Dihydroergotamine mesylate-loaded dissolving microneedle patch made of polyvinylpyrrolidone for management of acute migraine therapy

Accepted Manuscript Dihydroergotamine mesylate-loaded dissolving microneedle patch made of polyvinylpyrrolidone for management of acute migraine therapy

Cetin Tas, Jessica C. Joyce, Hiep X. Nguyen, Padmanabhan Eangoor, Jennifer S. Knaack, Ajay K. Banga, Mark R. Prausnitz PII: DOI: Reference:

S0168-3659(17)30908-2 doi:10.1016/j.jconrel.2017.10.021 COREL 9008

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

8 July 2017 4 October 2017 13 October 2017

Please cite this article as: Cetin Tas, Jessica C. Joyce, Hiep X. Nguyen, Padmanabhan Eangoor, Jennifer S. Knaack, Ajay K. Banga, Mark R. Prausnitz , Dihydroergotamine mesylate-loaded dissolving microneedle patch made of polyvinylpyrrolidone for management of acute migraine therapy. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi:10.1016/j.jconrel.2017.10.021

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ACCEPTED MANUSCRIPT Dihydroergotamine Mesylate-Loaded Dissolving Microneedle Polyvinylpyrrolidone for Management of Acute Migraine Therapy

Patch Made

of

Cetin Tas 1,2, Jessica C. Joyce1,3, Hiep X. Nguyen4, Padmanabhan Eangoor4, Jennifer S. Knaack4, Ajay K. Banga4, Mark R. Prausnitz 1,3,*

1

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School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332 USA 2

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Gülhane Education and Research Hospital, Department of Pharmaceutical Sciences, 06010, Etlik-Ankara, Turkey 3

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Wallace Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Georgia Institute of Technology, Atlanta, GA 30332 USA 4

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Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University, Atlanta, GA 30341 USA

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*To whom correspondance should be addressed: [email protected]

ACCEPTED MANUSCRIPT Abstract Migraine is a widespread neurological disease with negative effects on quality of life and productivity.

Moderate

to

severe

acute

migraine

attacks

can

be

treated

with

dihydroergotamine mesylate (DHE), an ergot derivative that is especially effective in nonresponders to triptan derivatives. To overcome limitations of current DHE formulations in subcutaneous injection and nasal spray such as pain, adverse side effects and poor bioavailability, a new approach is needed for DHE delivery enabling painless self-

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administration, quick onset of action, and high bioavailability. In this study, we developed a dissolving microneedle patch (MNP) made of polyvinylpyrrolidone, due to its high aqueous

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solubility and solubility enhancement properties, using a MNP design previously shown to be painless and simple to administer. DHE-loaded MNPs were shown to have a content

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uniformity of 108 ± 9 % with sufficient mechanical strength for insertion to pig skin ex vivo and dissolution within 2 min. In vivo pharmacokinetic studies were carried out on hairless rats, and DHE plasma levels were determined by liquid chromatography-tandem mass

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spectrometry (LC-MS/MS). The area under curve (AUC) value after DHE delivery by MNP (1259 ± 917 ng/mL min) was not significantly different (p0.05) as compared to

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subcutaneous injection, with a relative bioavailability of 97%. Also, appreciable plasma levels of DHE were seen within 5 min for both delivery methods and tmax value of MNPs (38 ± 23 min) showed no significant difference (p0.05) compared to subcutaneous injection (24 ± 13

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min). These results suggest that DHE-loaded MNPs have promise as an alternative DHE delivery method that can be painlessly self-administered with rapid onset and high

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bioavailability.

Keywords: Migraine, Dihydroergotamine mesylate, Microneedle patch, Polyvinylpyrrolidone,

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Liquid chromatography tandem mass spectrometry, Bioavailability, Rapid onset

ACCEPTED MANUSCRIPT 1. Introduction Approximately 50% of adults worldwide suffer from an active headache disorder, and according to a World Health Organization's survey, headaches rank 19th among the most disabling conditions 1. This sometimes-incapacitating health issue is among the top five most disabling conditions for women 2. Migraine headaches are the 3rd most prevalent illness in the world and affect about 11% of adults worldwide 3. Attacks are often accompanied by one or more disabling symptoms, and migraineurs consistently report

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reduced quality of life between attacks 4. The direct and indirect management of migraine therapy has an annual economic cost of approximately $13 billion just in the USA 5.

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There have been many encouraging developments in antimigraine medications over

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the past few decades, but currently available medical therapies are still far from optimal. The introduction of the "triptans" in the 1990s drastically changed prescribing patterns. Members of this antimigraine drug family are considered the first choice for moderate to severe attacks

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in migraine therapy unless there are contraindications 6. However, nearly one-third of patients taking triptans for acute migraine therapy discontinue this therapy because of lack of

managing

acute

migraine attacks

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efficacy, migraine recurrence, cost, and/or side effects 7. Thus, this subgroup has dificulties and often seeks

alternative drugs, such as

dihydroergotamine mesylate (DHE) 8.

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DHE is an ergot derivative that has been extensively utilized and studied in the treatment of episodic and chronic migraine. The mechanism of action is most likely

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vasoconstriction by stimulating -adrenergic and 5-HT receptors 9. Two pharmaceutical dosage forms of DHE, namely parenteral and nasal spray, were approved in United States in 1945 and 1990, respectively. An orally inhalable form of DHE was submitted to FDA for approval in 2013, but is not yet approved 10. Nasal administration of DHE exhibits

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inconsistent pharmacokinetic (PK) profiles, resulting in poor acceptance among patients and prescribers, whereas the parenteral route has the disadvantage of pain, needle phobia, risk of infections at the injection site, and requirement of specialized personnel for administration, which leads to poor patience compliance 11. Thus, there is a need for a new formulation design of DHE that enables easy self-administration, safe and effective delivery mimicking the parenteral (especially SC) PK profile. Such a new formulation could offer convenience as well as therapeutic advantages for migraineurs 12. In the last decade, advances in the field of transdermal delivery using microneedle patches (MNPs) have shown that the barrier function of stratum corneum can be overcome while retaining the advantages of patch-based delivery. Microneedles are typically a few hundred microns in width and up to 1 mm in length, and they are arranged as an array on a patch that is applied to the skin. The microneedles painlessly puncture the stratum corneum

ACCEPTED MANUSCRIPT and deliver drugs and vaccines to viable epidermis and dermis below 13, 14. MNPs can be self-administered and are strongly preferred over injection 15-17. Among the different types of microneedles, water-soluble polymer ones that dissolve in the skin have received great attention because they do not generated sharps waste and cannot be reused after removal from a patient’s skin 18, 19. Prior studies have investigated the delivery of triptans using microneedle patches for possible treatment of migraine, including sumatriptan 20-23, zolmitriptan 22 and rizatriptan 25.

