Lipidic cubic-phase leflunomide nanoparticles (cubosomes) as a potential tool for breast cancer management

Despite the fact of availability of several treatments for breast cancer, most of them fail to attain the desired therapeutic response due to their poor bioavailability, high doses, non-selectivity and as a result systemic toxicity. Here in an attempt made to study the transdermal effect of leflunomide (LEF) against breast cancer. In order to improve the poor physicochemical properties of LEF, it was loaded into cubosomes. Cubosomes were prepared by the emulsification method. Colloidal characteristics of cubosomes including particle size, ζ-potential, entrapment efficiency, in-vitro release profile and ex-vivo permeation were studied. In addition, morphology, stability, cytotoxicity and cell uptake in MDA-MB-231 cell line were carried out for the selected cubosomal formulation. The selected LEF loaded cubosomal formulation showed a small particle size (168 ± 1.08) with narrow size distribution (PI 0.186 ± 0.125) and negative ζ potential (–25.5 ± 0.98). Its Entrapment efficiency (EE%) was 93.2% and showed sustained release profile that extended for 24 h. The selected formulation showed stability when stored at 25 °C for three months in terms of size and EE%. TEM images illustrated the cubic structure of the cubosome. Cell culture results revealed the superiority of LEF cubosomes compared to LEF suspension in their cytotoxic effects with an IC50 close to that of doxorubicin. Furthermore, LEF cell uptake was significantly higher for LEF cubosomes. This may be attributed to the effect of nano-encapsulation on enhancing drug pharmacological effects and uptake indicating the potential usefulness of LEF cubosomes for breast cancer management.     

Formulation and evaluation of butenafine loaded PLGA-nanoparticulate laden chitosan nano gel

The present research work is designed to prepare and optimize butenafine (BT) loaded poly lactic co glycolic acid (PLGA) nanoparticles (BT-NPs). BT-NPs were prepared by emulsification probe sonication method using PLGA (A), PVA (B) as polymer and stabilizer, respectively. The optimum composition of BT-NPs was selected based on the point prediction method given by the Box Behnken design software. The optimized composition of BT-NPop showed a particle size of 267.21 ± 3.54 nm with an entrapment efficiency of 72.43 ± 3.11%. The optimum composition of BT-NPop was further converted into gel formulation using chitosan as a natural polymer. The prepared topical gel formulation (BT-NPopG) was further evaluated for gel characterization, drug release, permeation study, irritation, and antifungal studies. The prepared BT-NPopG formulation showed optimum pH, viscosity, spreadability, and drug content. The release and permeation study results revealed slow BT release (42.76 ± 2.87%) with significantly enhanced permeation across the egg membrane. The irritation study data showed negligible irritation with a cumulative score of 0.33. The antifungal study results conclude higher activity than marketed as well as pure BT. The overall conclusion of the results revealed BT-NPopG as an ideal delivery system to treat topical fungal infection.     

Nanocubosomal based in situ gel loaded with natamycin for ocular fungal diseases: development,optimization, in-vitro,and in-vivo assessment

Natamycin (NT) is a synthetic broad-spectrum antifungal used in eye drops. However, it has low solubility and high molecular weight, limiting its permeation, and generally causes eye discomfort or irritation when administered. Therefore, the present study aimed to develop an ophthalmic in situ gel formulation with NT-loaded cubosomes to enhance ocular permeation, improve antifungal activity, and prolong the retention time within the eye. The NT-loaded cubosome (NT-Cub) formula was first optimized using an I-optimal design utilizing phytantriol, PolyMulse, and NT as the independent formulation factors and particle size, entrapment efficiency %, and inhibition zone as responses. Phytantriol was found to increase particle size and entrapment efficiency %. Higher levels of PolyMulse slightly increased the inhibition zone whereas a decrease in particle size and EE% was observed. Increasing the NT level initially increased the entrapment efficiency % and inhibition zone. The optimized NT-Cub formulation was converted into an in situ gel system using 1.5% Carbopol 934. The optimum formula showed a pH-sensitive increase in viscosity, favoring prolonged retention in the eye. The in vitro release of NT was found to be 71 ± 4% in simulated tear fluid. The optimum formulation enhanced the ex vivo permeation of NT by 3.3 times compared to a commercial formulation and 5.2 times compared to the NT suspension. The in vivo ocular irritation test proved that the optimum formulation is less irritating than a commercial formulation of NT. This further implies that the developed formulation produces less ocular irritation and can reduce the required frequency of administration.     

