Limonene, a citrus monoterpene, non-complexed and complexed with hydroxypropyl-ß-cyclodextrin attenuates acute and chronic orofacial nociception in rodents: Evidence for involvement of the PKA and PKC pathway.

BACKGROUND: Chronic orofacial pain is a serious public health problem with a prevalence of 7-11% in the population. This disorder has different etiologies and characteristics that make pharmacological treatment difficult. Natural products have been shown to be a promising source of treatments for the management of chronic pain, as an example the terpenes. PURPOSE: The aim of this study was to evaluate the anti-nociceptive and anti-inflammatory effects of one of these terpenes, d-limonene (LIM - a common monoterpene found in citrus fruits) alone and complexed with hydroxypropyl-ß-cyclodextrin (LIM/HPßCD) in preclinical animal models. METHODS: Orofacial pain was induced by the administration of hypertonic saline on the corneal surface, the injection of formalin into the temporomandibular joint (TMJ), or chronic constriction injury of the infraorbital nerve (CCI-IoN). The study used male Wistar rats and Swiss mice treated with LIM (50 mg/kg), LIM/HPßCD (50 mg/kg), vehicle (control), gabapentin or morphine, and eyes wiping (induced by hypertonic saline), face rubbing (formalin-induced in TMJ) or mechanical hyperalgesia (provoked by CCI-IoN) were assessed. Additionally, ELISA was used to measure TNF-α, and western blot analysis to assess levels of PKAcα, NFκB, p38MAPK and phosphorylated PKC substrates. Serum levels of aspartate aminotransferase (AST) and alanine transferase (ALT) were also evaluated. RESULTS: LIM and LIM/HPßCD significantly reduced (p < 0.001) corneal nociception and formalin-induced TMJ nociception. In addition, both substances attenuated (p < 0.001) mechanical hyperalgesia in the CCI-IoN model. The antinociceptive effect induced by LIM and HPßCD/LIM was associated with decreased TNF-α levels, downregulation of the NFκB and p38MAPK signalling pathways and reduced PKC substrate phosphorylation and PKA immunocontent. Moreover, the results demonstrated that complexation with HPßCD was able to decrease the therapeutic dose of LIM. CONCLUSION: LIM was found to be a promising molecule for the treatment of orofacial pain due to its capacity to modulate some important mediators essential to the establishment of pain, and HPßCD can be a key tool to improve the profile of LIM.

Physicochemical characterization and cytotoxicity of articaine-2-hydroxypropyl-ß-cyclodextrin inclusion complex.

Articaine (ATC) is one of the most widely used local anesthetics in dentistry. Despite its safety, local toxicity has been reported. This study aimed to develop an ATC-2- hydroxypropyl-ß-cyclodextrin inclusion complex (ATC HPßCD) and to assess its toxicity in vitro. The inclusion complex was performed by solubilization, followed by a fluorimetric and job plot assay to determine the complex stoichiometry. Scanning electron microscopy, DOSY- 1 H-NMR, differential scanning calorimetry (DSC), and sustained release kinetics were used to confirm the inclusion complex formation. In vitro cytotoxicity was analyzed by MTT assay and immunofluorescence in HGF cells. Fluorimetric and job plot assay determined the inclusion complex stoichiometry (ATC:HPßCD = 1:1) and complex formation time (400 min), as indicated by a strong host/guest interaction (Ka = 117.8 M - 1), complexed fraction (f = 41.4%), and different ATC and ATC HPßCD melting points (172 °C e 235 °C, respectively). The mean of cell viability was 31.87% and 63.17% for 20-mM ATC and 20-mM ATC HPßCD, respectively. Moreover, remarkable cell toxicity was observed with free ATC by immunofluorescence. These results indicate the ATC HPßCD complex could be used to improve the safety of ATC. Further research are needed to establish the anesthetic safety and effectiveness in vivo .

Interaction with Modified Cyclodextrin as a Way to Increase the Antimalarial Activity of Primaquine.

