(351h) Transdermal Delivery of Biopharmaceuticals Using Dissolving Microneedles Patch

 SEQ CHAPTER \h \r 1TRANSDERMAL DELIVERY OF
BIOPHARMACEUTICALS USING DISSOLVING MICRONEEDLES PATCH

Jeong Woo Lee¹,
Seong-O Choi¹, Eric I. Felner^1,2, Mark R. Prausnitz¹

¹School
of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta,
GA 30332

²Division
of Pediatric Endocrinology, Hughes Spalding Children's Hospital, Emory
University School of Medicine,

 Atlanta, GA 30332

Introduction

            The delivery of
biopharmaceuticals via the oral route is not practically possible due to
enzymatic degradation in the gastrointestinal tract, low absorption through
mucous membranes of intestine, and hepatic degradation due to the first-pass
effect in the systemic circulation. Hypodermic injection can ensure higher bioavailability;
however, it has low patient compliance because of fear and pain caused by
hypodermic needles and it often requires patients to visit a clinic for correct
injection, disposal of used needles and maintaining the integrity of
biopharmaceuticals in the cold chain. As an alternative, transdermal delivery
with a dissolving microneedles patch can address these problems associated with
oral and injection delivery. Here, we present a dissolving microneedles patch loaded
with human growth hormone (hGH) by examining the functional activity of hGH and
in vivo pharmacokinetics of hGH to assess transdermal delivery of
biopharmaceuticals with a dissolving microneedles patch and ultimately for the
self-administration of biopharmaceuticals.

Methods and Materials

   Dissolving
microneedles patches
as shown in Figure 1A were prepared using
microfabrication technologies, molding
techniques, and modified solvent-casting methods.^[1] First, microneedle
master structures were fabricated using UV photolithography processes and an inverse mold was made by casting
master structures in polydimethylsiloxane (PDMS). To fabricate dissolving microneedles patches,
carboxymethylcellulose (CMC) was
dissolved in deionized water and then dehydrated to form a viscous hydrogel. In the case of CMC/trehalose
microneedles, CMC and trehalose were dissolved in deionized water at a ratio of 1:1. Then, recombinant hGH (Genotropin, Pfizer) was added to the concentrated hydrogel, which contains hGH and matrix at a mass ratio of 1:9 on a dry basis. Finally, the hydrogel containing hGH
was cast onto the mold and dried under centrifugation used to fill the microneedle
cavities.

Figure 1. Brightfield micrograph of (A)
CMC/trehalose dissolving microneedles patch encapsulating hGH. Scanning
electron micrographs of (B) CMC microneedles patch and (C) CMC/trehalose
microneedles patch after 24 h application to rat skin.
hGH functional activity was determined
by measurement of hGH-stimulated growth of Nb2 rat lymphoma cells.^[2] Nb2 cells in stationary culture
medium will
resume replication when stimulated by hGH. The
viable Nb2 cell
population at 3 days after the addition of
hGH was measured using a cell viability analyzer
(Vi-CELL, Beckman Coulter). Five different hGH solutions were added to the Nb2
cell culture; (i) CMC placebo solution (negative
control), (ii) hGH solution (Genotropin, positive control), (iii) hGH solution
mixed with CMC reconstituted from dissolved placebo microneedles,
(iv) a
reconstituted CMC microneedles patch encapsulating hGH, and (v) a reconstituted CMC microneedles patch encapsulating hGH after 3 months storage at
ambient conditions (23±2^oC and 38±5% relative humidity).
The pharmacokinetic study was performed with wild-type male hairless rats with approval by the
Institutional Animal Care and Use Committee of Georgia Tech. Four groups were involved; Group
1 - subcutaneous injection of hGH from the manufacturer (positive control), Group 2 - CMC microneedles patch
encapsulating hGH, Group 3 - CMC/trehalose microneedles patch encapsulating hGH, and Group
4 - subcutaneous injection of a reconstituted
CMC/trehalose microneedle patch encapsulating hGH Rat blood was drawn from the tail vein at various time points up
to 24 h after administration and
collected in a CAPIJECT tube (T-MG, Terumo Medical). The collected blood was
spun to isolate serum, which was stored at -70^oC until hGH concentration was determined by enzyme-linked
immunosorbent assay (ELISA). Areas under the serum hGH
concentration curve were computed to calculate bioavailability.

Results and Discussion

We
assessed the stability of hGH in
dissolving microneedles patches by determining the level of cell proliferation of each group at the saturation level of hGH stimulation. As shown in Figure 2A,
unprocessed hGH (positive control) stimulated cell growth resulting in almost 750% of
initial population. Addition of CMC to unprocessed
hGH stimulated cell growth to the statistically same level as the positive control, and reconstituted CMC microneedles (containing no
hGH, placebo) did not stimulate cell growth, suggesting that CMC had no effect on hGH activity or cell
proliferation, further indicating that CMC was inert. The cell population increase
by reconstituted hGH in a CMC microneedles patch was not statistically different
from the positive control, showing that the fabrication and reconstitution of hGH microneedles patches were not detrimental to hGH stability.
Moreover, storage of hGH microneedles patches for 3 months in air at ambient temperature (23°) and relative humidity (~40%), caused only a 15%
loss of hGH activity. This hGH functional activity study demonstrates the suitability of the formulation and
process to fabricate
hGH dissolving microneedles patches.
We next administered hGH into hairless rats and measured plasma concentration of hGH to assess
bioavailability
of hGH from a dissolving microneedles patch. As shown in Figure 2B, hGH administered by any group quickly had a peak hGH
plasma concentration (C_max) at t_max ≈ 0.7 h (ANOVA, p > 0.05) and
then decreased over the next 6 hours. Subcutaneous injection of hGH was used as
the positive control corresponding to 100% bioavailability. A reconstituted microneedles patch injected subcutaneously had the statistically same pharmacokinetic profile and bioavailability in comparison with the positive control, implying that the fabrication
process of hGH microneedles patch did not degrade hGH integrity.
We next studied hGH
administration using microneedle patches prepared with two different
formulations. The first formulation contained only CMC as the microneedle material.
In this case, the hGH serum concentration profile was significantly lower than the positive control, showing a C_max value that was 20% of the positive
control (Student's t-test, p < 0.05) and a bioavailability
of 13%. We hypothesized that this low bioavailability can be attributed to slow and incomplete dissolution of the CMC
microneedle matrix in the limited amount of interstitial fluid in the skin. To examine this, we prepared microneedles formulated
with a mixture of CMC and trehalose, which can dissolve more
quickly with less fluid than CMC. Consistent with this hypothesis, CMC/trehalose
microneedles achieved four times higher C_max and five times higher
bioavailability than CMC microneedles (Student's t-test, p < 0.05).

                                  
(A)                                                     
                              (B)

Figure 2. (A) Stability of hGH in a dissolving microneedles
patch. Asterisk indicates statistical comparison with positive control.
(two-way ANOVA, P>0.05 for * and P<0.05 for **) (B) Pharmacokinetic
profile of hGH in rat serum after administration.