Clinical comparative study of optimized metronidazole loaded lipid nanocarrier vaginal emulgel for management of bacterial vaginosis and its recurrence

The main focus of the current work was to design, evaluate and clinically compare the efficiency of novel metronidazole (MTD) loaded solid lipid nanoparticles (SLNs) vaginal emulgel with the marketed vaginal gel (Metron^®) against Bacterial vaginosis (BV). Eight formulations were fabricated using 2³ full factorial design and prepared by stearic acid and tween 80 as solid lipid and surfactant, respectively. Lipid and surfactant concentrations in addition to sonication amplitude were chosen as the independent variables (X₁–X₃). Then, the prepared MTD loaded SLNs were evaluated based on the dependent variables which were particle size, polydispersity index, zeta potential, entrapment efficiency, and cumulative % drug release for 24 h (Y₁–Y₅). The in vitro release study exhibited a sustained release of MTD from the SLNs up to 24 h. The optimal MTD loaded SLNs showed nanosized particles (256 nm) with EE% (52%), and an acceptable ZP value (−29.5 mV). Also, the optimized MTD-SLNs formulation was incorporated into Carbopol emulgel and investigated clinically for its effect against BV. Clinical studies recorded significant enhancement in therapeutic response of MTD from optimized SLNs vaginal emulgel formulation regarding the clinical treatment (p < .05) and low recurrence rate (p < .001) against the marketed product. In conclusion, our findings recommend that the fabricated MTD loaded SLNs vaginal emulgel have significant therapeutic effect in terms of BV management over commercially obtainable marketed vaginal gel (Metron^®).     

Nanophyto-gel against multi-drug resistant Pseudomonas aeruginosa burn wound infection

Burn wound is usually associated by antibiotic-resistant Pseudomonas aeruginosa infection that worsens and complicates its management. An effective approach is to use natural antibiotics such as cinnamon oil as a powerful alternative. This study aims to investigate topical nanostructured lipid carrier (NLC) gel loaded cinnamon oil for Pseudomonas aeruginosa wound infection. A 2⁴ full factorial design was performed to optimize the formulation with particle size 108.48 ± 6.35 nm, zeta potential −37.36 ± 4.01 mV, and EE% 95.39 ± 0.82%. FTIR analysis revealed no excipient interaction. Poloxamer 407 in a concentration 20% w/w NLC gel was prepared for topical application. Drug release exhibited an initial burst release in the first five hours, followed by a slow, sustained release of up to five days. NLC-cinnamon gel has a significant ability to control the drug release with the lowest minimum inhibitory concentration again P. aeruginosa compared to other formulations (p < .05). In vivo study also showed NLC-cinnamon gel effectively healed the infected burned wound after a six-day treatment course with better antibacterial efficacy in burned animal models. Histological examination ensured the tolerability of NLC-cinnamon gel. The results suggest that nanoparticle-based cinnamon oil gel is a promising natural product against antibiotic-resistant strains of P. aeruginosa in wound infection.     

Preparation of topical bimatoprost with enhanced skin infiltration and in vivo hair regrowth efficacy in androgenic alopecia

To prepare a topical formulation of bimatoprost (BIM) with high skin permeability, we designed a solvent mixture system composed of ethanol, diethylene glycol monoethyl ether, cyclomethicone, and butylated hydroxyanisole, serving as a volatile solvent, nonvolatile co-solvent, spreading agent, and antioxidant, respectively. The ideal topical BIM formulation (BIM–TF#5) exhibited 4.60-fold higher human skin flux and a 529% increase in dermal drug deposition compared to BIM in ethanol. In addition, compared to the other formulations, BIM–TF#5 maximally activated human dermal papilla cell proliferation at a concentration of 5 μM BIM, equivalent to 10 μM minoxidil. Moreover, BIM–TF#5 (0.3% [w/w] BIM) significantly promoted hair regrowth in the androgenic alopecia mouse model and increased the area covered by hair at 10 days by 585% compared to the vehicle-treated mice, indicating that entire telogen area transitioned into the anagen phase. Furthermore, at day 14, the hair weight of mice treated with BIM–TF#5 (5% [w/w] BIM) was 8.45- and 1.30-fold greater than in the 5% (w/w) BIM in ethanol and 5% (w/v) minoxidil treated groups, respectively. In the histological examination, the number and diameter of hair follicles in the deep subcutis were significantly increased in the BIM–TF#5 (0.3 or 5% [w/w] BIM)-treated mice compared to the mice treated with vehicle or 5% (w/w) BIM in ethanol. Thus, our findings suggest that BIM–TF#5 is an effective formulation to treat scalp alopecia, as part of a novel therapeutic approach involving direct prostamide F2α receptor-mediated stimulation of dermal papilla cells within hair follicles.     