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It is desirable to have rapid uptake of anti-migraine drugs for fast onset of relief to the patient. Rapid uptake is facilitated by the dense capillary bed in the superficial dermis where

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microneedle patches deliver drug and by formulation with suitable polymeric excipients that dissolve quickly, such as polyvinylpyrolidone (PVP) 26, fibroin 27, maltose 28 and

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chondroitin sulphate 29.

The aim of this study is to formulate dissolving MNPs for rapid release and capillary

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uptake of DHE in the skin. We therefore used highly water-soluble PVP as the microneedle matrix material to enable rapid delivery to treat acute migraine therapy and used DHE as the active pharmaceutical ingredient to provide relief to patients who suffer from unsufficient

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medication with triptan derivatives. We carried out in vitro tests measure content uniformity, mechanical strength of microneedle needles for complete insertion into skin, solubility and delivery efficiency of DHE from the MNPs, and optical microscopy imaging of MNPs. We also

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conducted in vivo studies of bioavailability of DHE delivered by MNPs in hairless rats. In this loaded MNPs.

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way, we introduce this alternative therapy approach to acute migraine therapy with DHE-

2. Materials and methods

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2.1. Materials

DHE (Tocris, Minneapolis, MN, USA), caroverine HCI (Sigma-Aldrich, St. Louis, MO, USA), PVP (10 kDa, Sigma-Aldrich) polyvinylalcohol (6 kDa, 78% hydrolyzed, Acros Organics, New Jersey, USA), Sucrose (Fluka Analytical, St. Louis, MO, USA), Gentian violet (Good Neighbor Pharma, Brawley, CA, USA), Optical Microscope (Olympus SZX16, Shinjuku, Tokyo, Japan). All reagents were of analytical grade and used as received.

2.2. Fabrication of DHE-loaded MNPs

ACCEPTED MANUSCRIPT MNPs were prepared using two separate solutions. A stock solution of DHE was prepared in methanol (adjusted to pH 4 with trifluoroacetic acid, TFA) at a concentration of 30 mg/mL. This stock solution was mixed with deionized (DI) water containing 10% w/v PVP to obtain a final solution of 7.15 mg/mL DHE and 7.5% w/v PVP (i.e., the drug solution). The backing solution consisted of polyvinyl alcohol, sucrose, and water in a mass ratio of 8:6:15. The production procedure consisted of the following: (a) 25 µL of drug solution was cast onto a polydimethylsiloxane (PDMS) microneedle mold, after which vacuum (27 mm Hg) was

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applied to help pull the drug solution into the microneedle mold cavities; (b) excess drug solution was removed from the mold surface with a flat blade; (c) the drug solution was

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allowed to dry into the tips of the microneedle mold cavities; (d) approximately 200 µL of backing solution was cast onto the microneedle mold under vacuum, thus forming a

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complete microneedle patch after drying based on methods published previously 30. Each patch was then stored in a desiccator at room temperature for 1 day before demolding with

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plastic backing material (polymethylmethacrylate, McMaster-Carr, Elmhurst, IL, USA). Demolded patches were stored in the desiccator until for further analysis.

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2.3. Assay of DHE in MNP

Each MNP loaded theoretically with 50 µg DHE was incubated into 50 mL DI water until dissolution was complete. The solution was then filtered through a membrane filter

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having a pore diameter of 0.45 µm and analyzed for DHE content with a validated HPLC coupled with fluorescence detector. Briefly, chromatographic separation was performed

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using a reverse-phase Zorbax C8 column (150 mm x 4.6 mm i.d., 5 µm particle size, Agilent, Santa Clara, CA, USA). The mobile phase consisted of acetonitrile and water (40:60 v/v) containing 0.1% TFA and 0.1% triethylamine degassed prior to use. The column temperature

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was 35°C and the flow rate was set at 1.2 mL/min. The excitation and emission wavelengths were 280 nm and 350 nm, respectively. The total run time of each analysis was 6 min, and the retention time of DHE was 4.2 min. The calibration curve was linear in the concentration range 25 – 5000 ng/mL, and the correlation coefficient was 0.999. 2.4 Ex vivo release of DHE from MNPs DHE-loaded MNPs were inserted into pig skin ex vivo with thumb force for 30 s. At predetermined time intervals (1, 2.5, 5 and 10 min), MNPs were removed from the skin and the insertion site was tape-stripped three times with an adhesive tape (3M TransporeTM, St. Paul, MN) to remove residual DHE on the skin surface. Used MNPs were imaged by optical microscopy to verify drug release into skin after insertion and then assayed for residual DHE content. The amount of DHE delivered into the skin was calculated by subtracting the

ACCEPTED MANUSCRIPT amount of DHE remaining in the MNPs after insertion and remaining on the skin surface from the amount originally encapsulated in the MNPs. To determine the residual amounts of DHE in the MNPs after skin insertion, MNPs were dissolved in DI water. The stripped tapes were soaked in methanol for 1 day at room temperature to recover the DHE on the tape. The amount of DHE extracted from the MNPs and the stripped tape was determined using HPLC, as described above.