BACKGROUND: Malaria is still a dangerous disease that impacts specifically Africa, Asia, and Latin America. The development of therapies to overcome the parasite infection is an important challenge nowadays. The medicine primaquine (PQ) is used in the treatment, although several side effects and low oral bioavailability are reported. OBJECTIVE: This work focused on the preparation and characterization of a complex between PQ and 2- hydroxypropyl-ß-cyclodextrin (HPCD), besides performing release tests of this formulation. METHODS: PQ:HPCD complexes were prepared at 1:1 and 1:2 molar ratios, by the lyophilization method. The association between PQ and HPCD was tested using UV-vis, infrared (FTIR), differential scanning calorimetry (DSC), scanning electron microscopy and NMR techniques (chemical shift, Job Plot, DOSY, and ROESY). Tests were also conducted to evaluate drug release before and after complexation with HPCD. RESULTS: Results showed that there was a weak interaction of PQ with HPCD, forming non-inclusion complexes. These results were supported by FTIR results and spatial correlations between hydrogens from PQ with the external HPCD hydrogens. A 1:2 PQ:HPCD preferred molar ratio was determined by DSC and Job Plot experiments and the time to release 96% of the drug was 21.2 h slower after complexation. CONCLUSION: Conclusion indicate that PQ interacts poorly with HPCD, probably due to its hydrophilic character, as well as to its interaction with the external rim of HPCD. Our results demonstrate that there was a significant improvement in the release time after the complexation process, which could lead to an increase in the activity of the drug.

Lipid nanoparticles as carriers of cyclodextrin inclusion complexes: A promising approach for cutaneous delivery of a volatile essential oil.

Solid inclusion complexes with cyclodextrins (CD) may be used to overcome volatility and solubility problems of essential oils of pharmacological interest. However, they lack the many dermatological advantages of lipid nanoparticles. This study intends to evaluate the ability of nanostructured lipid carriers (NLC) to encapsulate hydroxypropyl-ß-cyclodextrin inclusion complexes of Lippia origanoides essential oil (EO) and to maintain the desirable aspects of lipid colloids interaction with the skin, specifically follicular accumulation and controlled delivery. CD and NLC were also evaluated separately. Thymol (TML) was used as the essential oil marker and to produce control formulations. As expected, CD alone, though effective in overcoming volatility and low aqueous solubility of TML, were ineffective in controlling marker release (˜50% of EO released after 3 h, Hixson-Crowell kinetics). Even though NLC controlled drug release (˜20% EO released after 12 h, zero-order kinetics) enabling TML penetration into the skin (> 40 µg/cm2after 12 h), NLC alone were not efficient in preventing TML volatility, especially at higher temperatures (calculated shelf-life of 2 days at 35 °C). The combined approach resulted in a synergistic effect (˜20% EO released after 12 h; shelf life of 6 days). The lack of statistical difference of TML skin penetration from NLC and NLC-CD suggests the developed system maintained all skin interaction aspects of lipid colloids, including follicular accumulation forming a depot for controlled delivery. In conclusion, lipid nanoparticles demonstrated to be promising carriers for inclusion complexes of this particular volatile essential oil.

Development of morin/hydroxypropyl-ß-cyclodextrin inclusion complex: Enhancement of bioavailability, antihyperalgesic and anti-inflammatory effects.