Topical delivery of l-ascorbic acid spanlastics for stability enhancement and treatment of UVB induced damaged skin

l-Ascorbic acid (LAA) is considered a powerful antioxidant that protects skin from premature aging. Maintaining the stability of vitamin C remains the biggest challenge in cosmeceuticals. Our main aim is the entrapment of high dose of vitamin C in spanlastic vesicles to provide maximum stability and efficacy. LAA-loaded spanlastics were prepared by ethanol injection method and were characterized for entrapment efficiency (EE%), particles size (PS), polydispersity index (PDI), zeta potential, deformability index (DI) and in vivo skin permeation. Selected spanlastics formula composed of span 60 and tween 60 (5:1) showed highest EE% of 89.77 ± 3.61% (w/w), high deformability of 11.13 ± 1.145 as well as good physical and chemical stability for 6 months. Improved drug penetration into stratum corneum (SC) was obtained from spanlastics compared to topical LAA solution. Quantitative real time PCR revealed that MMP2 and MMP9 levels were significantly suppressed in response to LAA spanlastics treated rats by 30.4% and 65.3%, respectively, when compared to the control group after exposure to UV irradiation. Results were confirmed by western blot analysis. Histopathological study of rat skin after UV irradiation revealed that application of LAA-loaded spanlastics provided the highest skin protection compared to UVB and LAA solution treated group which was evident by the normal thick epidermal morphology and the densely arranged dermal collagen fibers. LAA-loaded spanlastics successfully improved LAA stability, skin permeation and antioxidant protection against skin photodamage.     

Formulation,optimization, and nephrotoxicity evaluation of an antifungal in situ nasal gel loaded with voriconazole‒clove oil transferosomal nanoparticles

Fungal infections of the paranasal cavity are among the most widely spread illnesses nowadays. The aim of the current study was to estimate the effectiveness of an in situ gel loaded with voriconazole‒clove oil nano-transferosomes (VRC-CO-NT) in enhancing the activity of voriconazole against Aspergillus flavus, which causes rhinosinusitis. The nephrotoxic side effects of voriconazole may be reduced through the incorporation of the clove oil, which has antioxidant activity that protects tissue. The Box‒Behnken design was applied to formulate the VRC-CO-NT. The particle size, entrapment efficiency, antifungal inhibition zone, and serum creatinine concentration were considered dependent variables, and the soybean lecithin, VRC, and CO concentrations were considered independent ones. The final optimized formulation was loaded into a deacetylated gellan gum base and evaluated for its gelation, rheological properties, drug release profile, permeation capabilities, and in vivo nephrotoxicity. The optimum formulation was determined to be composed of 50 mg/mL lecithin, 18 mg/mL VRC, and 75 mg/mL CO, with a minimum particle size of 102.96 nm, an entrapment efficiency of 71.70%, an inhibition zone of 21.76 mm, and a serum creatinine level of 0.119 mmol/L. The optimized loaded in situ gel released 82.5% VRC after 12 hours and resulted in a 5.4-fold increase in drug permeation. The in vivo results obtained using rabbits resulted in a nonsignificant differentiation among the renal function parameters compared with the negative control group. In conclusion, nasal in situ gel loaded with VRC-CO-NT is considered an efficient novel carrier with enhanced antifungal properties with no signs of nephrotoxicity.     