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2.5. In vitro dissolution of DHE from MNPs DHE-loaded MNPs were incubated in a beaker containing 20 mL DI water at 32°C. At

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predetermined time intervals, 500 µL of dissolution medium was removed and replenished with the same amount fresh DI water. The samples were filtered through a membrane filter

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having a pore diameter of 0.45 µm, and DHE content was analyzed by HPLC, as described

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above. 2.6. Determination of DHE solubility

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An excess of DHE was added to 5 mL of 10% w/v PVP in DI water, sonicated for 1 h, and agitated in a shaker with a temperature maintained at 37°C for 24 h. The suspension was filtered through a membrane fitler having a pore diameter of 0.45 μm, diluted with

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methanol and analyzed by HPLC with fluorescence detector. The average of three experiments was taken. Solubility of DHE in DI water and in 10% w/v PVP in DI water was

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found to be 0.64 ± 0.14 and 1.13 ± 0.19 mg / mL respectively. The DHE solubility significantly increased by the addition of PVP (p<0.05).

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2.7. Insertion of DHE-loaded MNPs into pig skin ex vivo. Succesful insertion of DHE-loaded MNPs was evaluated on pig skin ex vivo. Pig skin was carefully shaved with a razor to remove hair. DHE-loaded MNPs were applied to the skin by pressing them down with the thumb, left on skin for 1 min, and then removed. After that, the pierced skin was stained with gentian violet for 5 min. Excess dye was removed, and the skin was evaluated for the appearance of blue dots on the stratum corneum using an optical microscope. 2.8. In vivo bioavailability studies of DHE loaded MNPs All animal studies were conducted with approval by the Georgia Institute of Technology Institutional Animal Care and Use Committee (IACUC). Fifteen hairless male Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA, USA) weighing 500–550

ACCEPTED MANUSCRIPT g were equally divided into three groups (i.e., five rats per group). Parenteral formulations of DHE at a concentration of 1 mg/mL (containing alcohol, glycerin and DI water as excipients at a pH value 3.6 ± 0.4) were preparared under aseptic conditions. Group 1 received intravenous (IV) injection of DHE parenteral formulation as a positive control. Group 2 received SC injection of DHE parenteral formulation as a positive control. Group 3 received DHE by MNP delivery, where MNPs were left on the skin for 30 min. The administered DHE dose was 50 µg for each rat. The rats were anesthetized with isoflurane during drug

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administration and until the end of the experiment. Blood samples (250 µL) were collected by the jugular vein catheter using gel separator tubes containing lithium heparin (Vacuette,

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Greiner Bio-One, Monroe, NC, USA) at 0, 2, 5, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300 and 360 min after IV and SC administration and at 0, 5, 15, 30, 45, 60, 75, 90, 120, 150,

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180, 240, 300 and 360 min after MNPs were first applied to skin. All samples were centrifuged (Triac centrifuge, BD Diagnostic Systems, Hunt Valley, MD, USA) at 3500 rpm

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for 5 min to collect plasma.

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2.9. LC MS/MS Analysis

For analysis by LC-MS/MS, an Agilent 1200 series HPLC (Agilent) and 6410B triple quadrupole mass spectrometer (Agilent) were used for the study. A Waters XBridge C18 column (3.5 µm, 2.1 x 100 mm, Waters, Milford, MA, USA) was used for chromatographic

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separation and was maintained at 25°C. Mobile phase A was comprised of HPLC-MS grade water with 0.1% TFA and mobile phase B contained HPLC-MS grade acetonitrile with 0.1%

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TFA. The run time was 23 min with a gradient from 10% organic to 50% organic in 18 min at a flow rate of 0.5 mL/min. Ionization was performed using an electrospray ionization source with the nebulizer pressure set to 35 psi, a gas flow rate of 12 L/min, a source temperature of

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300°C and the capillary voltage set to 4000 V. Data acquisition was performed using Mass Hunter Data Acquisition B.04.01 software (Agilent) under positive polarity. The m/z transition of 584.3 → 270.2 was used to quantify DHE, and with the m/z transition of 584.3 → 253.2 was used to qualify the data. The fragmentor voltage was set to 165 V and the collision energy was set to 30 V for both the transitions of DHE. For the caroverine, internal standard, the m/z transition of 366 → 100.2 was used as the quantitative ion and 366 → 121.2 was used as the qualitative ion. The fragmentor voltage was set to 108 V for both the transitions of caroverine and the collision energies were 24 V and 30 V for the quantitative and qualitative ions of caroverine, respectively. Quantitative analysis was performed using Mass Hunter QQQ Quantitative Analysis Software. A calibration curve was plotted with relative response (peak areas) of DHE to the internal standard on the y-axis and the concentration (ng/mL) on the x-axis. The unknown concentration of the samples was calculated by

ACCEPTED MANUSCRIPT interpolating their respective relative responses to the x-axis. The calibration curve was linear in the concentration range 0.5 – 200 ng/mL, with a correlation coefficient of 0.998. 2.9.1. Sample preparation A 10 mg/mL caroverine stock solution and further dilutions of caroverine were made using 50:50 acetonitrile:water. A 1 mg/mL DHE stock solution was made in methanol. DHE calibrants of concentrations 0.5, 1, 5, 10, 50, 100 and 200 ng/mL, and quality controls of

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concentrations 2 ng/mL and 150 ng/mL, were made in rat plasma (Sprague Dawley Rat Plasma, Innovative Research, Novi, MI, USA). Calibrants and quality controls were prepared

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by serial dilution of the 1 mg/mL stock solution of DHE in blank plasma.

The calibrants, quality controls and samples from the study were all treated similarly.

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Aliquots (50 µL) of calibrants, quality controls and samples were spiked with 10 µL of 50 ng/mL caroverine internal standard solution (previously diluted from the caroverine stock

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using 50:50 water:acetonitrile).

Liquid-liquid extraction was used to separate calibrants, quality controls and samples

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after the addition of internal standard. Then, 5 µL of 1 M ammonium buffer (5.35 g NH4Cl dissolved in 100 mL aqueous ammonia) and 150 µL diethyl ether were added to the extracts and the mixture was vortexed for 30 s before centrifuging for 5 min at 11,300 x g. Approximately 150 µL of the supernatant was then pipetted out and dried in a chemical hood

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acetonitrile:water, 0.1% TFA).

