Dissolving microneedle patches-mediated percutaneous delivery of tetramethylpyrazine for rheumatoid arthritis treatment.

Recently, transdermal treatment of rheumatoid arthritis (RA) has received increasing attention due to the advantages of improving patient compliance and avoiding gastrointestinal side effects. However, the stratum corneum (SC) barrier limits the transdermal delivery of most substances. Therefore, we constructed tetramethylpyrazine-loaded dissolving microneedle patches (TMP-DMNPs) and investigated its anti-rheumatoid arthritis effect. The cone-shaped dissolving microneedle patch had complete, neatly arranged needles and great mechanical strength. It could effectively penetrate the stratum corneum when applied to the skin. In vitro transdermal experiment showed that DMNPs could significantly promote the transdermal penetration of TMP compared with TMP-cream. The needles were completely dissolved within 18 min and the applied skin recovered completely within 3 h. The excipients and blank DMNP had good safety and biocompatibility to human rheumatoid arthritis fibroblast synovial cells. To compare the therapeutic effects, the animal model was established. The experiments of paw swelling, histopathology and X-ray examination showed that dissolving microneedles significantly alleviated paw condition, reduced the serum concentrations of proinflammatory cytokines, and inhibited synovial tissue damage in AIA rats. These results indicate that the DMNPs we prepared can deliver TMP safely, effectively and conveniently, providing a basis for the percutaneous treatment of RA.

Dissolving microneedle patch-assisted transdermal delivery of methotrexate improve the therapeutic efficacy of rheumatoid arthritis.

Methotrexate (MTX) is a first-line treatment for rheumatoid arthritis (RA), but its clinical use is greatly limited by the adverse effects and poor patient compliance caused by traditional oral administration or injection. In recent years, some transdermal drug delivery systems have received considerable attention due to overcoming these shortcomings. In this study, we developed dissolving microneedle patch (DMNP) for transdermal delivery of MTX to treat RA safely and effectively. The morphology, mechanical strength, skin insertion, drug content, in vitro transdermal delivery, and other properties of DMNP were characterized. Meanwhile, the adjuvant-induced arthritis model of rats was established to investigate the therapeutic effect of MTX-loaded DMNP in vivo. The results showed that the microneedles had excellent morphology with neat array and complete needles, good puncture performance and mechanical strength, and rapid intradermal dissolution rate. In vitro transdermal delivery results indicated that microneedles could significantly increase drug transdermal permeation compared with the cream group. The pharmacological study showed that MTX-loaded DMNP significantly alleviated paw swelling, inhibit inflammatory response via downregulating the levels of TNF-α and IL-1ß, relieved synovium destruction with less cartilage erosion, and slowed the progression of RA in AIA rats. Besides, DMNP presented better therapeutic performance than cream or intragastric administration at the same dosage of MTX. In conclusion, the MTX-loaded dissolving microneedle patch has advantages of safety, convenience, and high efficacy over conventional administrations, laying a foundation for the transdermal drug delivery system treatment of rheumatoid arthritis.

High Efficacy Combined Microneedles Array with Methotrexate Nanocrystals for Effective Anti-Rheumatoid Arthritis.

Introduction: Methotrexate (MTX) is the first-line drug for the treatment of rheumatoid arthritis (RA) in several countries. However, MTX has an extremely low solubility in water, and the side effects caused by its delivery mode restrict its curative effect. In this study, we designed a dissolving microneedles array (DMNA) containing MTX nanocrystals (MTX-NCs) (MTX-NC@DMNA) to improve the treatment of RA. DMNA-based drug delivery combines the advantages of patient compliance with the use of transdermal drug delivery systems and high-efficiency injection administration; thus, it can mitigate the side effects that result from current administration routes. Carrier-free and surfactant-free MTX-NCs were prepared to overcome bioavailability limitations and poor drug loading problems. Methods: The MTX-NCs prepared by reverse solvent precipitation method was encapsulated in the DMNA. The morphology, mechanical properties, safety, stability and in vivo dissolution were evaluated, and its pharmacodynamic characteristics were assessed in a rat model of RA. Results: The particle size of the MTX-NCs was 148.1 ± 10.1 nm. The MTX-NC@DMNA were found to be rigid enough to penetrate the skin and deliver the drug successfully. The results indicated effective skin recovery after removal of the DMNA. It was found that the MTX-NC@DMNA significantly reduced foot swelling in the rats and regulated the balance in the levels of related cytokines. It also reduced pathological damage to the synovium, joint, and cartilage, and effectively alleviated organ injury in the rats. Conclusion: Transdermal administration of MTX-NC@DMNA may be an effective approach for treating RA.

