Haripriya Kalluri

Transdermal Delivery of Proteins

Transdermal delivery of peptides and proteins avoids the disadvantages associated with the invasive parenteral route of administration and other alternative routes such as the pulmonary and nasal routes. Since proteins have a large size and are hydrophilic in nature, they cannot permeate passively across the skin due to the stratum corneum which allows the transport of only small lipophilic drug molecules. Enhancement techniques such as chemical enhancers, iontophoresis, microneedles, electroporation, sonophoresis, thermal ablation, laser ablation, radiofrequency ablation and noninvasive jet injectors aid in the delivery of proteins by overcoming the skin barrier in different ways. In this review, these enhancement techniques that can enable the transdermal delivery of proteins are discussed, including a discussion of mechanisms, sterility requirements, and commercial development of products. Combination of enhancement techniques may result in a synergistic effect allowing increased protein delivery and these are also discussed.

Microchannels Created by Sugar and Metal Microneedles: Characterization by Microscopy, Macromolecular Flux and Other Techniques

The objective of this study was to investigate the feasibility of using microneedle technology to enhance transcutaneous permeation of human immunoglobulin G (IgG) across hairless rat skin. Microchannels created by maltose and metal (DermaRoller) microneedles were characterized by techniques such as methylene blue staining, histological examination, and calcein imaging. Methylene blue staining and histological sections of treated skin showed that maltose microneedles and DermaRoller breached the skin barrier by creating microchannels in the skin with an average depth of approximately 150 microm, as imaged by confocal microscopy. Calcein imaging and pore permeability index values suggested the uniformity of the created pores in microneedle-treated skin. Transdermal studies with IgG indicated a flux rate of 45.96 ng/cm(2)/h, in vitro, and a C(max) of 7.27 ng/mL, in vivo, for maltose microneedles-treated skin while a flux rate of 353.17 ng/cm(2)/h, in vitro, and a C(max) of 9.33 ng/mL, in vivo, was achieved for DermaRoller-treated skin. Transepidermal water loss measurements and methylene blue staining, in vivo, indicated the presence of microchannels for upto 24 h, when occluded. In conclusion, the microchannels created by maltose microneedles and DermaRoller resulted in the percutaneous enhancement of a macromolecule, human IgG.

The safety, immunogenicity, and acceptability of inactivated influenza vaccine delivered by microneedle patch (TIV-MNP 2015): a randomised, partly blinded, placebo-controlled, phase 1 trial

BACKGROUND Microneedle patches provide an alternative to conventional needle-and-syringe immunisation, and potentially offer improved immunogenicity, simplicity, cost-effectiveness, acceptability, and safety. We describe safety, immunogenicity, and acceptability of the first-in-man study on single, dissolvable microneedle patch vaccination against influenza. METHODS The TIV-MNP 2015 study was a randomised, partly blinded, placebo-controlled, phase 1, clinical trial at Emory University that enrolled non-pregnant, immunocompetent adults from Atlanta, GA, USA, who were aged 18-49 years, naive to the 2014-15 influenza vaccine, and did not have any significant dermatological disorders. Participants were randomly assigned (1:1:1:1) to four groups and received a single dose of inactivated influenza vaccine (fluvirin: 18 μg of haemagglutinin per H1N1 vaccine strain, 17 μg of haemagglutinin per H3N2 vaccine strain, and 15 μg of haemagglutinin per B vaccine strain) (1) by microneedle patch or (2) by intramuscular injection, or received (3) placebo by microneedle patch, all administered by an unmasked health-care worker; or received a single dose of (4) inactivated influenza vaccine by microneedle patch self-administered by study participants. A research pharmacist prepared the randomisation code using a computer-generated randomisation schedule with a block size of 4. Because of the nature of the study, participants were not masked to the type of vaccination method (ie, microneedle patch vs intramuscular injection). Primary safety outcome measures are the incidence of study product-related serious adverse events within 180 days, grade 3 solicited or unsolicited adverse events within 28 days, and solicited injection site and systemic reactogenicity on the day of study product administration through 7 days after administration, and secondary safety outcomes are new-onset chronic illnesses within 180 days and unsolicited adverse events within 28 days, all analysed by intention to treat. Secondary immunogenicity outcomes are antibody titres at day 28 and percentages of seroconversion and seroprotection, all determined by haemagglutination inhibition antibody assay. The trial is completed and registered with ClinicalTrials.gov, number NCT02438423. FINDINGS Between June 23, 2015, and Sept 25, 2015, 100 participants were enrolled and randomly assigned to a group. There were no treatment-related serious adverse events, no treatment-related unsolicited grade 3 or higher adverse events, and no new-onset chronic illnesses. Among vaccinated groups (vaccine via health-care worker administered microneedle patch or intramuscular injection, or self-administered microneedle patch), overall incidence of solicited adverse events (n=89 vs n=73 vs n=73) and unsolicited adverse events (n=18 vs n=12 vs n=14) were similar. Reactogenicity was mild, transient, and most commonly reported as tenderness (15 [60%] of 25 participants [95% CI 39-79]) and pain (11 [44%] of 25 [24-65]) after intramuscular injection; and as tenderness (33 [66%] of 50 [51-79]), erythema (20 [40%] of 50 [26-55]), and pruritus (41 [82%] of 50 [69-91]) after vaccination by microneedle patch application. The geometric mean titres were similar at day 28 between the microneedle patch administered by a health-care worker versus the intramuscular route for the H1N1 strain (1197 [95% CI 855-1675] vs 997 [703-1415]; p=0·5), the H3N2 strain (287 [192-430] vs 223 [160-312]; p=0·4), and the B strain (126 [86-184] vs 94 [73-122]; p=0·06). Similar geometric mean titres were reported in participants who self-administered the microneedle patch (all p>0·05). The seroconversion percentages were significantly higher at day 28 after microneedle patch vaccination compared with placebo (all p<0·0001) and were similar to intramuscular injection (all p>0·01). INTERPRETATION Use of dissolvable microneedle patches for influenza vaccination was well tolerated and generated robust antibody responses. FUNDING National Institutes of Health.

