Development, Evaluation, and Pharmacokinetic Assessment of Polymeric Microarray Patches for Transdermal Delivery of Vancomycin Hydrochloride.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause harmful and potentially deadly infections. Vancomycin remains the first-line antibiotic treatment for MRSA-derived infections. Nevertheless, as a peptide drug, it is poorly absorbed when administered orally because of its high molecular weight and low permeability in the gastrointestinal tract and is therefore administered intravenously for the treatment of systemic diseases. In order to circumvent some of the many drawbacks associated with intravenous injection, other routes of drug delivery should be investigated. One of the strategies which has been employed to enhance transdermal drug delivery is based on microarray patches (MAPs). This work, for the first time, describes successful transdermal delivery of vancomycin hydrochloride (VCL) using dissolving MAPs (DMAPs) and hydrogel-forming MAPs (HFMAPs). VCL was formulated into DMAPs and reservoirs [film dosage forms, lyophilized wafers, and compressed tablets (CSTs)] using excipients such as poly(vinyl pyrrolidone), poly(vinyl alcohol), sodium hyaluronate, d-sorbitol, and glycerol. In this study, HFMAPs were manufactured using aqueous blends containing poly(methylvinyl ether-co-maleic acid) cross-linked by esterification with poly(ethylene glycol). The VCL-loaded CSTs (60% w/w VCL) were the most promising reservoirs to be integrated with HFMAPs based on the physicochemical evaluations performed. Both HFMAPs and DMAPs successfully delivered VCL in ex vivo studies with the percentage of drug that permeated across the neonatal porcine skin recorded at 46.39 ± 8.04 and 7.99 ± 0.98%, respectively. In in vivo studies, the area under the plasma concentration time curve from time zero to infinity (AUC0-∞) values of 162.04 ± 61.84 and 61.01 ± 28.50 µg h/mL were achieved following the application of HFMAPs and DMAPs, respectively. In comparison, the AUC0-∞ of HFMAPs was significantly greater than that of the oral administration control group, which showed an AUC0-∞ of 30.50 ± 9.18 µg h/mL (p < 0.05). This work demonstrates that transdermal delivery of VCL is feasible using DMAPs and HFMAPs and could prove effective in the treatment of infectious diseases caused by MRSA, such as skin and soft tissue infections, lymphatic-related infections, and neonatal sepsis.

Control of Klebsiella pneumoniae Infection in Mice by Using Dissolving Microarray Patches Containing Gentamicin.

Using a murine model of Klebsiella pneumoniae bacterial infection, we demonstrate that gentamicin dissolving microarray patches, applied to murine ears, could control K. pneumoniae infection. Mice treated with microarray patches had reduced bacterial burden in the nasal-associated lymphoid tissue and lungs compared with their untreated counterparts. This proof of concept study represents the first published data on the in vivo delivery of the antibiotic gentamicin via dissolving microarray patches, resulting in the control of bacterial infection.

Novel Hydrogel-Forming Microneedle Array for Intradermal Vaccination in Mice Using Ovalbumin as a Model Protein Antigen.

Global vaccination strategies have traditionally relied on the hypodermic needle and syringe model. However, to facilitate increased immunization coverage and reduce costs, novel methods of vaccine delivery are warranted. Dissolving microneedle arrays (MNs) have been proposed as an alternative approach to the hypodermic needle, offering the prospect for self-vaccination and increased immunogenicity via direct targeting of skin dendritic cells. This study, for the first time, compares the use of novel hydrogel-forming MNs and dissolving MNs for the delivery of a model protein antigen ovalbumin (OVA). We provide comparative data on both MN types in terms of in vitro characteristics and in vivo immunogenicity. Herein, both MN platforms were tested and characterized in terms of mechanical integrity and insertion properties using a validated skin insertion model. A comparative in vivo vaccination study in BALB/c mice was conducted, whereby anti-OVA specific IgG was used as a measure of delivery efficacy and subsequent immune response. While vaccination of mice with both MN platforms resulted in IgG responses, those vaccinated with dissolving MNs had significantly higher IgG titers ( p < 0.0149), despite the quantity of OVA delivered being significantly less. This study highlights the importance of MN design and the potential impact of dissolving MN polymers on the immune response to vaccine antigens. Furthermore, detailed studies are therefore required to elucidate the effects of polymer-vaccine interactions and their subsequent effect on immune responses.