Morin is a flavonoid has been reported with several pharmacological effects such as, antioxidant, anti-inflammatory, anticancer, antidiabetic, etc. However, morin has low solubility in water, which decreases the bioavailability and limits its clinical application. In this way, to improve the pharmaceutical properties, morin was complexed in hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and its oral bioavailability and anti-inflammatory effects were evaluated. Initially, a phase solubility study was performed, which showed that HP-ß-CD would be the better cyclodextrin for the formation of complexes with morin. The morin/HP-ß-CD inclusion complex (1:1) was prepared by freeze-drying method. The sample obtained was characterized by DSC, FTIR, PXRD, SEM and 1H NMR techniques, evidencing the formation of morin/HP-ß-CD inclusion complex. In addition, complexation efficiency (98.3%) and loading content (17.63%), determined by HPLC demonstrated that morin was efficiently complexed in HP-ß-CD. In vitro dissolution study confirmed that morin/HP-ß-CD inclusion complex increased the solubility and dissolution rate of morin. The oral bioavailability of the morin/HP-ß-CD complex and free morin were evaluated through a pharmacokinetic study in rat plasma. The oral bioavailability of morin complexed with HP-ß-CD was increased by 4.20 times compared with the free morin. Hyperalgesia induced by carrageenan and carrageenan-induced pleurisy were carried out in mice to evaluate the antihyperalgesic and anti-inflammatory activities of free morin and inclusion complex. Morin/HP-ß-CD inclusion complex showed antihyperalgesic effect in inflammatory pain model and anti-inflammatory effect decreasing leukocyte migration and TNF-α levels at a lower dose than free morin. Therefore, the morin/HP-ß-CD inclusion complex improved the solubility, dissolution rate, oral bioavailability, antihyperalgesic and anti-inflammatory effects of morin. In this way, the morin/HP-ß-CD inclusion complex exhibits potential for development of new pharmaceutical product for future clinical applications.

Binary and ternary complexes of norfloxacin to improve the solubility of the active pharmaceutical ingredient.

AIM: Binary and ternary complexes with hydroxypropyl-ß-cyclodextrin (HPßCD), using glutamic acid (GA), proline or lysine as the third component, were developed to increase the solubility and the dissolution rate of norfloxacin (NOR). METHODS/RESULTS: Complexation was evaluated by phase solubility studies, obtaining the highest NOR solubility with GA and HPßCD. Thermal analysis suggested that different kinds of interactions occur among NOR, HPßCD and each amino acid, and when the systems were prepared by kneading or by means of freeze-drying technique. Dissolution studies, performed on simulated gastric fluid and subsequent simulated intestinal fluid, showed the highest rate of NOR from NOR-HPßCD-GA. CONCLUSION: NOR:HPßCD:GA was the best approach for improving the bioavailability of NOR.

Development of a new formulation of roflumilast for pulmonary drug delivery to treat inflammatory lung conditions.

The aim of this study was to develop roflumilast dry powder inhaler (DPI) formulations by spray drying using hydroxypropyl-ß-cyclodextrin (HPßCD) and to determine their suitability for pulmonary delivery. Different feed solution concentrations, solvent systems and spray drying parameters were used to obtain the formulations which were characterized using X-ray powder diffraction, thermal analysis, scanning electron microscopy, particle size distribution, bulk and tapped density, specific surface area, dynamic vapour sorption, in vitro deposition properties using a Next Generation Impactor (NGI) and transepithelial permeability. Microparticles spray dried from ethanol were wrinkled and amorphous, exhibiting high glass transition temperatures while those from methanol:n-butyl acetate consisted of irregularly shaped porous particles partially crystalline. All formulations presented low density, particle size and residual solvent content exhibiting high depositon in the lower stages of the NGI. Mass median aerodynamic diameters (MMADs) were in the range of 3.32-4.49 µm, with high fine particle fractions (FPF < 5 µm). Stability studies demonstrated no significant modifications in the solid-state nature and in the aerolisation performance of the selected formulation which presented a Papp of 8.73 × 10-6 ±â€¯4.70 × 10-7 cm/s. The developed roflumilast DPI formulations have potential therapeutic applications in the treatment of lung diseases.

Coumestrol/hydroxypropyl-ß-cyclodextrin association incorporated in hydroxypropyl methylcellulose hydrogel exhibits wound healing effect: in vitro and in vivo study.