Combination and efficiency: preparation of dissolving microneedles array loaded with two active ingredients and its anti-pigmentation effects on guinea pigs.

Hyperpigmentation is a common skin disorder caused by excessive melanogenesis and uneven dispersion of melanin in the skin. To combine multiple active agents with an efficient transdermal drug delivery system is an effective strategy to combat UV induced skin pigmentation. In this work, Arbutin (Arb) and Vitamin C (Vc) mixed in 1:1 were found to have the greatest inhibition effects on melanogenesis and tyrosinase activity in B16 murine melanoma cells. And hyaluronic acid (HA) based dissolving microneedles array (DMNA) was employed to overcome the skin barriers for improved topical drug delivery, which exhibited the most desirable features, including morphology, mechanical properties, dissolving ability, and the highest drug loading. Furthermore, DMNA could greatly increase the stability of Vc during storage without adding any antioxidant which is an important issue for Vc administration. Pharmacodynamics study showed that DMNA loaded with Arb and Vc could synergistically suppress UVB-induced hyperpigmentation in guinea pig skin. This work provides a promising treatment strategy and solution for skin pigmentation and other skin problems.

Transdermal Delivery of Salmon Calcitonin Using a Dissolving Microneedle Array: Characterization, Stability, and In vivo Pharmacodynamics.

Salmon calcitonin (sCT) is a polypeptide drug, possessing the ability to inhibit osteoclast-mediated bone resorption. Just like other bioactive macromolecules, sCT is generally administered to the patients by either injection for poor compliance or through nasal spray for low bioavailability, which limits its use as therapeutic drugs. In the present study, to overcome the limitations of the conventional routes, two new dissolving microneedle arrays (DMNAs) based on transdermal sCT delivery systems were developed, namely sCT-DMNA-1 (sCT/Dex/K90E) and sCT-DMNA-2 (sCT/Dex-Tre/K90E) with the same dimension, meeting the requirements of suitable mechanical properties. An accurate and reliable method was established to determine the needle drug loading proportion in sCT-DMNAs. The stability study exhibited that the addition of trehalose could improve the stability of sCT in DMNA under high temperature and humidity. Further, in vivo pharmacodynamic study revealed that DMNA patch could significantly enhanced relative bioavailability to approximately 70%, and the addition of trehalose was found to be beneficial for sCT transdermal delivery. Therefore, sCT-DMNA is expected to replace traditional dosage form, providing a secure, efficient, and low-pain therapeutic strategy for bone disorders.

Development and evaluation of new methods for protein quantification in dissolving microneedles formulations.

Dissolving microneedles (DMNs) are vital for the transdermal delivery of protein-based drugs due to their painless penetration, effective drug permeation, and safety characteristics. Thus, it is necessary to quantify the proteins encapsulated inside the DMNs. The classic BCA(c-BCA) assay is commonly employed for protein quantification. However, the presence of dextran (Dex, an excipient of DMNs) interferes with the quantification of ovalbumin (OVA) by the c-BCA assay, leading to erroneous results. Therefore, two new techniques, including modified BCA (m-BCA) and reversed-phase HPLC (RP-HPLC) were developed to accurately determine the protein concentrations in pharmaceutical formulations, compared with the c-BCA assay. The results showed that the m-BCA and RP-HPLC methods were reliable for protein quantification in DMNs, with linear responses for correlation coefficients of >0.99; the acceptable added samples recoveries of 100 ± 10 %; and the inter-day precisions for a relative standard deviation of <10 %. Herein, we describe these two methods for the rapid quantification of protein loaded in DMNs.

Dissolving Microneedle Arrays with Optimized Needle Geometry for Transcutaneous Immunization.

In this study, the feasibility of transcutaneous immunization using different needle-geometries dissolving microneedle array (DMNA) were investigated as drug carriers for ovalbumin (OVA) preparations. A two-step molding process was used in which needles were loaded with OVA. The microneedles displayed a geometry and dimensions consistent with the main molds. DMNA with different needle-geometries were compared and characterized. Drug loading of the prepared DMNAs reached ~100 µg measured via BCA assay. The stability of OVA in the DMNAs was investigated by SDS-PAGE electrophoresis and showed that the OVA encapsulated in the DMNAs was stable during preparation. The immune responses induced by the DMNAs and hypodermic needle-based injections were compared through in vivo immunoglobulin G (IgG) production assays. OVA-loaded DMNAs also induced stronger immune responses compared to hypodermic needle-based injections. In conclusion, these results suggest that: (1) the needle-morphology of DMNAs influences their mechanical properties, insertion capacity, and dissolution, thus affecting the immune response; (2) Cone-DMNAs are optimal for transcutaneous immunization. These data provide a theoretical basis for the use of transcutaneous immunization of DMNAs for vaccine development.

