Development and characterization of a topical gel formulation of

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Development and characterization of a topical gel formulation of adapalene-TyroSpheres and its clinical efficacy assessment Tannaz Ramezanli, and Bozena B. Michniak-Kohn Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00318 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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Molecular Pharmaceutics

Development and characterization of a topical gel formulation of adapaleneTyroSpheres and its clinical efficacy assessment Tannaz Ramezanli1,2, Bozena B. Michniak-Kohn1,2* 1

Ernest Mario School of Pharmacy, Rutgers - The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA

2

Center for Dermal Research, Rutgers - The State University of New Jersey, 145 Bevier Rd, Piscataway, New Jersey 08854, USA

*

Corresponding author: Bozena B. Michniak-Kohn, Tel.: + 1 (848) 445 3589; Fax: +1 732-4455006; Email: [email protected] Center for Dermal Research, Rutgers - The State University of New Jersey, 145 Bevier Rd, Piscataway, New Jersey 08854, USA

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1 2 3

Abstract

4

with adapalene to enhance the efficacy and improve the skin tolerability in acne therapy. An

5

amphiphilic and biocompatible copolymer that self-assembles to nanospheres (known as

6

TyroSpheres) was used to encapsulate adapalene and increase its solubility. A water soluble

7

viscous agent was applied to prepare gel formulation of adapalene loaded-TyroSpheres

8

(Adapalene-TyroSphere). Particle size, morphology, homogeneity and rheological characteristics

9

of the Adapalene-TyroSphere gel formulations were studied. The formulation with the preferred

10

physical and structural properties was further investigated for in vitro skin irritation and in vivo

11

comedolytic activity in a rhino mouse model. Based on the in vitro skin irritation study

12

encapsulation of adapalene in TyroSphere significantly decreased secretion of proinflammatory

13

cytokines (IL-1α and IL-8) confirming TyroSphere formulation of adapalene is less irritant than

14

the commercial gel (Differin ). TyroSphere gel formulation of adapalene improved the

15

comedolytic properties of the formulation by significantly reducing the size of open utricles in

16

rhino mice comparing to Differin . Using TyroSphere we were able to develop an alternative

17

topical formulation of adapalene, which is potentially less irritant and more potent than the

18

commercial product.

19 20

Key Words: adapalene, comedolytic, nanoparticles, rhino mice

In this study we aimed to develop a semisolid formulation of polymeric nanoparticles loaded

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1. Introduction

4

the most common skin disorders in the young population. It is associated with increased sebum

5

production, altered keratinization, inflammation, bacterial colonization in the hair follicles, and

6

immunological host reactions. The early and non-inflammatory stage of the acne usually features

7

comedones, where the abnormal desquamation of follicular epithelium, increased cohesiveness

8

of corneocytes, and androgen-driven activation of sebum secretion can occur. As a result the

9

follicular orifice is partially or completely clogged with excess keratin and dead cells that would

10 11

Acne is a chronic inflammatory disease involving the pilosebaceous unit and it is one of

obstruct the drainage of sebum to the surface of the skin.1, 2 Topical medications are preferred in the treatment of mild to moderate acne. Retinoids

12

are vitamin A derivatives, which are commonly prescribed for topical therapy of the comedonal

13

acne.3 They influence keratinocyte differentiation through their interaction with retinoic acid

14

nuclear receptors (RARs) that activate genes responsible for cellular differentiation, which

15

results in decreased microcomedone formation.4 Adapalene is a third generation retinoid with

16

anti-inflammatory, keratolytic and anti-seborrheic effects. Adapalene’s challenging properties

17

(pKa=4.23, logP=8.04, from SciFinder database) limit its local bioavailability. Erythema,

18

dryness, and scaling have been reported with commercial adapalene formulations.5 These topical

19

adverse effects may be derived from the active ingredient as well as other ingredients in the

20

formulation (e.g. alcohol or chemical penetration enhancers).