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for 1 h. After drying, samples were reconstituted in 50 µL of reconstitution solution (10:90

2.10. Data analysis and statistical evaluation Results are presented as the mean of n = 5 determinations with its associated

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standard deviation (S.D.). Noncompartmental pharmacokinetic analysis was carried out using the Pharmacologic Calculation System (version 4.1, Springer–Verlag, Philadelphia, PA, USA) computer program which calculates the AUC (area under the curve) of the plasma concentration as a function of time. The maximum plasma concentration (C max ) and the time to reach the maximum plasma concentration (tmax ) were obtained from the experimental data. Absolute (Fabs ; versus IV) and relative (Frel; versus SC) bioavailability were calculated according to following equations: Fabs = (AUCi * DoseIV) / (AUCIV * Dosei) * 100%

(1)

Frel = (AUCi * DoseSC ) / (AUCSC * Dosei) * 100%

(2)

where the subscript i corresponds to SC or MNP. All results are expressed as means ± S.D. Statistical differences between values were determined using SPSS 24.0 for Windows

ACCEPTED MANUSCRIPT software (IBM, Istanbul, Turkey), with Student’s t-test. The difference was regarded statistically significant when p < 0.05. 3. Results 3.1. Characterization of DHE-loaded MNPs MNPs were formulated using PVP as microneedle matrix material because of its suitable properties, such as biocompatibility, water solubility and rapid release of

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encapsulated drug after insertion into the skin. A two-step casting process under vacum was used to localize the drug in the tips of the needles, where the first cast was used to create

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the drug-loaded microneedles and the second cast, which contained no drug, was used to create the drug-free base of the microneedle array. MNPs were fabricated as a 10 x 10

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microneedle array in a  1 cm 2 area attached to a clear supporting plastic backing (Fig. 1a). MNPs formulated to contain 50 µg DHE were dissolved in 50 mL DI water and DHE

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content was found as 108 ± 9 % (Supplementary Information, Table S1). This shows that our lab-scale fabrication process was reproducible in loading the target drug amount into each

a

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MNP.

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b

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Figure 1: Microneelde patch for delivery of DHE. (a) Magnified view of a section of a representative microneedle patch. (b) A representative image of pig skin ex vivo after application of a patch containing a 10 x 10 array of microneedles and stained with gentian

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violet to identify sites of microneedle puncture into skin.

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3.2. Kinetics of in vitro dissolution of DHE from MNPs

The dissolution of DHE from MNPs was approximately 2 min when submerged in DI

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water (Fig. 2a) due in part to the high water solubility of PVP. The MNPs were designed for rapid dissolution of DHE in order to expedite onset of action for rapid relief of migraine when

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used in a future clinical senario.

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a

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b

Fig. 2. Dissolution profiles of DHE from microneedle patches. (a) Dissolution in DI water as a

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receptor medium. (b) Delivery study in pig skin ex vivo. Data show mean ± SD (n = 4).

3.3. MNP insertion and drug delivery into skin ex vivo MNPs must have sufficient mechanical strength to pierce the skin during application. MNPs were applied to pig skin ex vivo by pressing on the patch backing with the thumb. These MNPs were designed for manual insertion without the need for a high-velocity applicator. The skin insertion site was then exposed to a violet tissue-marking dye that selectively stains sites of skin puncture, which enabled visualization of microneedle insertion efficiency. After staining the skin, a complete array of violet spots (10 X 10) indicated that all microneedles were succesfully inserted into the skin (Fig. 1b).

ACCEPTED MANUSCRIPT To assess drug delivery efficiency, the amount of drug deposited in skin was measured and found to increase over time while the MNPs remained on the skin. The amount of DHE delivered in skin was 62% after 1 min and then steadily increased to 79% after 10 min (Fig. 2b). There was very little drug deposited on the skin surface (1.4 ± 0.9%, Supplementary Information, Table S2). Imaging of MNPs after removal from skin showed that the MNPs dissolved with similar kinetics, where the microneedle tips were dissolved within 1 min (Supplementary Information, Fig. S1). Because approximately 60% of the DHE

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loaded in MNPs was deposited in skin within 1 min and the microneedle tips dissolved within the same timeframe, these data indicate that most of the drug was localized towards the tips

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of the needles, which dissolved quickly, probably due to complete insertion into skin. Later time points up to 10 min only released an additional ~20% of the loaded drug, which

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indicates that this drug was located toward the base of the microneedle, which may not have been fully inserted into the skin and therefore dissolved more slowly.

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3.4. Pharmacokinetics and bioavailability of DHE in hairless rats

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3.4.1. IV and SC injection

Parenteral formulations of DHE were first injected at a dose of 50 µg (100 µg/kg) via IV and SC routes in hairless rats to determine the mean plasma concentration of DHE versus time profile (Fig. 3). The average peak concentration, C max , was 141 ng/mL and 11 ng/mL

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and the average area under the curve, AUC, was 1751 ng mL/min and 1304 ng mL/min for IV and SC injection, respectively (Table 1). There was a significant difference between Cmax

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values after IV versus SC injection (p  0.05), but the difference between AUC values after SC and IV injection was not significant (p  0.05). The average time until peak concentration, tmax , was 2 min after IV injection, which was significantly faster than after SC injection, which

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was 24 (p < 0.01, Table 1). Because the first data point after IV injection was taken at 2 min, it is possible that the tmax was even shorter and the C max was even higher for IV injection than reported.

Table 1 Pharmacokinetic parameters of DHE after application via different delivery routes in hairless rats (n=5). Delivery route a

Formulation AUC (ng/mL.min)

Cmax (ng / tmax (min) mL)

Fabs %b

Frel %c

ACCEPTED MANUSCRIPT IV

Solution

1751 ± 873

141 ± 113

2

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Solution

1304 ± 585

10.7 ± 2.3

23.8

± 74,5

12.5

33,4

37.5

± 71.9

± 96.5

22.6

52.3

70.3

MNPs

1259 ± 917

7.1 ± 5.5

IV: Intravenous; SC: subcutaneous.

b

Fabs . absolute bioavailability (relative to IV injection).

c

Frel . relative bioavailability (relative to SC injection).

