Models and methods to characterise levonorgestrel release from intradermally administered contraceptives

Microneedle (MN)-based technologies have been proposed as a means to facilitate minimally invasive sustained delivery of long-acting hormonal contraceptives into the skin. Intradermal administration is a new route of delivery for these contraceptives and therefore no established laboratory methods or experimental models are available to predict dermal drug release and pharmacokinetics from candidate MN formulations. This study evaluates an in vitro release (IVR) medium and a medium supplemented with ex vivo human skin homogenate (SH) as potential laboratory models to investigate the dermal release characteristics of one such hormonal contraceptive that is being tested for MN delivery, levonorgestrel (LNG), and provides details of an accompanying novel two-step liquid–liquid drug extraction procedure and sensitive reversed-phase HPLC–UV assay. The extraction efficiency of LNG was 91.7 ± 3.06% from IVR medium and 84.6 ± 1.6% from the medium supplemented with SH. The HPLC–UV methodology had a limit of quantification of 0.005 µg/mL and linearity between 0.005 and 25 µg/mL. Extraction and detection methods for LNG were exemplified in both models using the well-characterised, commercially available sustained-release implant (Jadelle®). Sustained LNG release from the implant was detected in both media over 28 days. This study reports for the first time the use of biologically relevant release models and a rapid, reliable and sensitive methodology to determine release characteristics of LNG from intradermally administered long-acting drug delivery systems

Formulation of hydrophobic peptides for skin delivery via coated microneedles

Microneedles (MNs) have been investigated as a minimally-invasive delivery technology for a range of active pharmaceutical ingredients (APIs). Various formulations and methods for coating the surface of MNs with therapeutics have been proposed and exemplified, predominantly for hydrophilic drugs and particulates. The development of effective MN delivery formulations for hydrophobic drugs is more challenging with dosing restrictions and the use of organic solvents impacting on both the bioactivity and the kinetics of drug release. In this study we propose a novel formulation that is suitable for MN coating of hydrophobic auto-antigen peptides currently being investigated for antigen specific immunotherapy (ASI) of type 1 diabetes. The formulation, comprising three co-solvents (water, 2-methyl-2-butanol and acetic acid) and polyvinylalcohol 2000 (PVA2000) can dissolve both hydrophilic and hydrophobic peptide auto-antigens at relatively high, and clinically relevant, concentrations (25 mg/ml or 12.5 mg/ml). The drug:excipient ratio is restricted to 10:1 w/w to maximise dose whilst ensuring that the dry-coated payload does not significantly impact on MN skin penetration performance. The coating formulation and process does not adversely affect the biological activity of the peptide. The delivery efficiency of the coated peptide into skin is influenced by a number of parameters. Electropolishing the metal MN surface increases delivery efficiency from 2.0 ± 1.0% to 59.9 ± 6.7%. An increased mass of peptide formulation per needle, from 0.37 μg to 2 μg peptide dose, resulted in a thicker coating and a 20% reduction in the efficiency of skin delivery. Other important performance parameters for coated MNs include the role of excipients in assisting dissolution from the MNs, the intrinsic hydrophobicity of the peptide and the species of skin model used in laboratory studies. This study therefore both exemplifies the potential of a novel formulation for coating hydrophobic and hydrophilic peptides onto MN devices and provides new insight into the factors that influence delivery efficiency from coated MNs. Importantly, the results provide guidance for identifying critical attributes of the formulation, coating process and delivery device, that confer reproducible and effective delivery from coated MNs, and thus contribute to the requirements of the regulators appraising these devices

An analysis of the relationship between microneedle spacing, needle force and skin strain during the indentation phase prior to skin penetration

Microneedle (MN) array patches present a promising new approach for the minimally invasive delivery of therapeutics and vaccines. However, ensuring reproducible insertion of MNs into the skin is challenging. The spacing and arrangement of MNs in an array are critical determinants of skin penetration and the mechanical integrity of the MNs. In this work, the finite element method was used to model the effect of MN spacing on needle reaction force and skin strain during the indentation phase prior to skin penetration. Spacings smaller than 2–3 mm (depending on variables, e.g., skin stretch) were found to significantly increase these parameters

TGFβ induces a SAMHD1-independent post-entry restriction to HIV-1 infection of human epithelial Langerhans cells