Dual boronate affinity nanoparticles-based plasmonic immunosandwich assay for specific and sensitive detection of ginsenosides.

Ginsenoside is a large family of triterpenoid saponins from Panax ginseng with various important biological functions. It is crucial to develop effective analytical approach for qualitative and quantitative analysis of ginsenosides. Herein, a dual boronate affinity nanoparticles-based plasmonic immunosandwich assay has been developed for analysis of ginsenosides. An imprinted Au NPs-coated glass slide was prepared via controllable oriented surface imprinting and used as specific extraction substrate for target molecules. In the meantime, Ag-cored Raman nanotags were used for specific labeling of target molecules. The MIP-based recognitions ensured the specificity of the assay, while enhanced Raman signal derived from the imprinted substrate-target-nanotags sandwich-like complexes provided high sensitivity. The proposed immunosandwich assay exhibited wide linear range (10 ng/mL to 10 µg/mL), high sensitive (LOD: 1.7 ng/mL, LOQ: 5 ng/mL) and good reproducibility (RSD: 8.6%). For real-world applications, successful quantitative analysis of ginsenoside Re in ginseng was performed. Therefore, this dual boronate affinity nanoparticles-based plasmonic immunosandwich assay holds great promise in many important applications such as pharmaceutical analysis.

Preparation of salbutamol imprinted magnetic nanoparticles via boronate affinity oriented surface imprinting for the selective analysis of trace salbutamol residues.

Salbutamol (SAL) is one of the most widely abused feed additives in animal husbandry. Selective enrichment of SAL from complex biological samples is a key step for the analysis of SAL. Herein, we present SAL-imprinted magnetic nanoparticles (MNPs) as an ideal sorbent for selective enrichment of SAL. The SAL-imprinted MNPs were prepared according to the newly reported boronate affinity oriented surface imprinting. The prepared imprinted MNPs exhibited several significant advantages, such as excellent selectivity towards SAL, superb tolerance to interferences, fast binding equilibrium and easy manipulation, which made the obtained imprinted MNPs an ideal sorbent for selective enrichment. The feasibility of SAL-imprinted MNPs for real samples was well demonstrated by the analysis of trace SAL in pig liver. Because of their highly desirable properties, the SAL-imprinted MNPs could find more applications in the analysis of SAL.

The Herb-Drug Interaction of Clopidogrel and Xuesaitong Dispersible Tablet by Modulation of the Pharmacodynamics and Liver Carboxylesterase 1A Metabolism.

OBJECTIVE: Clopidogrel and Xuesaitong dispersible tablet (XST) have been clinically proven to be effective for treating cardiocerebrovascular disease. The present study was to investigate the herb-drug interaction of Clopidogrel and XST by modulation of the pharmacodynamics and liver Carboxylesterase 1A(CES1A) metabolism. METHODS: 30 male SD rats were randomly divided into a control group (equal volumes of saline, 6 rats for mRNA analysis), a clopidogrel group (clopidogrel with dose 30 mg/kg), and a combination group (clopidogrel and XST, with dose 30 and 50 mg/kg respectively, each group continuous administration once daily for 30 days). The clopidogrel and combination group comprised 12 rats, with 6 designated for mRNA analysis and 6 for the pharmacokinetic study. The 2-bromo-3'-methoxyacetophenone- (MPB-) derivatized clopidogrel active thiol metabolite (CAMD) was measured by UHPLC-MS/MS for pharmacokinetics (n=6). The expression of CES1A mRNA was examined with real-time RT-PCR (n=6). Molecular simulation was used to investigate the inhibition effect of XST on the CES1A protein. The CAMD pharmacodynamics and CES1A metabolism were investigated to evaluated the herb-drug interaction. RESULTS: Clopidogrel and XST coadministration appreciably increased the Cmax, AUC, and MRT of CAMD. However, the expression of CES1A mRNA was decreased accordingly. It also indicated that the bioactive components in XST had good interaction with the CES1A metabolism target by molecular simulation. The animal study indicated that clopidogrel and XST coadministration produced significant herb-drug interactions at active CAMD pharmacokinetic and CES1A metabolic enzyme aspect. CONCLUSION: 30-days dose of coadministration altered hepatic CES1A protein and resulted in reduced plasma levels of active CAMD. both the decreased CES1A mRNA expression and the inhibition on the protein were due to the combination of XST, which accordingly upregulated the pharmacokinetics of plasma active CAMD.