21

Since locally acting dermal product has minimal penetration into the blood stream, the

22

systemic pharmacokinetic studies are not a feasible approach to evaluate the efficacy of the new

23

formulation of an existing drug against the reference standard. Therefore, clinical endpoint

24

studies are often recommended to assess the performance of locally acting drug products. The


2


1

rhino mouse (RH J/LeJ) is a strain of hairless mouse that carries the rh gene (recessive allele of

2

the hairless gene). At around 4 weeks, the coat hair of the mice is irreversibly lost and the upper

3

part of the follicular unit forms epidermal pseudocomedone known as an utriculus or utricle.6

4

These utricles (utriculi) become filled with solid impactions of horny cells and sebum and

5

enlarge as the animal ages. At 7-8 weeks, these sebaceous follicles progressively distended due

6

to accumulation of horny material and histologically resemble human microcomedones.6

7

Therefore, utriculi reduction assay using the rhino mouse is a well-characterized preclinical

8

model for evaluation of comedolysis and epithelial differentation7, 8 and can be predictive of

9

retinoid efficacy.9

10

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Nanotechnology platforms were first applied to develop injectables and oral dosage

11

forms and later it also drew attention for potential use in the field of topical and transdermal

12

delivery. In order to develop a successful nanoparticle-based topical delivery system, it is critical

13

to understand nanoparticles’ interactions with the skin and their transport mechanisms.

14

Depending on their size and physicochemical properties and surface charge, nanoparticles may

15

be able to translocate intact into the skin or can be degraded on skin surface and release the

16

incorporated therapeutic agents into the skin. Nevertheless, there is convincing evidence in the

17

literature that irrespective of nanomaterial used, most particles (especially particles larger than

18

20 nm) do not penetrate to viable tissues in healthy skin and the transappendageal pathway

19

seems to be the dominant route of nanoparticle entry into skin.10,15 One of the nanocarriers that

20

have been studied for topical drug delivery is tyrosine-derived polymeric nanoparticles (known

21

as TyroSpheres), which was developed at New Jersey Center for Biomaterials at Rutgers, The

22

State University of New Jersey. TyroSpheres has shown to be a suitable carrier system for

23

several hydrophobic compounds including paclitaxel11, Nile Red12, curcumin, diclofenac sodium,


3

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1

vitamin D313, cyclosporine A14, and adapalene15. In our previous study, we showed that

2

TyroSpheres effectively encapsulated adapalene and substantially enhanced its aqueous

3

solubility, while decreasing the crystallinity of the drug in the formulation. Skin distribution of

4

adapalene via TyroSphere formulation was evaluated ex vivo using human cadaver and porcine

5

ear skin and this was compared with the commercial adapalene formulation, Differin®.

6

Sustained drug release across stratum corneum in 51 h was observed from the TyroSphere liquid

7

dispersion.15 Adapalene-TyroSphere aqueous suspension, have the flow properties and viscosity

8

very similar to that of water and thus it is not ideal for topical administration. This has been

9

addressed in the previous studies and several pharmaceutically acceptable thickening agents,

10

such as Carbopol and hydroxypropyl methylcellulose (HPMC) were used to prepare viscous

11

formulations for TyroSpheres.12, 16 The gel system not only can serve as a stabilizer, but can also

12

form a uniform dispersion of the carriers in the matrix and increase the contact time of the

13

nanoparticles on the skin resulting in enhanced skin penetration of the payload.12 There are

14

several studies in literature, where hydrophilic gels have been used as an effective and inert

15

environment for drug-loaded nanocarriers including nanospheres16, nanocapsules17, liposomes18,

16

and solid lipid nanoparticles19.