Lipid-based nano-formulation platform for eplerenone oral delivery as a potential treatment of chronic central serous chorioretinopathy: in-vitro optimization and ex-vivo assessment

PurposeEplerenone (EPL) is a selective mineralocorticoid receptor antagonist used for treatment of chronic central serous chorioretinopathy which characterized by accumulation of subretinal fluid causing a localized area of retinal detachment. unfortunately, EPL suffers from poor oral bioavailability due to poor aqueous solubility in addition to high hepatic first pass metabolism.MethodAiming to improve its oral bioavailability, EPL-loaded nanostructured lipid carriers (NLCs) were prepared by the emulsification solvent evaporation method and in-vitro evaluated for particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (EE%). A D-optimal design was used for study the effect of liquid lipid to solid lipid ratio, surfactant type and percentage on PS, PDI, EE%, and for data optimization. The optimized EPL-loaded NLCs system was further evaluated using in-vitro drug release and ex-vivo permeation studies through rabbit intestine in comparison to EPL aqueous suspension. The physicochemical properties of the drug in the optimized system were further examined using FT-IR and X-ray diffraction studies.ResultsThe resultant NLCs showed small PS (100.85–346.60 nm), homogenous distribution (0.173–0.624), negatively charged particles (ZP −20.20 to −36.75 mV), in addition to EE% (34.31–70.64%). The optimized EPL-loaded NLCs system with a desirability value of 0.905 was suggested through the Design expert^® software, containing liquid to solid lipid ratio (2:1) in presence of 0.43%w/v Pluronic^® F127 as a surfactant. The optimized EPL-loaded NLCs system showed a PS of 134 nm and PDI of 0.31, in addition to high EE% (76 ± 6.56%w/w), and ZP (-32.37 mV). The ex-vivo permeation study showed two-fold higher drug permeation through rabbit intestine compared to that from the aqueous drug suspension after 24 h, confirming the ability of optimized EPL-loaded NLCs system as successful oral targeting delivery carrier.ConclusionOur results pave the way for a new oral nanotherapeutic approach toward CSCR treatment. In-vivo study is currently under investigation.     

Statistical optimization of bile salt deployed nanovesicles as a potential platform for oral delivery of piperine: accentuated antiviral and anti-inflammatory activity in MERS-CoV challenged mice

The objective of this paper is to confine piperine, a poor oral bioavailable herbal drug into bile salt based nano vesicles for improving its aqueous solubility, hence, its therapeutic activity. Piperine-loaded bilosomes were fabricated adopting thin film hydration technique according to 3².2¹ full factorial design to investigate the impact of different formulation variables on the characters of bilosomes: entrapment efficiency (EE%), particle size, and % of drug released post 8 h (Q8hr). The selected optimum formula was F2 (enclosing 1% bile salt, brij72 as a surfactant, and ratio of surfactant:cholesterol was 9:1) with desirability value 0.801, exhibiting high EE% (97.2 ± 0.8%) nanosized spherical vesicles (220.2 ± 20.5 nm) and Q8hr (88.2%±5.6). The superiority of the optimized formula (F2) over the drug suspension was revealed via ex vivo permeation study, also pharmacokinetic study denoted to the boosted oral bioavailability of piperine-loaded bilosome compared to piperine suspension. Moreover, antiviral activity and safety margin of F2 was significantly higher than that of the drug suspension. The ability of piperine to interact with the key amino acids in the receptor binding domain 4L3N as indicated by its docking configuration, rationalized its observed activity. Furthermore, F2 significantly reduce oxidant markers, inflammatory cytokines in MERS-CoV-infected mice. Hence, bilosomes can be considered as a carrier of choice for piperine with potential antiviral and anti-inflammatory activities.     