Laminaria japonica increases plasma exposure of glycyrrhetinic acid following oral administration of Liquorice extract in rats.

The present study was designed to investigate the effects of Laminaria japonica (Laminaria) on pharmacokinetics of glycyrrhetinic acid (GA) following oral administration of Liquorice extract in rats. Following oral administrations of single-dose and multi-dose Liquorice extract and Liquorice-Laminaria extract, respectively, plasma samples were obtained at various times and the concentrations of GA, liquiritigenin, and isoliquiritigenin were measured by LC-MS. The effects of Laminaria extract on pharmacokinetics of GA were also investigated, following single-dose and multidose of glycyrrhizic acid (GL). The effects of Laminaria extract on intestinal absorption of GA and GL were studied using the in situ single-pass intestinal perfusion model. The metabolism of GL to GA in the contents of small and large intestines was also studied. The results showed Liquorice-Laminaria extract markedly increased the plasma concentration of GA, accompanied by a shorter Tmax. Similar alteration was observed following multidose administration. However, pharmacokinetics of neither liquiritigenin nor isoliquiritigenin was affected by Laminaria. Similarly, Laminaria markedly increased concentration and decreased Tmax of GA following oral GL were observed. The data from the intestinal perfusion model showed that Laminaria markedly increased GL absorption in duodenum and jejunum, but did not affect the intestinal absorption of GA. It was found that Laminaria enhanced the metabolism of GL to GA in large intestine. In conclusion, Laminaria increased plasma exposures of GA following oral administration of liquorice or GL, which partly resulted from increased intestinal absorption of GL and metabolism of GL to GA in large intestine.

Paroxetine decreased plasma exposure of glyburide partly via inhibiting intestinal absorption in rats.

Accumulating evidences have shown that diabetes is often accompanied with depression, thus it is possible that oral antidiabetic agent glyburide and antidepressive agent paroxetine are co-administered in diabetic patients. The aim of this study was to assess interactions between glyburide and paroxetine in rats. Effect of paroxetine on pharmacokinetics of orally administered glyburide was investigated. Effect of naringin (NAR), an inhibitor of rat intestinal organic anion transporting polypeptides 1a5 (Oatp1a5), on pharmacokinetics of glyburide was also studied. The results showed that co-administration of paroxetine markedly reduced plasma exposure and prolonged Tmax of glyburide, accompanied by significant increase in fecal excretion of glyburide. Co-administration of naringin also significantly decreased plasma exposure of glyburide. Data from intestinal perfusion experiments showed that both paroxetine and naringin significantly inhibited intestinal absorption of glyburide. Caco-2 cells were used to investigate whether paroxetine and naringin affected intestinal transport of glyburide and fexofenadine (a substrate of Oatp1a5). The results showed that both paroxetine and naringin greatly inhibited absorption of glyburide and fexofenadine. All results gave a conclusion that co-administration of paroxetine decreased plasma exposure of glyburide in rats via inhibiting intestinal absorption of glyburide, which may partly be attributed to the inhibition of intestinal Oatp1a5 activity.

Aggravation of clozapine-induced hepatotoxicity by glycyrrhetinic acid in rats.

Clozapine (CLZ) was reported to be associated with hepatotoxicity. Glycyrrhetinic acid (GA) has a liver protective effect. Our preliminary experiments showed that GA aggravated rather than attenuated CLZ-induced hepatotoxicity in primary cultured rat hepatocytes. The study aimed to describe the enhancing effect of GA on CLZ-induced hepatotoxicity in vivo and in vitro. Data from primary cultured rat hepatocytes showed the decreased formation of metabolites demethylclozapine (nor-CLZ) and clozapine N-oxide (CLZ N-oxide). The results in vivo showed that 7-day CLZ treatment led to marked accumulation of triglyceride (TG) and increase in γ-glutamyl transpeptidase (γ-GT) activity, liver weight, and serum AST in rats. Co-administration of GA enhanced the increases in hepatic TG, γ-GT, liver weight, and serum total cholesterol induced by CLZ. GA decreased plasma concentrations of nor-CLZ and CLZ N-oxide. Compared with control rats, hepatic microsomes of GA rats exhibited the decreased formations of nor-CLZ and CLZ N-oxide, accompanied by decreases in activities of CYP2C11 and CYP2C19 and increased activity of CYP1A2. QT-PCR analysis demonstrated that GA enhanced expression of CYP1A2, but suppressed expression of CYP2C11 and CYP2C13. All these results support the conclusion that GA aggravated CLZ-induced hepatotoxicity, which was partly via inhibiting CYP2C11 and CYP2C13 or inducing CYP1A2.