17

Two different water-soluble thickening agents that are commonly used in many topical

18

formulations were chosen for this study. Cellulose ether derivatives, such as HPMC and

19

hydroxyethyl cellulose are not only popular in the pharmaceutical industry to make sustained

20

release oral dosage forms (tablets), but are also widely used for thickening topical products and

21

adjusting their rheology.20, 21 Poloxamer 407 is an amphiphilic synthetic copolymer that consists

22

of hydrophobic poly(oxypropylene) block between two hydrophilic poly(oxyethylene) blocks.22


4


1

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The aqueous solution of poloxamer 407 shows thermoreversible properties: it is in a fluid

2

state at room temperature and transitions to a gel at temperatures above 30°C (The sol-gel

3

transition temperature decreases with increase in polymer concentration). In Food and Drug

4

Administration (FDA) guidelines, poloxamer 407 is presented as an “inactive” ingredient for the

5

preparation of various dosage forms, including ophthalmic and topical formulations. This

6

copolymer has been shown to stabilize proteins and micro/nanoparticle-based formulations.23

7

In the present study we report on the development and characterization of topical gel

8

formulations of adapalene-TyroSpheres. The rheological behavior of the TyroSphere gels was

9

assessed and their potential skin irritation and comedolytic efficacy in comparison with the

10

commercial adapalene gel (Differin ) were studied using the well-established in vitro and in vivo

11

models.

12 13

2. Materials and Methods

14

2.1. Materials

15

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Suberic acid (SA), poly(ethylene glycol) monomethyl ether MW 5000 (PEG5K),

16

trifluoroacetic acid (TFA), Tween-80, dimethylformamide (DMF), neutral buffered formalin

17

solution, high viscosity hydroxypropyl methyl cellulose (HV HMPC) viscosity 2,600-5,600 cP,

18

2% in H2O (20°C), 10% neutral buffered formalin solution, adapalene, and Dulbecco's

19

phosphate buffered saline (PBS) were purchased from Sigma Aldrich (St. Louis, MO).

20

Dulbecco’s modified Eagle medium, high glucose (DMEM), gentamycin (50mg/mL), trypsin

21

(0.25% Trypsin-EDTA), fetal bovine serum (FBS), trypsin-EDTA solution, and Dulbecco’s

22

phosphate buffered saline without calcium chloride and magnesium chloride (DPBS) were

23

purchased from Life Technologies (Grand Island, NY). HPLC grade water, acetonitrile, and


5

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1

methanol, and the reagents used for tissue dehydration were obtained from Fisher Scientific

2

(Pittsburgh, PA). Uranyl acetate was purchased from Electron Microscopy Sciences (Hatfield,

3

PA). Micronized poloxamer 407 (MP407) was a generous gift from BASF (Florham Park, NJ).

4

EpiDerm skin kit, EpiDerm culture medium, and MTT assay kit were purchased from MatTek

5

Corporation (Ashland, MA). Differin® topical gel, 0.1% by Galderma containing adapalene,

6

Carbomer 940, edetate disodium, methylparaben, poloxamer 124, propylene glycol, purified

7

water and sodium hydroxide. Rhino mice (male Homozygous, 5-6 weeks old) were obtained

8

from Jackson Laboratory (Bar Harbor, ME). The reagents used for tissue staining were

9

purchased from Biosystems (Buffalo Grove, IL). PEG -b-oligo(desaminotyrosyl-tyrosine octyl

™

™

5K

10

ester suberate)-b-PEG ((DTO-SA/5K) M = 22.9 kDa, Mw = 31.9 kDa, PD = 1.39 obtained from

11

gel permeation chromatography) was synthesized according to previously published and

12

established procedures in the New Jersey Center for Biomaterials and their chemical structure

13

and purity were confirmed by H NMR.24, 25

14

2.2. Preparation of adapalene-TyroSphere gel formulation

15

5K

n

1

ABA triblock copolymers composed of hydrophilic A blocks of poly(ethylene glycol)

16

and hydrophobic B blocks of desaminotyrosyl-tyrosine octyl ester and subaric acid were used for