Design and optimization of cranberry extract loaded bile salt augmented liposomes for targeting of MCP-1/STAT3/VEGF signaling pathway in DMN-intoxicated liver in rats

Cranberry extract (CBE) is a major source of the antioxidant polyphenolics but suffers from limited bioavailability. The goal of this research was to encapsulate the nutraceutical (CBE), into bile salt augmented liposomes (BSALs) as a promising oral delivery system to potentiate its hepatoprotective impact against dimethylnitrosamine (DMN) induced liver injury in rats. The inclusion of bile salt in the liposomal structure can enhance their stability within the gastrointestinal tract and promote CBE permeability. CBE loaded BSALs formulations were fabricated utilizing a (2³) factorial design to explore the impact of phospholipid type (X₁), phospholipid amount (X₂), and sodium glycocholate (SGC) amount (X₃) on BSALs properties, namely; entrapment efficiency percent, (EE%); vesicle size, (VS); polydispersity index; (PDI); zeta potential, (ZP); and release efficiency percent, (RE%). The optimum formulation (F1) exhibited spherical vesicles with EE% of 71.27 ± 0.32%, VS; 148.60 ± 6.46 nm, PDI; 0.38 ± 0.02, ZP; −18.27 ± 0.67 mV and RE%; 61.96 ± 1.07%. Compared to CBE solution, F1 had attenuated DMN-induced hepatic injury, as evidenced by the significant decrease in serum level of ALT, AST, ALP, MDA, and elevation of GSH level, as well as SOD and GPX activities. Furthermore, F1 exhibited an anti-inflammatory character by suppressing TNF-α, MCP-1, and IL-6, as well as downregulation of VEGF-C, STAT-3, and IFN-γ mRNA levels. This study verified that when CBE was integrated into BSALs, F1, its hepatoprotective effect was significantly potentiated to protect the liver against DMN-induced damage. Therefore, F1 could be deliberated as an antioxidant, antiproliferative, and antifibrotic therapy to slow down the progression of hepatic damage.     

Efficacy and safety of nebulized glycopyrrolate for administration using a high efficiency nebulizer in patients with chronic obstructive pulmonary disease

AimsTo establish the dose−response for pharmacodynamics (bronchodilatation), safety and pharmacokinetics for a nebulized formulation of the long acting muscarinic antagonist glycopyrrolate (EP-101) with a high efficiency nebulizer in patients with chronic obstructive pulmonary disease (COPD).MethodsPatients with moderate to severe COPD (GOLD II/III), with reversible lung function, were enrolled into this randomized, double-blind, placebo-controlled, six period crossover study (n = 42). Patients received single doses of EP-101 (12.5–400 μg) and placebo via a high efficiency nebulizer (eFlow® PARI nebulizer), with washout between treatments. Plasma pharmacokinetics were assessed in a subset of patients (n = 11).ResultsAll treatments were well tolerated with similar adverse event rates reported with placebo and at all doses. There were no clinically relevant changes in heart rate, systolic and diastolic blood pressure or in ECG parameters including QT_c interval. Following treatment with EP-101 at all doses there was a rapid bronchodilator response within 5 min. Significant improvements in mean change from baseline FEV₁ at 24 h were reported at doses ≥50 μg compared with placebo, with a clear dose−response relationship. Mean changes in FEV₁ were 0.10 l (95% CI 0.06, 0.14) and 0.12 l (95% CI 0.08, 0.16) for 100 μg and 200 μg, respectively.ConclusionSingle doses of EP-101 ranging from 12.5 μg to 400 μg were well tolerated. EP-101 delivered by high efficiency nebulizer device produced a rapid onset of bronchodilatation with clinically meaningful improvements in lung function maintained over a 24 h period at all doses >50 μg.     

Ethosomal gel for rectal transmucosal delivery of domperidone: design of experiment,in vitro,and in vivo evaluation

Despite high efficiency of domperidone (DOM) in prophylaxis of emesis accompanied with radiotherapy and chemotherapy, it still can bother cancer patients by its powerful side effects and difficulty of its oral administration. The study was designed to develop and optimize DOM loaded ethosomal gel for rectal transmucosal delivery. Ethosomal formulations were prepared using a 2¹, 5¹ full-factorial design where the impact of lecithin concentration and additives were investigated. The optimum ethosomal vesicles were subsequently incorporated in Carbopol gel base where rheological behavior, spreadability, mucoadhesion, and in vivo pharmacokinetic parameters were studied. Based on Design Expert^® software (Stat Ease, Inc., Minneapolis, MN), the optimum formulation illustrated entrapment efficiency of 70.02%±5.52%, and vesicular size of 112 ± 3.3 nm, polydispersity index of 0.32 ± 0.01, zeta potential of −59 ± 0.28 mV, and % drug released after 6 h of 76.30%±2.45%. Moreover, ex vivo permeation through rabbit intestinal mucosa increased four times compared to free DOM suspension. The gel loaded with ethosomes showed excellent mucoadhesion to rectal mucosa. DOM ethosomal gel showed a raise in C_max and AUC_0–48 of DOM by twofolds compared to free DOM gel. The study suggested that ethosomes incorporated in gels could be an efficient candidate for rectal transmucosal delivery of DOM.     

