual moisture of the lyophilisates was analysed by Karl Fischer titration. It was demonstrated that 1% sucrose or 2% trehalose were suitable to maintain particle integrity after reconstitution of lyophilised PLGA nanoparticles. The storage stability study over 3 months showed notable changes in mean particle size, size distribution, and residual moisture content, depending on the composition of the formulation.     

Effect of Divalent Cations on Pore Formation and Degradation of Poly(D,L-lactide-co-glycolide)

Poly(d,l-lactide-co-glycolide) (PLG) is probably the biodegradable polymer most often used for polymeric controlled-release formulations. Different salts have been shown to affect the swelling and degradation of PLG, which, in turn, affect the release of encapsulated drugs. In this investigation the effect of divalent cations was especially investigated. Films of PLG were incubated in phosphate buffer saline (PBS), a buffer containing salts similar to plasma, Hepes buffer, and Hepes buffer with ZnCl₂, CaCl₂, MgCl₂, or Na₂CO₃ added. Pore formation at the surface and inside the film was analyzed by scanning electron microscopy. The samples were also analyzed gravimetrically at predetermined intervals to determine the mass loss, and for some samples the pH within the PLG films was determined by confocal microscopy. Pores were formed faster in the presence of all divalent cations, and the results indicated a greater degradation rate in the presence of Zn²⁺. The catalyzing effect of the divalent cations on degradation was attributed to their ability to act as Lewis acids. Pores were formed more slowly in PBS than in a buffer containing salts similar to plasma, which should be considered when choosing the in vitro release medium.     

Three weeks release BCNU loaded hydrophilic-PLGA microspheres for interstitial chemotherapy: Development and activity against human glioblastoma cells

The aim of this study is the development of microspheres of BCNU for intracranial administration, as an alternative to marketed novel Gliadel® Implant in the treatment of brain tumours. H poly-lactide-co-glycolide biodegradable microspheres of BCNU with a mean size of 33.5 ± 1.8 µm were obtained by an oil-in-water emulsion solvent evaporation method. Their small size would allow their intracranial administration through a needle by cerebral stereotaxia if tumour recurrence occurs, without a surgical intervention, as Gliadel® needs. BCNU was released from these microspheres during 21 days, mainly by a mechanism of diffusion from the polymer matrix (K = 2.91 mg days^?½). The cytotoxic effects of these microspheres on human glioblastoma cells were demonstrated all through 21 days and the value of percentage of viable cells was less than 40%. These microspheres should be commercialized as a freeze-dried product to keep at ?20°C. Three hundred and twenty milligrams of microspheres contain 61.6 mg of BCNU, the same amount of BCNU contained in 1600 mg or eight wafers of Gliadel usually implanted after the tumour resection.     

Effect of preparative variables on small interfering RNA loaded Poly(D,L-lactide-co-glycolide)-chitosan submicron particles prepared by emulsification diffusion method

In this study, poly(D,L-lactide-co-glycolide) (PLGA)-chitosan particles were investigated as an effective delivery system for small interfering RNA (siRNA) by emulsification diffusion method. The type, molecular weight and concentration of chitosan, PLGA type as well as centrifugation and freeze-drying process were amongst the investigated variables. PLGA-chitosan particles obtained were positively charged with particle size between ～0.4–1 µm depending on type, molecular weight and concentration of chitosan as well as type of PLGA. A better siRNA loading capacity was observed when a higher degree of ‘uncapped end groups’ were used. The addition of trehalose has also been shown to stabilize these particles from severe aggregation induced by freeze-drying. It was found that physical properties of PLGA-chitosan particles and their siRNA binding capacity were highly influenced by certain preparation parameters. The desired positive charge of submicron size range PLGA-chitosan particles could therefore be obtained by adjusting and optimizing these preparative and formulation parameters.     

Neuroprotectant effects of iso-osmolar D-mannitol to prevent Pacific ciguatoxin-1 induced alterations in neuronal excitability: a comparison with other osmotic agents and free radical scavengers

The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 mM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-clamp recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 mM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 mM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Na(v) channels, as well as the increased rate of recovery from inactivation of TTX-resistant Na(v) channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant I(Na) amplitude by P-CTX-1. Additional experiments using hyper- and iso-osmolar D-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na(v) channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Na(v) channel subtype, possibly to alter or reduce toxin association.     

Effect of propionyl-l-carnitine,l-arginine and nicotinic acid on the efficacy of vardenafil in the treatment of erectile dysfunction in diabetes

ABSTRACT

Objective: The association of diabetes-related vascular damage and the role of metabolic factors in erectile dysfunction are well known in the literature. The compounds propionyl-l-carnitine (PLC), l-arginine (l-Arg) and nicotinic acid have numerous metabolic actions which have been reported to improve endothelial function. This study investigated the administration of the combination of these three compounds alone and in association with an inhibitor of 5-phosphodiesterase (5PDE), vardenafil, on endothelial function in diabetic patients with erectile dysfunction.