Microneedle-Mediated Transdermal Delivery of Bevacizumab.

Bevacizumab is a recombinant humanized monoclonal antibody used clinically as a combination chemotherapeutic. Antibody therapeutics are usually formulated as parenteral injections, owing to their low oral bioavailability. Microneedle technology provides a transdermal alternative for drug-delivery using micron-scale needle structures to penetrate directly through the stratum corneum into the dermal interstitium. This study describes the design, formulation, and in vitro characterization of both dissolving and hydrogel-forming microneedle array platforms for transdermal delivery of bevacizumab. Bevacizumab recovery and transdermal permeation studies were conducted and analyzed using bevacizumab specific ELISA. Prototype microneedle-patches were tested in vivo in Sprague-Dawley rats with serum, exterior lumbar and axial lymph nodes, spleen, and skin tissue concentrations of bevacizumab reported. This work represents the first example of high dose transdermal delivery of an antibody therapeutic in vivo using dissolving and hydrogel-forming microneedle platforms. Basic pharmacokinetic parameters are described including hydrogel-forming microneedles: Cmax 358.2 ± 100.4 ng/mL, Tmax 48 h, AUC 44357 ± 4540, and Css 942 ± 95 ng/mL, highlighting the potential for these devices to provide sustained delivery of antibody therapeutics to the lymph and systemic circulation. Targeted delivery of chemotherapeutic agents to the lymphatic system by MN technology may provide new treatment options for cancer metastases.

TNF-α-induced p38MAPK activation regulates TRPA1 and TRPV4 activity in odontoblast-like cells.

The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α-induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts.

Hydrogel-Forming Microneedle Arrays Made from Light-Responsive Materials for On-Demand Transdermal Drug Delivery.

We describe, for the first time, stimulus-responsive hydrogel-forming microneedle (MN) arrays that enable delivery of a clinically relevant model drug (ibuprofen) upon application of light. MN arrays were prepared using a polymer prepared from 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) by micromolding. The obtained MN arrays showed good mechanical properties. The system was loaded with up to 5% (w/w) ibuprofen included in a light-responsive 3,5-dimethoxybenzoin conjugate. Raman spectroscopy confirmed the presence of the conjugate inside the polymeric MN matrix. In vitro, this system was able to deliver up to three doses of 50 mg of ibuprofen upon application of an optical trigger over a prolonged period of time (up to 160 h). This makes the system appealing as a controlled release device for prolonged periods of time. We believe that this technology has potential for use in "on-demand" delivery of a wide range of drugs in a variety of applications relevant to enhanced patient care.

Microneedles: A New Frontier in Nanomedicine Delivery.

This review aims to concisely chart the development of two individual research fields, namely nanomedicines, with specific emphasis on nanoparticles (NP) and microparticles (MP), and microneedle (MN) technologies, which have, in the recent past, been exploited in combinatorial approaches for the efficient delivery of a variety of medicinal agents across the skin. This is an emerging and exciting area of pharmaceutical sciences research within the remit of transdermal drug delivery and as such will undoubtedly continue to grow with the emergence of new formulation and fabrication methodologies for particles and MN. Firstly, the fundamental aspects of skin architecture and structure are outlined, with particular reference to their influence on NP and MP penetration. Following on from this, a variety of different particles are described, as are the diverse range of MN modalities currently under development. The review concludes by highlighting some of the novel delivery systems which have been described in the literature exploiting these two approaches and directs the reader towards emerging uses for nanomedicines in combination with MN.

Microneedle applications in improving skin appearance.