Sterile alpha motif (SAM) and histidine-aspartic (HD) domains protein 1 (SAMHD1) was previously identified as a critical post-entry restriction factor to HIV-1 infection in myeloid dendritic cells. Here we show that SAMHD1 is also expressed in epidermis-isolated Langerhans cells (LC), but degradation of SAMHD1 does not rescue HIV-1 or vesicular stomatitis virus G-pseudotyped lentivectors infection in LC. Strikingly, using Langerhans cells model systems (mutz-3-derived LC, monocyte-derived LC [MDLC], and freshly isolated epidermal LC), we characterize previously unreported post-entry restriction activity to HIV-1 in these cells, which acts at HIV-1 reverse transcription, but remains independent of restriction factors SAMHD1 and myxovirus resistance 2 (MX2). We demonstrate that transforming growth factor-β signaling confers this potent HIV-1 restriction in MDLC during their differentiation and blocking of mothers against decapentaplegic homolog 2 (SMAD2) signaling in MDLC restores cells’ infectivity. Interestingly, maturation of MDLC with a toll-like receptor 2 agonist or transforming growth factor-α significantly increases cells’ susceptibility to HIV-1 infection, which may explain why HIV-1 acquisition is increased during coinfection with sexually transmitted infections. In conclusion, we report a SAMHD1-independent post-entry restriction in MDLC and LC isolated from epidermis, which inhibits HIV-1 replication. A better understanding of HIV-1 restriction and propagation from LC to CD4+ T cells may help in the development of new microbicides or vaccines to curb HIV-1 infection at its earliest stages during mucosal transmission

Targeting proinsulin to local immune cells using an intradermal microneedle delivery system; a potential antigen-specific immunotherapy for type 1 diabetes

Antigen-specific immunotherapy (ASI) has been proposed as an alternative treatment strategy for type 1 diabetes (T1D). ASI aims to induce a regulatory, rather than stimulatory, immune response in order to reduce, or prevent, autoimmune mediated β-cell destruction, thus preserving endogenous insulin production. The abundance of immunocompetent antigen presenting cells (APCs) within the skin makes this organ an attractive target for immunotherapies. Microneedles (MNs) have been proposed as a suitable drug delivery system to facilitate intradermal delivery of autoantigens in a minimally invasive manner. However, studies to date have employed single peptide autoantigens, which would restrict ASI to patients expressing specific Human Leukocyte Antigen (HLA) molecules, thus stratifying the patient population. This study aims to develop, for the first time, an intradermal MN delivery system to target proinsulin, a large multi-epitope protein capable of inducing tolerance in a heterogenous (in terms of HLA status) population of T1D patients, to the immunocompetent cells of the skin. An optimized three component coating formulation containing proinsulin, a diluent and a surfactant, facilitated uniform and reproducible coating of >30 μg of the active pharmaceutical ingredient on a stainless steel MN array consisting of thirty 500 μm projections. When applied to a murine model these proinsulin-coated MNs efficiently punctured the skin and after a limited insertion time (150 s) a significant proportion of the therapeutic payload (86%) was reproducibly delivered into the local tissue. Localized delivery of proinsulin in non-obese diabetic (NOD) mice using the coated MN system stimulated significantly greater proliferation of adoptively transferred antigen-specific CD8+ T cells in the skin draining lymph nodes compared to a conventional intradermal injection. This provides evidence of targeted delivery of the multi-epitope proinsulin antigen to skin-resident APCs, in vivo, in a form that enables antigen presentation to antigen-specific T cells in the local lymph nodes. The development of an innovative coated MN system for highly targeted and reproducible delivery of proinsulin to local immune cells warrants further evaluation to determine translation to a tolerogenic clinical outcome

Evaluation of student engagement with differential media for Flipped Classroom teaching

Flipped learning, where the teaching content is delivered prior to the contact session, is an effective pedagogy. Most commonly, video is used for flipped approaches, although many other media are also effective, such as written, audio or electronic resources. The ideal medium used for information delivery is likely to vary, depending on the needs and learning approaches of each individual learner. Is there, therefore, a medium which is ideal for most students, or would flipped learning best be delivered using a suite of multimedia sources for the core information? This project aims to investigate the preferences shown by undergraduate students for different media in a series of flipped pedagogic settings. The project also aims to investigate whether there are any correlations between media preferences and student personality types and study approaches. Initial findings suggest that students engage readily with flipped learning pedagogies, although it is rare for students to engage with teaching materials well in advance of the class session. Early findings suggests that students show a preference for video as a medium of delivery for taught content

Topical steroid therapy induces pro-tolerogenic changes in Langerhans cells in human skin