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a

±

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Skin

±

3.4.2. Delivery using MNPs

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The pharmacokinetics of DHE delivery using MNPs showed that tmax was not significantly different from SC injection (p  0.05), whereas that Cmax was significantly lower (p  0.01, Fig. 3, Table 1). Cmax was lower and tmax was longer for MNP delivery compared to

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a

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IV injection (p  0.01, Fig. 3, Table 1). There was no significant difference of AUC value after

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b

Fig. 3. Plasma concentration of DHE (a) after IV administration and (b) after MNP and SC

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administration to hairless rats in vivo. Data show mean ± SD (n=5). MNP delivery compared to SC injection (p > 0.05, Table 1). Both MNP and SC delivery

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yielded AUC values significantly lower than IV injection (p < 0.05, Table 1). The absolute bioavailability of DHE after MNP delivery was 72%, and the relative bioavailability after MNP delivery was 97%, which supports the use of MNPs as an alternative drug delivery route to

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SC injection. Imaging by optical microscopy of MNPs after application to the rat skin shows that the tips of the microneedles dissolved, with only the base part of MNPs remaining (Fig. 4). Measurement of residual DHE in used MNPs indicates that the dose delivery efficiency of

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MNPs in vivo was 66% (Supplementary information, Table S3), which is in reasonable

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agreement with ex vivo findings (Fig. 2b).

Fig. 4: Representative optical microscopic image of a section of a microneedle patch loaded with DHE after application to hairless rat skin in vivo for 30 min. We did not observe local skin irritation such as erythema or edema, or any other adverse effects during the experiment.

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4. Discussion Migraine can cause significant negative effects on social activities and relationships, including decreased school and work attainment and productivity 5. There is a need for effective and acceptable migraine treatments to decrease impact and disability associated with the disease. The formulation of DHE in MNPs can have a significant effect on migraine therapy as a patient-friendly delivery method that serves as an alternative approach for

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migraneurs, especially triptan non-responders 31, 32.

Migraine is one of the most common chronic illness, associated with recurrent

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headache attacks and related symptoms in the gastrointestinal and autonomic nervous system 33. Migraines affect approximately 18% of women and 6% of men in the United

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States and nearly half of these people complain of reduced work or school productivity 34. Triptan derivatives are the first choice as the most effective therapy for acute migraine, but

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almost one-third of migraneurs fail to achieve adequate pain relief and seek alternative medication to alleviate migraine pain 12. DHE is a semisynthetic ergot alkaloid derivative

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with 5-HT1 agonist activity that is used to treat migraine. Parenteral and nasal dosage forms of DHE are on the market, but disadvantages of these routes of administration have motivated research to develop alternative dosage forms 35. An ideal dosage form for

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migraine treatment should have rapid onset of action, enable simple self-administration, offer high bioavailability, and provide strong efficacy and safety. While parenteral delivery has rapid onset with high bioavailability, it is not simple to administer. Nasal delivery, in contrast,

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is relatively simple to administer and offers rapid onset, but bioavailability is ~40% 36. DHE administered as an oral inhaled powder is a new pharmaceutical dosage form that has been under investigation for several years and offers fast pharmacologic response, patient friendly

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use, and bioavailability of ~80% 12, 37. However, it has not been approved by FDA due to technical concerns about the manufacturing and functioning of the device 38. In the last decade, there has been significant progress in the field of transdermal drug delivery, notably in the development of MNPs that increase skin permeability by creating micro-scale pathways across the skin barrier and combine the simple self-administration of transdermal patches and the delivery effectiveness of hypodermic needles 14, 39. MNPs have been shown to be suitable for many vaccines and drugs, with clinical trials completed on delivery of zolmitriptan 40, parathyroid hormone 41 , glucagon 42 and influenza vaccine 43, 44. MNPs have the advantages of painless self-administartion, no sharp biohazardous waste, control over the drug delivery rate using formulations, and cost-effective production technology 19.

ACCEPTED MANUSCRIPT In this study, we aimed to formulate DHE in MNPs as an improved alternative to marketed parenteral and nasal dosage forms. PVP was chosen as the microneedle matrix material to enable rapid and efficient transdermal delivery of DHE 45. PVP was chosen for four main reasons. First, vinyl pyrrolidone monomer has a ring structure chemical backbone that gives mechanical strength by increasing intramolecular rigidity 26, which is important for insertion of microneedles into skin. Second, PVP is higly water soluble at concentrations up to 50% 46 which can enable rapid dissolution of encapsulated drug molecules after

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insertion into skin. Third, PVP has been used as a plasma expander clinically for decades 47 Finally, PVP has been shown to increase solubility of drugs that exhibit poor water

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solubility, such as acetaminophen and gidazepam 48. The molecular weight of PVP used to make MNPs was 10 kDa, because PVP with molecular weight less than 20 kDa is known to

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be cleared effectively by the kidney, even after parenteral administration 47. One drawback of PVP that is hygroscopic, and water absorption in a humid environment can reduce

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microneedle mechanical strength 49. This issues was addressed by storing MNPs with desiccant.