Delivery of salmon calcitonin using a microneedle patch

Peptides and polypeptides have important pharmacological properties but only a limited number have been exploited as therapeutics because of problems related to their delivery. Most of these drugs require a parenteral delivery system which introduces the problems of pain, possible infection, and expertise required to carry out an injection. The aim of this study was to develop a transdermal patch containing microneedles (MNs) coated with a peptide drug, salmon calcitonin (sCT), as an alternative to traditional subcutaneous and nasal delivery routes. Quantitative analysis of sCT after coating and drying onto microneedles was performed with a validated HPLC method. In vivo studies were carried out on hairless rats and serum levels of sCT were determined by ELISA. The AUC value of MNs coated with a trehalose-containing formulation (250 ± 83 ng/mL min) was not significantly different as compared to subcutaneous injections (403 ± 253 ng/mL min), but approximately 13 times higher than nasal administration (18.4 ± 14.5 ng/mL min). T(max) (7.5 ± 5 min) values for MN mediated administration were 50% shorter than subcutaneous injections (15 min), possibly due to rapid sCT dissolution and absorption by dermal capillaries. These results suggest that with further optimization of coating formulations, microneedles may enable administration of sCT and other peptides without the need for hypodermic injections.

Microneedles and transdermal drug delivery

Transdermal delivery has become a popular route of drug delivery in recent years. However, permeation of drugs through skin is limited to small lipophilic molecules. Microneedle technology involves the creation of micron-sized channels in the skin, which can allow the delivery of hydrophilic molecules including large proteins which do not pass the skin barrier passively. Microneedles can be manufactured in different geometries from various materials such as silicon, glass, sugars and polymers and this technology has been shown to effectively increase the permeation rates of a wide range of therapeutic compounds, including proteins, in several studies on animal models and humans. Combination with other techniques such as iontophoresis and sonophoresis may result in synergistic effects. New products involving microneedle technology are currently being developed in the industry and the prospects are highly promising.

Tolerability, usability and acceptability of dissolving microneedle patch administration in human subjects

To support translation of microneedle patches from pre-clinical development into clinical trials, this study examined the effect of microneedle patch application on local skin reactions, reliability of use and acceptability to patients. Placebo patches containing dissolving microneedles were administered to fifteen human participants. Microneedle patches were well tolerated in the skin with no pain or swelling and only mild erythema localized to the site of patch administration that resolved fully within seven days. Microneedle patches could be administered by hand without the need of an applicator and delivery efficiencies were similar for investigator-administration and self-administration. Microneedle patch administration was not considered painful and the large majority of subjects were somewhat or fully confident that they self-administered patches correctly. Microneedle patches were overwhelmingly preferred over conventional needle and syringe injection. Altogether, these results demonstrate that dissolving microneedle patches were well tolerated, easily usable and strongly accepted by human subjects, which will facilitate further clinical translation of this technology.