Microneedles (MNs) are micron-sized, minimally invasive devices that breach the outermost layer of the skin, the stratum corneum (SC), creating transient, aqueous pores in the skin and facilitating the transport of therapeutic molecules into the epidermis. Following many years of extensive research in the area of MN-mediated trans- and intra-dermal drug delivery, MNs are now being exploited in the cosmeceutical industry as a means of disrupting skin cell architecture, inducing elastin and collagen expression and deposition. They are also being used as vehicles to deliver cosmeceutic molecules across the skin, in addition to their use in combinatorial treatments with topical agents or light sources. This review explores the chronology of microneedling methodologies, which has led to the emergence of MN devices, now extensively used in cosmeceutical applications. Recent developments in therapeutic molecule and peptide delivery to the skin via MN platforms are addressed and some commercially available MN devices are described. Important safety and regulatory considerations relating to MN usage are addressed, as are studies relating to public perception of MN, as these will undoubtedly influence the acceptance of MN products as they progress towards commercialisation.

Hydrogel-forming microneedle arrays can be effectively inserted in skin by self-application: a pilot study centred on pharmacist intervention and a patient information leaflet.

PURPOSE: To investigate, for the first time, the influence of pharmacist intervention and the use of a patient information leaflet on self-application of hydrogel-forming microneedle arrays by human volunteers without the aid of an applicator device. METHODS: A patient information leaflet was drafted and pharmacist counselling strategy devised. Twenty human volunteers applied 11 × 11 arrays of 400 µm hydrogel-forming microneedle arrays to their own skin following the instructions provided. Skin barrier function disruption was assessed using transepidermal water loss measurements and optical coherence tomography and results compared to those obtained when more experienced researchers applied the microneedles to the volunteers or themselves. RESULTS: Volunteer self-application of the 400 µm microneedle design resulted in an approximately 30% increase in skin transepidermal water loss, which was not significantly different from that seen with self-application by the more experienced researchers or application to the volunteers. Use of optical coherence tomography showed that self-application of microneedles of the same density (400 µm, 600 µm and 900 µm) led to percentage penetration depths of approximately 75%, 70% and 60%, respectively, though the diameter of the micropores created remained quite constant at approximately 200 µm. Transepidermal water loss progressively increased with increasing height of the applied microneedles and this data, like that for penetration depth, was consistent, regardless of applicant. CONCLUSION: We have shown that hydrogel-forming microneedle arrays can be successfully and reproducibly applied by human volunteers given appropriate instruction. If these outcomes were able to be extrapolated to the general patient population, then use of bespoke MN applicator devices may not be necessary, thus possibly enhancing patient compliance.

LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid.

AIM: To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease. MATERIALS AND METHODS: Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS: The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites. CONCLUSIONS: LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis.

Enhancement strategies for transdermal drug delivery systems: current trends and applications.

Transdermal drug delivery systems have become an intriguing research topic in pharmaceutical technology area and one of the most frequently developed pharmaceutical products in global market. The use of these systems can overcome associated drawbacks of other delivery routes, such as oral and parenteral. The authors will review current trends, and future applications of transdermal technologies, with specific focus on providing a comprehensive understanding of transdermal drug delivery systems and enhancement strategies. This article will initially discuss each transdermal enhancement method used in the development of first-generation transdermal products. These methods include drug/vehicle interactions, vesicles and particles, stratum corneum modification, energy-driven methods and stratum corneum bypassing techniques. Through suitable design and implementation of active stratum corneum bypassing methods, notably microneedle technology, transdermal delivery systems have been shown to deliver both low and high molecular weight drugs. Microneedle technology platforms have proven themselves to be more versatile than other transdermal systems with opportunities for intradermal delivery of drugs/biotherapeutics and therapeutic drug monitoring. These have shown that microneedles have been a prospective strategy for improving transdermal delivery systems.

Novel transdermal bioadhesive surfactant-based system for release and solubility improvement of antimalarial drugs artemether-lumefantrine.