This study showed that DHE can be formulated in MNPs and administered to hairless

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rats to generate a pharmacokinetic profile similar to SC injection. The AUC (absolute bioavailability ~75%) and tmax (~30 min) were not significantly different from each other with comparable Cmax (~10 ng/mL) which suggests that MNP administration could be used to

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provide rapid relief from migraines with the kinetics of injection, but without the pain and expertise needed to administer DHE parenterally. Moreover the absolute bioavailability of

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DHE administered by MNPs was much higher than nasal liquid formulations of DHE reported previously, which was in the range of 16-50 % in the rabbit model 36. Dose uniformity is also important for the development of MNPs 50. In this study,

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DHE dose uniformity in MNPs was 108 ± 9%. This shows that even in our manual, laboratory-scale processes, each step in fabrication such as preparing the drug solution with PVP, casting the solution onto molds, applying vacuum and drying MNPs, was designed and controlled to reach an acceptable dose uniformity range. In this study, DHE delivery with MNPs had similar pharmacokinetics to SC injection, achieving a high relative bioavailability of 96% (Table 1). This result is similar to bioavailability values determined for MNPs loaded with exenatide 51 and insulin 18. MNPs were fabricated with 50 µg DHE and administered the drug to skin with a delivery efficiency of ~80% within 10 min ex vivo (Fig. 2b) and ~65% within 30 min in vivo (Table S3) with only ~1% of drug left on the skin surface (Table S2). This shows that MNPs can release the majority of loaded drug in a short period of time, which reduces patch

ACCEPTED MANUSCRIPT wearing time, thereby minimizing risk of skin irritation related to long-term contact with transdermal patches 52. In prior studies, drug delivery efficiency from MNPs into skin ex vivo has not always correlated with drug delivery in vivo. For example, insulin formulated with starch gelatin in MNPs could be be released into skin within 5 min ex vivo, but in vivo studies in rats yielded an insulin tmax value of 2 h 18. This suggests that after application of MNPs to the skin, insulin was entrapped in a gelatinous polymer matrix that slowed its release and diffusion to

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capillaries for systemic distribution. In contrast, MNPs formulated with sodium hyaluronate were able to deliver encapsulated exenatide with a tmax value of 15 min, which was

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comparable to SC injection 51. Like sodium hyaluronate, the PVP used in our MNP formulation exhibits high water solubility, which may explain the statistical equivalence of t max

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after MNP and SC delivery. However, further studies are needed to better determine t max values, because of large error bars and small number of animals used in this study.

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Drug molecules must dissolve and diffuse away from the delivery site to reach the systemic blood circulation. Most drugs have either weak acid or basic characteristics and exhibit poor aqueous solubility 53. Similarly, DHE is poorly soluble in water and can be

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classifed in Biopharmaceutics Classification System as group II, which characteristically exhibits low solubility and high permeability which typically results in longer tmax values 54. Increased drug dissolution at the delivery site of absorption can lead higher bioavailability

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with shorter tmax values. A common method to enhance drug solubility is to formulate them in solid dispersion using the solvent evaporation method. Solid dispersions are solid products

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consisting of at least two constituents, typically a hydrophilic carrier and a hydrophobic active substance. PVP, polyethylene glycols (PEG) and Plasdone-S630 are commonly incorporated in solid dispersions as hydrophilic carriers 55. The production of DHE-loaded MNPs made

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of PVP involves a process similar to the preparation of solid dispersions by solvent evaporation. The solubility enhancement mechanism of poorly soluble drugs in solid dispersion may be related to increased area of solid-solvent contact due to reduced particle size, improved wettability and particles with higher porosity 56. Thus, the similar pharmacokinetic properties of MNPs formulated with PVP compared to SC injection may be attributed in part to the MNP production process that resembles a solid dispersion form of DHE. 5. Conclusion DHE delivery using MNPs had a pharmacokinetic profile similar to SC injection, as determined by no statistically significant difference in tmax and AUC but comparable Cmax values. DHE delivery was facilitated by formulation with PVP, which is believed to enable

ACCEPTED MANUSCRIPT rapid dissolution of microneedles due to the high water solubility of PVP and to increase DHE solubility in a manner similar to solid dispersions used in other types of dosage forms. These findings suggest that DHE-loaded MNPs offer a promising new approach to treat acute migraine attack with rapid onset of action, simple self-administration and high bioavailability using a drug with well-established safety and efficacy, which can represent a significant improvement over

conventions DHE delivery by parenteral and nasal routes of

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administration. Acknowledgements

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Mark Prausnitz is an inventor of patents licensed to companies developing microneedle-based products, is a paid advisor to companies developing microneedle-based

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products, and is a founder/shareholder of companies developing microneedle-based products (Micron Biomedical). This potential conflict of interest has been disclosed and is

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managed by Georgia Tech and Emory University. The authors would like to thank Donna Bondy of Georgia Tech for administrative assisstance. This work was supported by a grant

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from The Scientific and Technological Research Council of Turkey (TUBITAK) with the

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reference number of B.14.2.TBT.0.06.01-219-19961.

References

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1 The World Health Report 2001. World Health Organization WHO, Geneva: 2001. 19-45 2 C.B. Britton, Stress and headache, in M.W. Green, P.R. Muskin (Eds.), The Neuropsychiatry of Headache, Cambridge University Press, 2013, pp. 54-62.

AC C

3 T.J. Steiner, G.L. Birbeck, R.H. Jensen, Z. Katsarava, L.J. Stovner, P. Martelleti, Headache disorders are third cause of disability worldwide, J Headache Pain 16:58 (2015) 1-3.

4 D.C. Buse, M.F. Rupnow, R.B. Lipton, Assessing and managing all aspects of migraine: migraine attacks, migraine-related functional impairment, common comorbidities, and quality of life, Mayo Clin. Proc. 84 (2009) 422-435. 5 W.N. Burton, S.H. Landy, K.E. Downs, M.C. Runken, The impact of migraine and the effect of migraine treatment on workplace productivity in the United States and suggestions for future research, Mayo Clin Proc. 84 (2009) 436-445.

ACCEPTED MANUSCRIPT 6 C.M. Villalon, D. Centurion, L.F. Valdivia, P.De Vries, P.R. Saxena, An introduction to migraine: from ancient treatment to functional pharmacology and antimigraine therapy, Proc. West. Pharmacol. Soc. 45 (2002) 199-210. 7 M. Viana, A.A. Genazzani, S. Terrazzino, G. Nappi, P.J. Goadsby, Triptan nonresponders: do they exist and who are they? Cephalalgia 33 (2013) 891-896. 8 J.A. Morren, N. Galvez-Jimenez, Where is dihydroergotamine mesylate in the changing landscape of migraine therapy? Expert Opin. Pharmacother. 11 (2010) 3085-3093.