Several beneficial effects on the skin have been reported for coumestrol (COU), such as protection against photoaging and improvement of skin elasticity and thickness in postmenopausal women. However no reports on the effect of COU on wound healing were found. Nevertheless, COU has low aqueous solubility, which is a crucial limitation for biological tests. The present study was designed as a two-step experiment to evaluate the wound healing effect of COU. First, we used fibroblasts and the experimental in vitro artificial wound model, scratch assay, to compare the effects of COU free, dissolved in dimethyl sulfoxide (DMSO) or Dulbecco's modified Eagle's medium (DMEM), or associated with hydroxypropyl-ß-cyclodextrin (HPßCD). The 50 µM (66.1%) and 10 µM (56.3%) COU/HPßCD association induced cell proliferation and migration in inflicted wounds. Subsequently, the in vivo wound healing experimental model (Wistar rats) revealed that COU/HPßCD incorporated into hypromellose (HPMC) hydrogel had similar efficacy in wound healing in comparison to the positive control (Dersani®), with the advantage that 50% wound healing was achieved within a shorter period. In summary, the results successfully demonstrated, for the first time, the wound healing effect of COU/HPßCD incorporated into HPMC hydrogel and describe the feasibility of the biological tests with the use of HPßCD instead DMSO.

Development of carvedilol-cyclodextrin inclusion complexes using fluid-bed granulation: a novel solid-state complexation alternative with technological advantages.

OBJECTIVES: This study sought to evaluate the achievement of carvedilol (CARV) inclusion complexes with modified cyclodextrins (HPßCD and HPγCD) using fluid-bed granulation (FB). METHODS: The solid complexes were produced using FB and spray drying (SD) and were characterised by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction, SEM, flowability and particle size analyses and in vitro dissolution. KEY FINDINGS: The DSC, FTIR and powder X-ray diffraction findings suggested successful CARV inclusion in the modified ß- and γ-cyclodextrins, which was more evident in acidic media. The CARV dissolution rate was ~7-fold higher for complexes with both cyclodextrins prepared using SD than for raw CARV. Complexes prepared with HPßCD using FB also resulted in a significant improvement in dissolution rate (~5-fold) and presented superior flowability and larger particle size. CONCLUSIONS: The findings suggested that FB is the best alternative for large-scale production of solid dosage forms containing CARV. Additionally, the results suggest that HPγCD could be considered as another option for CARV complexation because of its excellent performance in inclusion complex formation in the solid state.

Characterization, dissolution and in vivo evaluation of solid acetazolamide complexes.

The effects of binary and ternary systems of acetazolamide (ACZ) with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) alone or with triethanolamine (TEA) on the crystalline properties, dissolution and intraocular pressure (IOP)-lowering effect were investigated. It was found that the crystal structure of ACZ powder could be modified by the processing conditions. Freeze-drying ACZ powder affected not only the particle morphology but also its polymorphic form and the starting ACZ was converted to pure form A upon freeze-drying treatment. Results provided by DSC/TGA, XRPD, SEM and FT-IR suggested the formation of inclusion complexes between ACZ with HP-ß-CD alone or with TEA, obtained by the freeze-drying method and the conversion of the drug into the amorphous state. Binary and ternary systems of ACZ obtained by freeze-drying exhibited significantly enhanced ACZ dissolution rates. The IOP-lowering effects of ACZ and its complexes with HP-ß-CD alone or with TEA were studied in normotensive rabbits. Whereas the maximum IOP-lowering effect (~4 mmHg, ~33%), obtained with these binary and ternary lyophilized ACZ systems occurred at around 90 min, the ternary system exhibited a longer maximum IOP-lowering effect peak compared with that of the binary system. These results are in line with those obtained from the dissolution studies, where the ternary system exhibited longer dissolution times compared to the lyophilized binary one. Results obtained from the dissolution studies, also showed that freeze-drying the native crystalline form of ACZ significantly increased the dissolution rate of ACZ, thus improving the IOP-lowering effect of this drug.