Pectin Micro- and Nano-capsules of Retinyl Palmitate as Cosmeceutical Carriers for Stabilized Skin Transport

Retinyl palmitate (RP)-loaded pectinate micro- and nano-particles (PMP and PNP) were designed for stabilization of RP that is widely used as an anti-wrinkle agent in anti-aging cosmeceuticals. PMP/PNP were prepared with an ionotropic gelation method, and anti-oxidative activity of the particles was measured with a DPPH assay. The stability of RP in the particles along with pectin gel and ethanolic solution was then evaluated. In vitro release and skin permeation studies were performed using Franz diffusion cells. Distribution of RP in each skin tissue (stratum corneum, epidermis, and dermis) was also determined. PMP and PNP could be prepared with mean particle size diameters of 593~843 μm (PMP) and 530 nm (i.e., 0.53 μm, PNP). Anti-oxidative activity of PNP was greater than PMP due largely to larger surface area available for PNP. The stability of RP in PMP and PNP was similar but much greater than RP in pectin bulk gels and ethanolic solution. PMP and PNP showed the abilities to constantly release RP and it could be permeated across the model artificial membrane and rat whole skin. RP was serially deposited throughout the skin layers. This study implies RP loaded PMP and PNP are expected to be advantageous for improved anti-wrinkle effects.     

Role of ATP in fast excitatory synaptic potentials in locus coeruleus neurones of the rat

1. Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). The pressure application of α,β-methylene ATP (α,β-meATP) caused reproducible depolarizations which were depressed by suramin (30 μM) and abolished by suramin (100 μM). Pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10, 30 μM) also concentration-dependently inhibited the α,β-meATP-induced depolarization, although with a much slower time-course than suramin. Almost complete inhibition developed with 30 μM PPADS. Reactive blue 2 (30 μM) did not alter the effect of α,β-meATP, while reactive blue 2 (100 μM) slightly depressed it.
2. Pressure-applied (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also depolarized LC neurones. Kynurenic acid (500 μM) depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM) abolished the response to AMPA. Suramin (100 μM) potentiated the AMPA effect.
3. Pressure-applied noradrenaline hyperpolarized LC neurones. Suramin (100 μM) did not alter the effect of noradrenaline.
4. Focal electrical stimulation evoked biphasic synaptic potentials consisting of a fast depolarization (p.s.p.) followed by a slow hyperpolarization (i.p.s.p.). A mixture of D(−)-2-amino-5-phosphonopentanoic acid (AP-5; 50 μM), CNQX (50 μM) and picrotoxin (100 μM) depressed both the p.s.p. and the i.p.s.p. Under these conditions suramin (100 μM) markedly inhibited the p.s.p., but did not alter the i.p.s.p. In the combined presence of AP-5 (50 μM), CNQX (50 μM), picrotoxin (100 μM), strychnine (0.1 μM), tropisetron (0.5 μM) and hexamethonium (100 μM), a high concentration of suramin (300 μM) almost abolished the p.s.p. without changing the i.p.s.p.
5. In the presence of kynurenic acid (500 μM) and picrotoxin (100 μM), PPADS (30 μM) depressed the p.s.p. Moreover, the application of suramin (100 μM) to the PPADS (30 μM)-containing medium failed to cause any further inhibition. Neither PPADS (30 μM) nor suramin (100 μM) altered the i.p.s.p.
6. It was concluded that the cell somata of LC neurones are endowed with excitatory P2-purinoceptors. ATP may be released either as the sole transmitter from purinergic neurones terminating at the LC or as a co-transmitter of noradrenaline from recurrent axon collaterals or dendrites of the LC neurones themselves.