Effects of Chemical and Physical Enhancement Techniques on Transdermal Delivery of Cyanocobalamin (Vitamin B12) In Vitro

Vitamin B12 deficiency, which may result in anemia and nerve damage if left untreated, is currently treated by administration of cyanocobalamin via oral or intramuscular routes. However, these routes are associated with absorption and compliance issues which have prompted us to investigate skin as an alternative site of administration. Delivery through skin, however, is restricted to small and moderately lipophilic molecules due to the outermost barrier, the stratum corneum (SC). In this study, we have investigated the effect of different enhancement techniques, chemical enhancers (ethanol, oleic acid, propylene glycol), iontophoresis (anodal iontophoresis) and microneedles (soluble maltose microneedles), which may overcome this barrier and improve cyanocobalamin delivery. Studies with different chemical enhancer formulations indicated that ethanol and oleic acid decreased the lag time while propylene glycol based formulations increased the lag time. The formulation with ethanol (50%), oleic acid (10%) and propylene glycol (40%) showed the maximum improvement in delivery. Iontophoresis and microneedle treatments resulted in enhanced permeation levels compared to passive controls. These enhancement approaches can be explored further to develop alternative treatment regimens.

In Vivo Iontophoretic Delivery of Salmon Calcitonin Across Microporated Skin

The purpose of this study was to determine the effect of microneedle (MN) technology and its combination with iontophoresis (ITP) on the in vivo transdermal delivery of salmon calcitonin (sCT). Maltose MNs (500 µm) were used to porate skin prior to application of the drug, with or without ITP. Micropores created by maltose MNs were characterized by histological sectioning and calcein imaging studies, which indicated uniformity of the created micropores. In vivo studies were performed in hairless rats to assess the degree of enhancement achieved by ITP (0.2 mA/cm² for 1 h), MNs (81 MNs), and their combination. In vivo studies indicate a serum maximal concentration of 0.61 ± 0.42 ng/mL, 1.79 ± 0.72 ng/mL, and 5.51 ± 0.32 ng/mL for ITP, MNs, and combination treatment, respectively. MN treatment alone increased serum concentration 2.5-fold and the combination treatment increased the concentration ninefold as compared with iontophoretic treatment alone. Combination treatment of ITP and MNs resulted in the highest delivery of sCT and therapeutic levels were achieved within 5 min of administration.

Evaluation of Microneedles in Human Subjects

Microneedles have been studied extensively in vitro and in animals. Here we review the growing collection of microneedle studies involving human subjects. Human studies seek to validate microneedle performance and safety, including microneedle insertion into the skin, liquid infusion via hollow microneedles, pain associated with solid and hollow microneedles, safety, and skin resealing. Human studies have also assessed drug and vaccine delivery, including small molecules like naltrexone, methyl nicotinate, lidocaine and dyclonine, and aminolevulinic acid and methyl aminolevulinate; peptides and proteins like parathyroid hormone and insulin; and vaccines, especially influenza. Patient and provider preferences have been collected on topics such as general opinion of microneedles, willingness to vaccinate using microneedles, willingness to self-administer microneedles, and concerns about microneedles. Overall, these studies validate preclinical findings that microneedles offer a powerful new approach to human medical applications.

Microneedle patch delivery of influenza vaccine during pregnancy enhances maternal immune responses promoting survival and long-lasting passive immunity to offspring

Influenza virus causes life-threatening infections in pregnant women and their newborns. Immunization during pregnancy is the most effective means of preventing maternal and infant mortality/morbidity; however, influenza vaccination rates of pregnant women remain under 50%. Furthermore, the availability of vaccines in low-resource populations is limited. Skin immunization with microneedle patches (MN) is a novel and safe vaccination platform featuring thermostable vaccine formulations. Cold-chain independence and the potential for self-administration can expand influenza vaccination coverage in developing countries. In this study of pregnant BALB/c mice immunized with subunit H1N1 influenza vaccine, we demonstrate the advantage of skin vaccination over intramuscular delivery of a two-fold higher vaccine dose. MN vaccine induced superior humoral immune responses and conferred protective immunity against a lethal challenge dose of homologous influenza virus. Importantly, MN vaccination of mice at mid-gestation resulted in enhanced and long-lasting passive immunity of the offspring, measured by neutralizing antibody titers and survival rates after virus challenge. We conclude that skin vaccination using MN is a superior immunization approach with the potential to overcome immune tolerance observed in pregnancy, and lower vaccination costs through antigen dose-sparing, which is especially relevant in underserved countries.