Methods: A total of 40 patients aged between 50 and 60 years with insulin-dependent diabetes (IDDM) for 3–4 years were selected from 509 patients presenting with erectile dysfunction. The patients were randomly subdivided into four groups of ten to be treated for 12 weeks. Group A was administered one sachet each day of test formulation containing PLC, l-Arg and nicotinic acid (Ezerex); group B with one 20 mg capsule of vardenafil (Levitra) twice a week; group C was treated with one sachet each day of the test formulation plus vardenafil 20 mg twice a week. Group D was administered placebo capsules twice weekly. Endothelial function was evaluated by examining flow-mediated dilation (FMD) and erectile function was estimated with the International Index of Erectile Function (IIEF5) questionnaire in all subjects.

Results: At the end of treatment group A showed an increment of 2 points in the IIEF5; group B showed an increment of 4 points; group C, the group which was administered all the treatments, showed an increment of 5 points, and group D, treated with placebo, showed no increment in the IIEF5.

Conclusion: Although there was a small number of subjects in this study the data suggest that the test formulation may improve the endothelial situation in diabetes. The test formulation together with vardenafil was better than the 5PDE inhibitor alone, but further studies are needed to confirm these findings.     

Phenylalanine monooxygenase and the sulfur oxygenation of S-carboxymethyl-l-cysteine in mice

1.?The extent of sulfoxidation of the drug, S-carboxymethyl-l-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-l-cysteine has been investigated.

2.?Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased C_max, AUC_{(0–180?min)} and AUC_{(0–∞?min)} values (15?- to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-l-cysteine (13?- to 22-fold lower).

3.?Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-l-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.     

Preparation and characterization of D,L-PLA loaded 17-β-Estradiol valerate by emulsion/evaporation methods

PLA microparticles containing 17-β-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718–880 nm (inert micro-particles) and 3–4 µm (drug loaded microparticles). The encapsulation efficiency was ～80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-β-estradiol.     

Angiotensin-converting enzyme (ACE) inhibitory potential of standardized Mucuna pruriens seed extract

Abstract

Context: Mucuna pruriens Linn. (Fabaceae) is a tropical legume, traditionally used for controlling blood pressure. Inhibition of angiotensin-converting enzyme (ACE) is one of the successful strategies for controlling hypertension.

Objective: The present study evaluated the ACE inhibition potential of the standardized extract of M. pruriens seeds.

Materials and methods: Standardization of the extract and its fractions were carried out by RP-HPLC method [methanol and 1% v/v acetic acid in water (5:95 v/v)] using levodopa as a marker. The ACE inhibition activity of the extract and fractions was evaluated at different concentrations (20, 40, 60, 80, and 100?µg/mL) using the HPLC-DAD and the UV spectrophotometric method. The liberation of hippuric acid (HA) from hippuryl-l-histidyl-l-leucine (HHL) was estimated in the spectrophotometric method and RP-HPLC assay at 228?nm.

Results: Methanol extract and aqueous fraction showed a maximum activity with IC₅₀ values of 38.44?±?0.90 and 57.07?±?2.90?µg/mL (RP-HPLC), and 52.68?±?2.02 and 67.65?±?2.40?µg/mL (spectrophotometry), respectively.

Discussion: The study revealed that the aqueous extract contains the highest amount of levodopa. Eventually the methanol extract showed highest ACE inhibition activity except levodopa alone. It was further observed that the inhibition was altered with respect to the change in the content of levodopa in the extract. Thus, it can be assumed that levodopa may be responsible for the ACE inhibition activity of M. pruriens seeds.

Conclusion: It can be concluded that M. pruriens seed is a potential ACE inhibitor can be explored further as an effective antihypertensive agent.     

The synthesis of pennogenin 3-O-β-d-glucopyranosyl-(1 → 3)-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside

Pennogenin 3-O-β-d-glucopyranosyl-(1 → 3)-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside, a monodesmosidic saponin isolated from Paris polyphylla Smith var. yunnanensis with promised antitumor activities, was firstly synthesized from glucoside thiol via nine steps and with 27% overall yield.     

Two new flavonoid glycosides from the whole herbs of Hyssopus officinalis

Two new flavonoid glycosides, quercetin 7-O-β-d-apiofuranosyl-(1 → 2)-β-d-xylopyranoside (1) and quercetin 7-O-β-d-apiofuranosyl-(1 → 2)-β-d-xylopyranoside 3′-O-β-d-glucopyranoside (2), together with nine known flavonoids were isolated from the whole herbs of Hyssopus officinalis L. cultivated in Xinjiang Uygur Autonomous Region of China. All structures were characterized by the spectroscopic methods including UV, IR, ESI-MS, 1D, and 2D NMR. Their potent free radical scavenging activity against the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was evaluated.     