17

preparation of TyroSpheres. Adapalene loaded-TyroSpheres were prepared by dissolving

18

adapalene and DTO/SA-5K copolymer in DMF. The combination of drug and polymer solution

19

was added dropwise to PBS, pH 7.4 under constant stirring. The nanoparticles were formed and

20

added slight turbidity to PBS. The nanosuspension was filtered using 0.22 µm PVDF filters

21

(Millipore, Ireland) and subjected for ultra-centrifugation (Beckman L8-70M ultracentrifuge,

22

Beckman Coulter, Fullerton, CA) at 65000 rpm (290 000 × g) for 3 h at 18 C. The supernatant

23

was then discarded; the pelleted nanospheres were rinsed and re-suspended in PBS overnight. At

°


6


1

least three replicates were made for each batch of adapalene-TyroSpheres. The viscous

2

formulations were prepared by dissolving the thickening agent in TyroSphere liquid dispersion

3

under constant stirring overnight. The polymers used to form gel-like formulations of

4

TyroSphere were HV HPMC (at 1.5 and 2% w/w), and MP407 (at 15 and 20% w/w). In case of

5

MP407, the TyroSphere dispersion was cooled down to 4°C before addition of the thickening

6

agent.

7

2.3. Size and morphology of TyroSpheres in the gel formulation

8 9

The particle size and morphology of nanoparticles in gel formulations were determined by transmission electron microscopy (TEM) followed by image analysis using Image J software.

10

For sample preparation, small aliquots of 10 times diluted TyroSphere formulations were

11

deposited onto Formvar/Carbon-coated grid and 1% uranyl acetate (Electron Microscopy

12

Sciences, Hatfield, PA) aqueous solution was used for background staining. Electron

13

micrographs were taken on a model JEM 100CX TEM (JEOL Ltd).

14

2.4. Homogeneity analysis

15

To analyze the content uniformity of adapalene in TyroSphere gel formulations, five

16

samples were taken from each gel. The samples were weighed and then lyophilized. Adapalene

17

in dried samples was extracted with DMF and the amount of drug in each sample (w/w) was

18

quantified using HPLC. The test was performed in triplicate. The variability of adapalene

19

content in TyroSphere gels were reported as % RSD, % RSD = (mean drug content/standard

20

deviation)×100.

21

2.4.1 Adapalene quantification using an HPLC method

22 23

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An Agilent 1100 high-performance liquid chromatography (HPLC) system (Agilent Technologies, USA) equipped with a UV/Vis detector and reverse phase column (Promonax


7

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1

Luna 5µm C18(2) ,4.6 × 150 mm) was used for chromatographic separations at 25 °C. A mixture

2

of acetonitrile: water (0.1% TFA) 87:13 (v/v) was applied as the mobile phase with flow rate of 1

3

mL/min. The detection wavelength was set at 309 nm and injection volume was 20 µL. Standard

4

calibration curve was prepared at adapalene concentrations ranging from 0.1 to 100 µg/mL.

5 6 7

2.5 Rheological Characteristics Rheological measurements were performed on the TyroSphere gel formulations and

8

Differin gel using a Kinexus rheometer (Malvern, Worcestershire, UK) equipped with a parallel

9

plate temperature control geometry (diameter: 40 mm, gap: 1 mm). TyroSphere viscous

®

10

formulations were prepared from HV HPMC (1.5 and 2 wt%) and MP407 (15 and 20 wt%) and

11

stored at room temperature for 24-48 h before the study. The samples were equilibrated on the

12

plate for 5 min to reach the running temperature before each measurement. All the rheological

13

studies were performed in triplicate.

14 15 16

2.5.1. Oscillation test The frequency sweep test was performed with constant shear stress of 0.1 at 25 and 32 °C

17

(temperature of the surface of the skin). Rheological test parameters, including storage/elasticity

18

(G′) and loss/viscosity (G″) moduli, were obtained over non-destructive frequency range of

19

0.628-62.8 rad/s.