PT

9 E.B. Marcelo, S.J. Tepper, Ergotamine and dihydroergotamine: A review, Curr. Pain and Headache Rep. 7 (2003) 55-62.

RI

10 S.J. Tepper, Orally inhaled dihydroergotamine: A review, Headache 53 (S2) (2013) 4353.

SC

11 J.R. Saper, S. Silberstein, D. Dodick, A. Rapoport, DHE in the pharmacotherapy of migraine: potential for a larger role, Headache 46 (2006) 212-220.

NU

12 S.B. Shrewsbury, R.O. Cook, G. Taylor, C. Edwards, N.M. Ramadan, Safety and pharmacokinetics of dihydroergotamine mesylate administered via a novel (TEMPO) inhaler, Headache 48 (2008) 355–367.

MA

13 B. Bediz, E. Korkmaz, R. Khilwani, C. Donahue, G. Erdos, L.D. Falo, O.B. Ozdoganlar, Dissolvable microneedle arrays for Intradermal delivery of biologics: fabrication and application, Pharm. Res. 31 (2014) 117-135.

ED

14 R.F. Donnelly, T.R.R. Singh, A.D. Woolfson, Microneedle-based drug delivery systems: microfabrication, drug delivery, and safety, Drug Deliv. 17 (2010) 187-207.

EP T

15 K. van der Maaden, W. Jiskoot, J. Bouwstra, Microneedle technologies for (trans)dermal drug and vaccine delivery, J.Control. Release 161 (2012) 645-665. 16 J.C. Birchall, Microneedle array technology: the time is right but is the science ready?

AC C

Expert Rev. Med. Devices 3 (2006) 1-4. 17 X. Chen, G.J.P. Fernando, A.P. Raphael, S.R. Yukiko, E.J. Fairmaid, C.A. Primeiro, I.H. Frazer, L.E. Brown, M.A.F. Kendall, Rapid kinetics to peak serum antibodies is achieved following influenza vaccination by dry-coated densely packed microprojections to skin, J.Control. Release 158 (2012) 78-84. 18 M.H. Ling, M.C. Chen, Dissolving polymer microneedle patches for rapid and efficient transdermal delivery of insulin to diabetic rats, Acta Biomater. 9 (2013) 8952-8961. 19 Y-C. Kim, J-H. Park, M.R. Prausnitz, Microneedles for drug and vaccine delivery, Adv. Drug Deliver. Rev. 64 (2012) 1547-1568. 20 Y. Ito, S. Kashiwara, K. Fukushima, K. Takada, Two-layered dissolving microneedles for percutaneous delivery of sumatriptan in rats, Drug Dev. Ind. Pharm. 37 (2011) 1387-93.

ACCEPTED MANUSCRIPT 21 D. Wu, Y.S. Quan, F. Kamiyama, K. Kusamori, H. Katsumi, T. Sakane, A. Yamamoto, Improvement of transdermal delivery of sumatriptan succinate using a novel selfdissolving microneedle array fabricated from sodium hyaluronate in rats, Biol. Pharm. Bull. 38 (2015) 365-373. 22 B.N. Nalluri, S.S. Anusha, S.R. Bramhini, J. Amulya, A.S. Sultana, C.U. Teja, D.B. Das, In vitro skin permeation enhancement of sumatriptan by microneedle application, Curr Drug Deliv. 12 (2015) 761-769.

PT

23 D. Wu, H. Katsumi, Y.S. Quan, F. Kamiyama, K. Kusamori, , T. Sakane, A. Yamamoto, Permeation of sumatriptan succinate across human skin using multiple types of self-

RI

dissolving microneedle arrays fabricated from sodium hyaluronate, J. Drug Target. 24 (2016) 752-758.

SC

24 C.T. Uppuluri, J. Devineni, T. Han, A. Nayak, K.J. Nair, B.R. Whiteside, D.B. Das, B.N. Nalluri, Microneedle-assisted transdermal delivery of zolmitriptan: effect of microneedle

NU

geometry, in vitro permeation experiments, scaling analyses and numerical simulations, Drug Dev Ind Pharm. 43 (2017) 1292-1303.

25 C. Uppuluri, A.S. Shaik, T. Han, A. Nayak, K.J. Nair, B.R. Whiteside, B.N. Nalluri, D.B.

MA

Das, Effect of microneedle type on transdermal permeation of rizatriptan, AAPS PharmSciTech. 18 (2017) 1495-1506.

26 S.P. Sullivan, N. Murthy, M.R. Prausnitz, Minimally invasive protein delivery with rapidly

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dissolving polymer microneedles, Adv. Mater. 20 (2008) 933-938. 27 X. You, J-H Chang, B-K Ju, J.J. Pak, Rapidly dissolving fibroin microneedles for

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transdermal drug delivery, Mater. Sci. Eng. C 31 (2011) 1632-1636. 28 K. Lee, C.Y. Lee, H. Jung, Dissolving microneedles for transdermal drug administration prepared by stepwise controlled drawing of maltose, Biomaterials 32 (2011) 3134-3140.

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29 Y. Ito, J. Ohta, K. Imada, S. Akamatsu, N. Tsuchida, G. Inoue, N. Inoue, K. Takada, Dissolving microneedles to obtain rapid local anesthetic effect of lidocaine at skin tissue, J. Drug Target. 21 (2013) 770-775. 30 E.V. Vassilieva, H. Kalluri, D. McAllister, M.T. Taherbhai, E.S. Esser, W.P. Pewin, J.A. Pulit-Penaloza, M.R. Prausnitz, R.W. Compans, I. Skountzou, Improved immunogenecity of individual influenza vaccine components delivered with a novel dissolving microneedle patch stable at room temperature, Drug Deliv. Transl. Res. 5 (2015) 360-371. 31 H.S. Gill, D.D. Denson, B.A. Burris, M.R. Prausnitz, Effect of microneedle design on pain in human subjects, Clin. J. Pain 24 (2008) 585-594. 32 M. Bozoghlanian, S.V. Vasudevan, Overview of migraine treatment, Pain Manag. 2 (2012) 399-414.