Artemether (ART) and lumefantrine (LUM) are the gold standard antimalarial drugs used for the treatment of malaria in children and pregnant women. Typically, ART and LUM are delivered orally in the form of a combined tablet, however, the appropriateness of this route of administration for these drugs is questionable due to the poor absorption and therefore bioavailability observed unless administered alongside lipid-rich foods. Transdermal drug delivery in the form of a patch-type system has been identified as a viable alternative to the conventional tablet-based therapy. A novel, surfactant-based ART-LUM formulation (S3AL), developed for transdermal delivery, may eliminate the shortcomings associated with oral delivery; namely poor drug absorption which is caused by the inherently low solubility of ART and LUM. Moreover, by successfully delivering these antimalarials transdermally, first-pass metabolism will be avoided leading to enhanced drug bioavailability in both cases. The S3AL formulation contained ART and LUM at equal concentrations (2.5% w/w of each) as well as Procetyl® AWS (30% w/w), oleic acid (10% w/w), 1-methyl-2-pyrrolidone (10% w/w), and water (45% w/w). The addition of LUM to the formulation changed the system from a striae structure to a dark field structure when visualized by a polarized light microscope. Additionally, this system possessed higher viscosity and superior skin bioadhesion, as evidenced by mechanical characterization, when compared to a similar formulation containing ART alone. S3AL was also proven to be biocompatible to human keratinocyte cells. Finally,in vitrostudies demonstrated the propensity of S3AL for successful delivery via the transdermal route, with 2279 ± 295 µg cm-2of ART and 94 ± 13 µg cm-2of LUM having permeated across dermatomed porcine skin after 24 h, highlighting its potential as a new candidate for the treatment of malaria.

Influence of molecular weight on transdermal delivery of model macromolecules using hydrogel-forming microneedles: potential to enhance the administration of novel low molecular weight biotherapeutics.

With a view to improve the current monoclonal antibody-based therapies dominating the pharmaceutical market, low molecular weight (MW) protein-based macromolecules, such as recombinant antibody fragments, typically within the range of 10-70 kDa, have been developed. Previously, our group successfully delivered Avastin®, a monoclonal antibody (mAb) across the skin using hydrogel-forming microneedles (MN). However, it is thought that this delivery system can be further enhanced using novel, lower MW biomolecules. To address this perception, in the current study, FITC-dextran of different MWs (10, 70 and 150 kDa) was used to model the transdermal delivery of low MW biotherapeutics and mAbs with MWs of approximately 150 kDa. Conversely, fluorescein sodium was the compound selected to model hydrophilic, low MW drugs. As expected, fluorescein sodium produced the greatest cumulative permeation (637.4 ± 42.69 µg). The amounts of FITC-dextran 10 kDa and 150 kDa which permeated across neonatal porcine skin in vitro were 462.17 ± 65.85 µg and 213.54 ± 15.19 µg after 24 h, respectively. The results collated here suggest that the delivery of emerging novel biotherapeutics, via'super swelling' hydrogel-forming MNs, have the potential to result in greater permeation across human skin, compared to the delivery of mAbs delivered via the same route.

Hydrogel-forming microneedle arrays as a therapeutic option for transdermal esketamine delivery.

Treatment resistant depression is, by definition, difficult to treat using standard therapeutic interventions. Recently, esketamine has been shown as a viable rescue treatment option in patients in depressive crisis states. However, IV administration is associated with a number of drawbacks and advanced delivery platforms could provide an alternative parenteral route of esketamine dosing in patients. Hydrogel-forming microneedle arrays facilitate transdermal delivery of drugs by penetrating the outer layer of the skins surface, absorbing interstitial skin fluid and swelling. This subsequently facilitates permeation of medicines into the dermal microcirculation. This paper outlines the in vitro formulation development for hydrogel-forming microneedle arrays containing esketamine. Analytical methods for the detection and quantitation of esketamine were developed and validated according to International Conference on Harmonisation standards. Hydrogel-forming microneedle arrays were fully characterised for their mechanical strength and skin insertion properties. Furthermore, a series of esketamine containing polymeric films and lyophilised reservoirs were assessed as drug reservoir candidates. Dissolution testing and content drug recovery was carried out, followed by permeation studies using 350 µm thick neonatal porcine skin in modified Franz cell apparatus. Lead reservoir candidates were selected based on measured physicochemical properties and brought forward for testing in female Sprague-Dawley rats. Plasma samples were analysed using reverse phase high performance liquid chromatography for esketamine. Both polymeric film and lyophilised reservoirs candidate patches achieved esketamine plasma concentrations higher than the target concentration of 0.15-0.3 µg/ml over 24 h. Mean plasma concentrations in rats, 24 h post-application of microneedle patches with drug reservoir F3 and LW3, were 0.260 µg/ml and 0.498 µg/ml, respectively. This developmental study highlights the potential success of hydrogel-forming microneedle arrays as a transdermal drug delivery platform for ESK and supports moving to in vivo tests in a larger animal model.