     

Spironolactone hyaluronic acid enriched cerosomes (HAECs) for topical management of hirsutism: in silico studies,statistical optimization,ex vivo,and in vivo studies

Spironolactone (SP) is a potassium sparing diuretic with antiandrogenic properties. This study aimed at formulating SP into hyaluronic acid enriched cerosomes (HAECs) for topical management of hirsutism. HAECs were prepared by ethanol injection method, according to D-optimal design, after a proper in silico study. HAECs were evaluated by measuring their entrapment efficiency (EE%), particle size (PS), and polydispersity index (PDI). Optimal hyaluronic acid enriched cerosomes (OHAECs) were subjected to further in vitro and ex-vivo and in-vivo studies. The in silico study concluded better interactions between SP and phosphatidyl choline in presence of hyaluronic acid (HA) and high stability of their binding in water. The prepared HAECs had acceptable EE%, PS, and PDI values. The statistical optimization process suggested OHAEC containing 10.5 mg ceramide III and 15 mg HA, utilizing Kolliphor^® RH40. OHAEC had EE% and PS of 89.3 ± 0.3% and 261.8 ± 7.0 nm, respectively. OHAEC was stable for up to 3 months. It also showed a mixed tubular and vesicular appearance under transmission electron microscope. The ex vivo and in vivo studies concluded better skin deposition and accumulation of SP from OHAEC. The histopathological study demonstrated the safety of OHAEC for topical application. Therefore, OHAEC could be considered as effective system for topical application of SP to manage hirsutism, with prolonged action, coupled with minimized side effects.     

The role of tachykinin NK1 and NK2 receptors in atropine-resistant colonic propulsion in anaesthetized guinea-pigs

1. The role of endogenous tachykinins on guinea-pig colonic propulsion was investigated by using potent and selective tachykinin NK₁ and NK₂ receptor antagonists. Colonic propulsion and contractions were determined by means of a balloon-catheter device, inserted into the rectum of guanethidine (68 μmol kg⁻¹, s.c., 18 and 2 h before)-pretreated, urethane-anaesthetized guinea-pigs. Propulsion of the device (dynamic model) was determined by measuring the length of the catheter expelled during 60 min filling of the balloon (flow rate 5 μl  min⁻¹).
2. In control conditions the tachykinin NK₁ receptor antagonist SR 140333 (1 μmol kg⁻¹, i.v.) did not affect either colonic propulsion or the amplitude of contractions. The tachykinin NK₂ receptor antagonists MEN 10627 and MEN 11420 (1 μmol kg⁻¹, i.v.) increased colonic propulsion at 10 min (+120% and 150%, respectively) but at 60 min the effect was significant only for MEN 10627 (+84%). SR 48968 (1 μmol kg⁻¹, i.v.) did not significantly enhance the colonic propulsion. None of these tachykinin NK₂ receptor antagonists modified the amplitude of colonic contractions. In contrast, both atropine (6 μmol kg⁻¹, i.v., plus infusion of 1.8 μmol h⁻¹) and hexamethonium (55 μmol kg⁻¹, i.v., plus infusion of 17 μmol h⁻¹) abolished propulsion (81% and 87% inhibition, respectively) and decreased the amplitude of contractions (68% inhibition for either treatment).
3. In atropine-treated animals (6 μmol kg⁻¹, i.v., plus infusion of 1.8 μmol h⁻¹), apamin (30 nmol kg⁻¹, i.v.) restored colonic propulsion (+416%) and increased the amplitude of contractions (+367% as compared to atropine alone). Hexamethonium (55 μmol kg⁻¹, i.v., plus infusion of 17 μmol h⁻¹) abolished the apamin-induced, atropine-resistant colonic propulsion (97% inhibition) and reduced the amplitude of the atropine-resistant contractions (52% inhibition).
4. The apamin-induced, atropine-resistant colonic propulsion was inhibited by SR 140333 (−69% at 1 μmol kg⁻¹), SR 48968 (−78% at 1 μmol kg⁻¹), MEN 11420 (−59% at 1 μmol kg⁻¹) and MEN 10627 (−50% at 1 μmol kg⁻¹), although the latter effect was not statistically significant. The combined administration of SR 140,333 and MEN 10,627 (1 μmol kg⁻¹ for each antagonist) almost completely abolished colonic propulsion (90% inhibition). The amplitude of colonic contractions was also reduced by SR 140333 (−42%), SR 48968 (−29%), MEN 11420 (−45%) but not by MEN 10627 (−16%). The combined administration of SR 140333 and MEN 10,627 reduced the amplitude of contractions by 47%. SR 140603 (1 μmol kg⁻¹, i.v.), the less potent enantiomer of SR 140333, was inactive.
5. In control animals, apamin (30 nmol kg⁻¹, i.v.) enhanced colonic propulsion (+84%) and increased the amplitude of contractions (+68%), as compared to the vehicle. Hexamethonium (55 μmol kg⁻¹, i.v. plus infusion of 17 μmol h⁻¹) inhibited propulsion (86% inhibition) and decreased the amplitude of contractions (49% inhibition). SR 140333, SR 48968, MEN 11420, MEN 10627, or the coadministration of SR 140333 and MEN 10627 had no effect.
6. In a separate series of experiments, the mean amplitude of colonic contractions was also recorded under isovolumetric conditions through the balloon-catheter device kept in place at 75 mm from the anal sphincter (static model). In control conditions, neither SR 140333 nor MEN 11420 modified the amplitude of contractions. In atropine-pretreated guinea-pigs, SR 140333 and MEN 11420 (0.1–1 μmol kg⁻¹) dose-dependently decreased the amplitude of contractions. In apamin- and atropine-pretreated animals, only the highest (1 μmol kg⁻¹) dose of SR 140333 or MEN 11420 significantly decreased the amplitude of contractions. The inhibitory potency of atropine (0.3–1 μmol kg⁻¹) was similar in apamin-pretreated animals and in controls.
7. It was concluded that, in anaesthetized guinea-pigs, endogenous tachykinins, acting through both NK₁ and NK₂ receptors, act as non-cholinergic excitatory neurotransmitters in promoting an apamin-evoked reflex propulsive activity of the distal colon.