We have investigated the efficacy of conditioning skin Langerhans cells (LCs) with agents to promote tolerance and reduce inflammation, with the goal of improving the outcomes of antigen-specific immunotherapy. Topical treatments were assessed ex vivo, using excised human breast skin maintained in organ bath cultures, and in vivo in healthy volunteers by analysing skin biopsies and epidermal blister roof samples. Following topical treatment with a corticosteroid, TNF-α levels were reduced in skin biopsy studies and blister fluid samples. Blister fluid concentrations of MCP-1, MIP-1α, MIP-1β and IP-10 were also reduced, while preserving levels of IL-1α, IL-6, IL-8 and IL-10. Steroid pre-treatment of the skin reduced the ability of LCs to induce proliferation, whilst supernatants showed an increase in the IL-10/IFN-γ ratio. Phenotypic changes following topical steroid treatment were also observed, including reduced expression of CD83 and CD86 in blister derived LCs, but preservation of the tolerogenic signalling molecules ILT3 and PD-1. Reduced expression of HLA-DR, CD80 and CD86 were also apparent in LCs derived from excised human skin. Topical therapy with a vitamin D analogue (calcipotriol) and steroid, calcipotriol alone or Vitamin A elicited no significant changes in the parameters studied. These experiments suggest that pre-conditioning the skin with topical corticosteroid can modulate LCs by blunting their pro-inflammatory signals and potentially enhancing tolerance. We suggest that such modulation prior to antigen specific immunotherapy might provide an inexpensive and safe adjunct to current approaches to treat autoimmune diseases

Skin vaccination using microneedles coated with a plasmid DNA cocktail encoding nucleosomal histones of Leishmania spp.

Vaccine delivery using microneedles (MNs) represents a safe, easily disposable and painless alternative to traditional needle immunizations. The MN delivery of DNA vaccines to the dermis may result in a superior immune response and/or an equivalent immune response at a lower vaccine dose (dose-sparing). This could be of special interest for immunization programs against neglected tropical diseases such as leishmaniasis. In this work, we loaded a MN device with 60 μg of a plasmid DNA cocktail encoding the Leishmania infantum nucleosomal histones H2A, H2B, H3 and H4 and compared its immunogenicity and protective capacity against conventional s.c. or i.d. injection of the plasmid. Mice immunized with MNs showed increased ratios of IFN-γ/IL-10, IFN-γ/IL-13, IFN-γ/IL-4, and IFN-γ/TGF-β in the spleens and lymph nodes compared with mice immunized by s.c. and i.d. routes. Furthermore, CCXCL9, CXCL10 and CCL2 levels were also higher. These data suggest that the nucleic acid immunization using MNs produced a better bias towards a Th1 response. However, none of the immunizations strategies were able to control Leishmania major infection in BALB/c mice, as illustrated by an increase in lesion size and parasite burden

Using gold nanoparticles for enhanced intradermal delivery of poorly soluble auto-antigenic peptides

Ultra-small 1-2 nm gold nanoparticles (NP) were conjugated with a poorly-soluble peptide auto-antigen, associated with type 1 diabetes, to modify the peptide pharmacokinetics, following its intradermal delivery. Peptide distribution was characterized, in vivo, after delivery using either conventional intradermal injection or a hollow microneedle device. The poorly-soluble peptide was effectively presented in distant lymph nodes (LN), spleen and draining LN when conjugated to the nanoparticles, whereas peptide alone was only presented in the draining LN. By contrast, nanoparticle conjugation to a highly-soluble peptide did not enhance in vivo distribution. Transfer of both free peptide and peptide-NPs from the skin to LN was reduced in mice lacking lymphoid homing receptor CCR7, suggesting that both are actively transported by migrating dendritic cells to LN. Collectively, these data demonstrate that intradermally administered ultra-small gold nanoparticles can widen the distribution of poorly-soluble auto-antigenic peptides to multiple lymphoid organs, thus enhancing their use as potential therapeutics

Microneedle delivery of autoantigen for immunotherapy in type 1 diabetes

Antigen specific immunotherapy mediated via the sustained generation of regulatory T cells arguably represents the ideal therapeutic approach to preventing beta cell destruction in type 1 diabetes. However, there is a need to enhance the efficacy of this approach to achieve disease modification in man. Previous studies suggest that prolonged expression of self-antigen in skin in a non-inflammatory context is beneficial for tolerance induction. We therefore sought to develop a dry-coated microneedle (MN) delivery system and combine it with topical steroid to minimise local inflammation and promote prolonged antigen presentation in the skin. Here we show that a combination of surface-modified MNs coated with appropriate solvent systems can deliver therapeutically relevant quantities of peptide to mouse and human skin even with hydrophobic peptides. Compared to conventional “wet” intradermal (ID) administration, “dry” peptide delivered via MNs was retained for longer in the skin and whilst topical hydration of the skin with vehicle or steroid accelerated loss of ID-delivered peptide from the skin, MN delivery of peptide was unaffected. Furthermore, MN delivery resulted in enhanced presentation of antigen to T cells in skin draining lymph nodes (LNs) both 3 and 10 days after administration. Repeated administration of islet antigen peptide via MN was effective at reducing antigen-specific T cell proliferation in the pancreatic LN, although topical steroid therapy did not enhance this. Taken together, these data show auto-antigenic peptide delivery into skin using coated MNs results in prolonged retention and enhanced antigen presentation compared to conventional ID delivery and this approach may have potential in individuals identified as being at a high risk of developing type 1 diabetes and other autoimmune diseases