S-carboxymethyl-l-cysteine and it (R/S)-S-oxides in beagle dog plasma and hepatic cytosol

Abstract

1. Incubation of beagle hepatic cytosol, under conditions promoting phenylalanine hydroxylase activity, led to the formation of the sulfoxide derivatives of S-carboxymethyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine, S-methyl-l-cysteine and N-acetyl-S-methyl-l-cysteine. Thiodiglycolic acid was not a substrate. Enzyme kinetic parameters (K_m, V_max) were derived indicating S-carboxymethyl-l-cysteine had the greatest clearance; no enantioselective preference was observed for this S-oxygenation reaction.

2. Following oral administration of S-carboxymethyl-l-cysteine to beagle dogs, the parent substance and its sulfoxide were the only compounds identified in the plasma. Pharmacokinetic data have been obtained indicating that the small amount of sulfoxide formed persisted within the body for longer than the parent material, but that the majority of the ingested dose remained in the administered sulfide form.

3. The sulfide moiety within the muco-regulatory drug, S-carboxymethyl-l-cysteine, is thought to be vital as it acts as a free radical scavenger, resulting in the inactive sulfoxide. Additional extensive enyzme-mediated sulfoxidation would decrease the amount of active sulfide available. In the dog this appears to not be an issue, signalling possible exploitation for therapeutic benefit in treating airway disease.     

Chlorogenic acid protects d-galactose-induced liver and kidney injury via antioxidation and anti-inflammation effects in mice

Context Oxidative stress and inflammation are implicated in the aging process and its related hepatic and renal function decline. Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in the human diet. Recently, CGA has shown in vivo and in vitro antioxidant properties.

Objective The current study investigates the effects of protective effects of chlorogenic acid (CGA) on d-galactose-induced liver and kidney injury.

Materials and methods Hepatic and renal injuries were induced in a mouse model by subcutaneously injection of d-galactose (d-gal; 100?mg/kg) once a day for 8 consecutive weeks and orally administered simultaneously with CGA included in the food (200?mg/kg of diet). The liver and renal functions were examined. Histological analyses of liver and kidney were done by haematoxylin and eosin staining. The oxidative stress markers and pro-inflammatory cytokines in the liver and the kidney were measured.

Results CGA significantly reduced the serum aminotransferase, serum creatinine (SCr) and blood urea nitrogen (BUN) levels in d-gal mice (p?<0.05). CGA also restored superoxide dismutase, catalase, and malondialdehyde levels and decreased glutathione content in the liver and kidney in d-gal mice (p?<0.05). Improvements in liver and kidney were also noted in histopathological studies. CGA reduced tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) protein levels in the liver and kidney in d-gal mice (p?<0.05).

Discussion and conclusion These findings suggest that CGA attenuates d-gal-induced chronic liver and kidney injury and that this protection may be due to its antioxidative and anti-inflammatory activities.     

Two new terpenoid glucosides from Aster flaccidus

Two new terpenoid glucosides, namely 2-O-β-d-glucopyranoside-vicodiol (1) and 10-O-β-d-glucopyranoside-oplopanone (2), along with seven known compounds, were isolated from the aerial part of Aster flaccidus (composite), a traditional Chinese herb medicine. The structures of 1 and 2 were established by spectroscopic methods, especially 2D NMR experiments.     

Three new isoflavone glycosides from Tongmai granules

Three new isoflavone glycosides, 3′-methoxydaidzein-7,4′-di-O-β-d-glucopyranoside (1), biochanin A-8-C-β-d-apiofuranosyl-(1 → 6)-O-β-d-glucopyranoside (2), daidzein-7-O-β-d-glucopyranosyl-(1 → 4)-O-β-d-glucopyranoside (3), and a new natural isoflavone glycoside, daidzein-7-O-α-d-glucopyranosyl-(1 → 4)-O-β-d-glucopyranoside (4) were isolated along with 18 known isoflavones from the EtOAc and n-BuOH fractions of the aqueous extraction of Tongmai granules. All the isoflavones were obtained and determined for the first time from Tongmai granules. The structures of these compounds were elucidated by spectral methods. It was confirmed that the compounds 1–4 were originally from Puerariae Lobatae Radix based on HPLC-DAD analysis of the crude drug extract. The isoflavones isolated were tested for their antioxidative activities by measuring the capacities of scavenging the 2,2′-diphenyl-1-picrylhydrazyl radical.