     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Synthesis of nanocapsules blended polymeric hydrogel loaded with bupivacaine drug delivery system for local anesthetics and pain management

Local anesthetics are used clinically for the control of postoperative pain management. This study aimed to develop chitosan (CS) with genipin (GP) hydrogels as the hydrophilic lipid shell loaded poly(ε-caprolactone) (PC) nanocapsules as the hydrophobic polymeric core composites (CS-GP/PC) to deliver bupivacaine (BPV) for the prolongation of anesthesia and pain relief. The swelling ratio, in vitro degradation, and rheological properties enhancement of CS-GP/PC polymeric hydrogel. The incorporation of PC nanocapsules into CS-GP hydrogels was confirmed by SEM, FTIR, and XRD analysis. Scanning electron microscopy results demonstrated that the CS-GP hydrogels and CS-GP/PC polymeric hydrogels have a porous structure, the pore dimensions being non-uniform with diameters between 25 and 300 μm. The in vitro drug release profile of CS-GP/PC polymeric hydrogel has been achieved 99.2 ± 1.12% of BPV drug release in 36 h. Cellular viability was evaluated using the CCK-8 test on 3T3 fibroblast cells revealed that the obtained CS-GP/PC polymeric hydrogel with BPV exhibited no obvious cytotoxicity. The CS-GP/PC polymeric hydrogel loaded with BPV showed significant improvement in pain response compared to the control group animals for at least 7 days. When compared with BPV solution, CS-GP hydrogel and CS-GP/PC polymeric hydrogel improved the skin permeation of BPV 3-fold and 5-fold in 24 h, respectively. In vitro and in vivo results pointed out PC nanocapsules loaded CS-GP hydrogel can act as effective drug carriers, thus prolonging and enhancing the anesthetic effect of BPV. Histopathological results demonstrated the excellent biodegradability and biocompatibility of the BPV-loaded CS-GP/PC polymeric hydrogel system on 7, 14, and 21 days without neurotoxicity.

HIGHLIGHTS

• Preparation and characterization of CS-GP/PC polymeric hydrogel system.
• BPV-loaded CS-GP/PC exhibited prolonged in vitro release in PBS solution.
• Cytotoxicity of BPV-loaded CS-GP/PC polymeric hydrogel against fibroblast (3T3) cells.
• Development of CS-GP/PC a promising skin drug-delivery system for local anesthetic BPV.