Evaluation of the clinical impact of repeat application of hydrogel-forming microneedle array patches.

Hydrogel-forming microneedle array patches (MAPs) have been proposed as viable clinical tools for patient monitoring purposes, providing an alternative to traditional methods of sample acquisition, such as venepuncture and intradermal sampling. They are also undergoing investigation in the management of non-melanoma skin cancers. In contrast to drug or vaccine delivery, when only a small number of MAP applications would be required, hydrogel MAPs utilised for sampling purposes or for tumour eradication would necessitate regular, repeat applications. Therefore, the current study was designed to address one of the key translational aspects of MAP development, namely patient safety. We demonstrate, for the first time in human volunteers, that repeat MAP application and wear does not lead to prolonged skin reactions or prolonged disruption of skin barrier function. Importantly, concentrations of specific systemic biomarkers of inflammation (C-reactive protein (CRP); tumour necrosis factor-α (TNF-α)); infection (interleukin-1ß (IL-1ß); allergy (immunoglobulin E (IgE)) and immunity (immunoglobulin G (IgG)) were all recorded over the course of this fixed study period. No biomarker concentrations above the normal, documented adult ranges were recorded over the course of the study, indicating that no systemic reactions had been initiated in volunteers. Building upon the results of this study, which serve to highlight the safety of our hydrogel MAP, we are actively working towards CE marking of our MAP technology as a medical device.

A sensitive HPLC-UV method for quantifying vancomycin in biological matrices: Application to pharmacokinetic and biodistribution studies in rat plasma, skin and lymph nodes.

Vancomycin (VCN) is an antibiotic used in the treatment of methicillin-resistant Staphylococcus aureus (MRSA)-derived infections. As it has a relatively narrow therapeutic window, it is imperative to develop a sensitive and reliable analytical method for drug monitoring and pharmacokinetic purposes. In the present study, quick sample preparations and a sensitive high-performance liquid chromatography method using UV detection (HPLC-UV) have been developed and validated. The analytical method for detection and quantification of VCN in rat plasma, skin and lymph node samples was validated based on the Food and Drug Administration (FDA) and European Medicine Agency (EMEA) bioanalytical method validation guidelines. The optimised plasma sample preparation involved a simple protein precipitation step, with extraction recovery of 100.3 ±â€¯0.92 %. VCN in all biological matrices was analysed in a HPLC-UV system (215 nm) using a Cortecs® C18 column (4.6 × 150 mm, 2.7 µm particle size) fitted with a guard cartridge set at 20 °C. Reverse phase HPLC under gradient conditions, with mobile phase consisting of 20 mM phosphate buffer containing 0.5 % v/v of triethylamine and a mixture of methanol - acetonitrile (70:30, v/v), and runtime of 12 min/sample was employed. The calibration standards used for plasma ranged between 0.1-50 µg/ml, while in the skin and lymph node matrices, standards ranged between 0.05-50 µg/ml with correlation coefficients (R2) of ≥ 0.9995 for all matrices. The method was selective, sensitive, accurate and precise for detecting and quantifying VCN in the biological matrices used. The validated method was successfully utilised in the detection of VCN in a pharmacokinetic and organ biodistribution study carried out in rats following oral and IV bolus administration of the drug. This validated bioanalytical method offers a wide range of potential applications in clinical therapeutic drug monitoring, pharmacokinetics and toxicology.

The contribution of key hydrophobic residues in ecotin to enzyme-inhibitor complex stability.