ACCEPTED MANUSCRIPT 33 M. Schürks, Dihydroergotamine: role in the treatment of migraine, Expert Opin. Drug Metab. Toxicol. 5 (2009) 1141-1148. 34 B. Gilmore, M. Michael, Treatment of acute migraine headache, Am. Fam. Physician, 83 (2011) 271-280. 35 A. Brayfield, Martindale: The complete drug reference – 38 th edition, Pharmaceutical Press, 2016, pp. 672-673. 36 E. Marttin, S.G. Romeijn, J.C. Verhoef, F.W.H.M. Merkus, Nasal absorption of

PT

dihydroergotamine from liquid and powder formulations in rabbits, J.Pharm.Sci. 86 (1997) 802-807.

RI

37 A.M. Rapoport, New acute treatments for headache, Neurol. Sci. 31 (2010) 129–132.

SC

38 Headache and Migraine News, Whatever happened to Levadex? 13 Feb. 2015, http://headacheandmigrainenews.com/whatever-happened-to-levadex

NU

07.06.2017)

(accessed

39 D. Wu, Y.S. Quan, F. Kamiyama, K. Kusamori, H. Katsumi, T. Sakane, A. Yamamoto,

MA

Improvement of transdermal delivery of sumatriptan succinate using a novel selfdissolving microneedle array fabricated from sodium hyaluronate in rats, Biol. Pharm. Bull. 38 (2015) 365-73.

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40 First subject treated in Zosano Pharma’s pivotal efficacy trial for M207 patch for acute migraine. News Release, 25 July 2016, Zosano Pharma, Fremont. 07.06.2017)

EP T

http://ir.zosanopharma.com/ releasedetail.cfm?ReleaseID=980904, (accessed 41 M. Ameri, P.E. Daddona, Y.F. Maa, Demonstrated solid-state stability of parathyroid

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hormonePTH(1–34) coated on a novel transdermal microprojection delivery system, Pharm. Res. 26 (2009) 2454–2463. 42 Zosano Pharma. 2015. Zosano Pharma announces positive phase 2 results for its ZPglucagon patch program for treatment of severe hypoglycemia. News Release, 13 Oct. 2015, Zosano Pharma, Fremont. http://ir.zosanopharma.com/releasedetail.cfm?ReleaseID=936338 (accessed 07.06.2017) 43 S. Hirobe, H. Azukizawa, T. Hanafusa, K. Matsuo, Y.S. Quan, Clinical study and stability assessment of a novel transcutaneous influenza vaccination using a dissolving microneedle patch, Biomaterials 57 (2015) 50–58. 44 N.G. Rouphael, M. Paine, R. Mosley, S. Henry, D.V. McAllister, H. Kalluri, W. Pewin, P.M. Frew, T. Yu, N.J. Thornburg, S. Kabbani, L. Lai, E.V. Vassilieva, I. Skountzou, R.W.

ACCEPTED MANUSCRIPT Compans, M.J. Mulligan, M.R. Prausnitz, TIV-MNP 2015 Study Group. 2017. The safety, immunogenicity and acceptability of inactivated influenza vaccine delivered by microneedle patch (TIV-MNP 2015): a randomized, partially blind, placebo-controlled phase 1 trial, Lancet. In press 45 R.R.S. Thakur, A.I.A. Tekko, F. Al-Shammari, A.A. Ali, H. McCarthy, R.F. Donnelly. Rapidly dissolving polymeric microneedles for minimally invasive intraocular drug delivery, Drug Deliv. and Transl. Res. 6 (2016) 800–815.

PT

46 F. Haaf, A. Sanner, F. Straub. Poymers of N-vinylpyrrolidone: synthesis, characterization and uses, Polym. J. 17 (1985) 143-152.

RI

47 S. Wolfgang, PVP: A critical review of the kinetics and toxicology of polyvinylpyrrolidone (Povidone), CRC Press, 1990.

SC

48 V.G. Kadajji, G.V. Betageri, Water Soluble Polymers for Pharmaceutical Applications,

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Polymers, 2011, 3, 1972-2009.

49 W. Chen, C. Wang, L. Yan, L. Huang, X. Zhu, B. Chen, H.J. Sant, X. Niu, G. Zhu, K.N.

MA

Yu, V.A.L. Roy, B.K. Gale, X. Chen, Improved polyvinylpyrrolidone microneedle arrays with non-stoichiometric cyclodextrin, J. Mater. Chem. B 2 (2014) 1699-1705. 50 R.E.M. Lutton, J. Moore, E. Larraneta, S. Ligett, A.D. Woolfson, R.F. Donnelly,

ED

Microneedle characterisation: the need for universal acceptance criteria and GMP specifications when moving towards commercialisation, Drug Deliv. Transl. Res. 5 (2015) 313-331.

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51 Z. Zhu, H. Luo, W. Lu, H. Luan, Y. Wu, J. Luo, Y. Wang, J. Pi, C.Y. Lim, H. Wang, Rapidly dissolvable microneedle patches for transdermal delivery of exenatide, Pharm. Res. 12 (2014) 3348-3360.

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52 A.L. Musel, E.M. Warshaw, Cutaneous reactions to transdermal therapeutic systems, Dermatitis 17 (2006) 109-122. 53 K.T. Savjani, A.K. Gajjar, J.K. Savjani, Drug solubility: importance and enhancement techniques, ISRN Pharm. (2012) 1-10. 54 Y.G. Jadhav, U.C. Galgatte, P.D. Chaudhari, Challenges in formulation development of fast dissolving oral films, Indo Am.J.Pharm.Res. 3 (2013) 6391-6407. 55 G. Murtaza, Solubility enhancement of simvastatin: a review, Acta Pol. Pharm. 69 (2012) 581-590. 56 J. Singh, M. Walia, S.L. Harikumar, Solubility enhancement by solid dispersion method: a review, J. Drug Deliv. Ther. 3 (2013) 148-155.

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Graphical abstract