The Escherichia coli protease inhibitor ecotin is believed to be implicated in the evasion of host defenses during infection. The protein has also attracted attention as a scaffold for the design of novel, specific protease inhibitors. Ecotin interacts with its targets through two sites. Key hydrophobic residues in both sites (Leu-64, Trp-67, Tyr-69, Met-84, and Met-85) were mutated to alanine and the effects on the inhibition of trypsin, chymotrypsin, and elastase were assessed. Each of these mutant ecotin proteins tested in kinetic assays with these enzymes exerted less inhibitory potency compared to wild-type ecotin. However, these effects were relatively small and not additive.

Enhanced Intradermal Delivery of Nanosuspensions of Antifilariasis Drugs Using Dissolving Microneedles: A Proof of Concept Study.

Conventional oral administration of antifilariasis drugs results in nonspecific targeting of the drugs and the intradermal delivery of nanoparticles with sizes of <100 nm could be used to improve lymphatic uptake. This study investigated the combination of nanosuspension and dissolving microneedles (MN-NS) as an alternative intradermal delivery approach for the delivery of antifilariasis drugs, namely doxycycline, albendazole, and ivermectin. NS were fabricated and optimized using a bottom-up technique. The NS were then incorporated into the MN arrays. The optimized NS were <100 nm in diameter. Furthermore, MN-NS had suitable mechanical strength and insertion capabilities. The dermatokinetic study revealed that the delivery of drugs into the dermis of excised neonatal porcine skin by MNs was significantly higher than that from a needle-free patch, with 29.29 ± 4.65%, 31.54 ± 5.35%, and 34.54 ± 4.98% of doxycycline, albendazole sulfoxide, and ivermectin retained in the dermis after 24 h. The results presented here serve as proof of concept for the significant enhancement of drug retention times in the dermis, following their formulation into NS and delivery via MN. Leading on from these studies, future work must investigate in vivo lymphatic pharmacokinetic profiling of drugs formulated into NS, in a suitable animal model.

Solid lipid nanoparticle-based dissolving microneedles: A promising intradermal lymph targeting drug delivery system with potential for enhanced treatment of lymphatic filariasis.

Conventional oral therapy of lymphatic filariasis drugs is only effective to kill microfilariae in the bloodstream, but is often ineffective to kill adult filarial (macrofilariae) in the complex anatomy of the lymphatic system. The encapsulation of drugs into lipid-based nanoparticles with sizes of <100nm, and administration intradermally, could be used to enhance lymphatic uptake. Therefore, we developed an innovative approach, using solid lipid nanoparticles (SLNs) and dissolving microneedles (MNs) to deliver antifilariasis drugs, namely doxycycline, diethylcarbamazine and albendazole, intradermally. The SLNs were prepared from Geleol® and Tween®80 as a lipid matrix and stabilizer, respectively. The formulations were optimized using a central composite design, producing SLNs with sizes of <100nm. Drug release was sustained over 48h from SLNs, compared to pure drugs. The SLNs were then incorporated into a polymeric hydrogel which was casted to form SLNs-loaded MNs. SLNs-loaded MNs exhibited sufficient mechanical and insertion properties. Importantly, dermatokinetic studies showed that>40% of drugs were retained in the dermis of excised neonatal porcine skin up to 24h post-MN application, indicating the high possibility of the SLNs to be taken by the lymphatic system. In in vivo studies, the maximal lymph concentrations of the three drugs in rat, achieved following intradermal delivery, ranged between 4- and 7-fold higher than that recorded after oral administration. Additionally, compared to oral administration, despite the lower plasma Cmax and organ-distribution, the AUC and relative bioavailability of the three drugs in rat plasma was also higher using our delivery approach. Accordingly, this delivery approach could maximize the drugs concentrations in the lymph system without essentially increasing their plasma concentrations. This could potentially deliver the drugs efficiently to the bloodstream, where the microfilariae reside, while also targeting drug to the lymph nodes, where filarial nematodes reside in infected patients, leading to an effective therapy for lymphatic filariasis.

Functional domains of the human epididymal protease inhibitor, eppin.

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin's domains are involved in the protein's antibacterial activity, only the Kunitz domain is required for selective protease inhibition.