20 21 22

2.5.2 Strain sweep test The strain sweep test was conducted to determine linear viscoelastic regime of the

23

formulations. The test was run at a constant frequency of 1 rad/s with % shear stress ranging

24

from 0.01 to 10.


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1 2

2.5.3. Temperature sweep test

3

Shear viscosity was measured as a function of temperature under constant frequency of 1

4

rad/s and 0.1% shear strain. The test was performed for a range of 4-40°C. The sol-gel transition

5

temperature was determined for MP407.

6

2.6. Adapalene-TyroSphere MP407 gel microscopy characterization

7

2.6.1. Fluorescence microscopy

8

A small aliquot of adapalene-TyroSpheres in 15 wt% MP407 gel and Differin gel was ®

9

added on the glass slide and covered with cover slid. Fluorescent images were taken from the

10

slides using Zeiss fluorescent microscope in the Dapi channel equipped with AxioCam MRm

11

camera.

12 13 14

2.6.2. Field emission scanning electron microscopy (FE SEM) TyroSpheres in 15 wt% MP407 gel medium with and without adapalene were dispersed

15

lightly on a double-sided sticky tapes mounted to aluminum stubs (sample grid) and air-dried for

16

2 days. Then, the samples were sputter-coated with 15nm of gold using an Electron Microscopy

17

Sciences EMS150T ES coater and imaged with a field emission scanning electron microscope

18

(Zeiss Sigma).

19

2.7. In vitro skin irritation analysis of adapalene-TyroSphere gel and the adapalene

20

commercial product on a three-dimensional (3D) epidermal skin model

21 22

Fully differentiated human epidermis (EpiDerm ) specimens were purchased from ™

MatTek Corporation (Ashland, MA). The in vitro irritation study was conducted according to a


9

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1

protocol provided by the manufacturer. Upon arrival, the tissues were transferred to 6-well plates

2

and incubated at 37°C overnight in 0.9 ml assay medium (EPI-100-ASY, MatTek) at the air-

3

liquid interface. The next day, the surface of EpiDerm™ was treated with Differin gel or

4

adapalene-TyroSphere MP407 gel at drug concentration of 40 µg/cm in triplicate. The tissues

5

were fed from the bottom with the fresh assay medium and incubated at 37°C and 5% CO for 3

6

h or 24 h. EpiDerm™ samples treated with PBS pH 7.4 were used as a negative control. The

7

irritation potential of adapalene formulations was evaluated by MTT cell viability assay and

8

inflammatory mediators/cytokine analysis (to determine IL-1α and IL-8 content in the medium),

9

after the dosing period was completed.

®

2

2

10 11 12

2.7.1. MTT assay on the 3D epidermal skin model In the in vitro skin irritation analysis using three-dimensional (3D) epidermal skin model,

13

the viability of the tissues after dosing was analyzed by MTT. The tissue specimens were rinsed

14

with PBS and transferred to 24-well plates containing 300 µL MTT solution (provided by

15

MatTek Corp.) and incubated for 3 h. Next, the tissues were immersed in a 24-well plate

16

containing 2 ml extracting solution per well. The plate was sealed and incubated at room

17

temperature overnight. The optical density (OD) of the extracted samples was measured at 570

18

nm. Percent tissue viability was calculated using the following formula: % Viability=100 ×OD (sample)OD (negative control)

19 20 21 22 23

2.7.2 ELISA assay Release of IL-1α and IL-8 in the EpiDerm™ culture medium was quantified using ELISA kits from BioLegend (San Diego, CA) according to the assay protocol provided by the


10


Page 12 of 35

1

manufacturer. UV/Vis spectrophotometric measurements were performed with a Biotek

2

PowerWave X microplate reader.

3

2.8. Rhino mouse study

4

2.8.1. Animals Male Homozygous rhino mice (RH J/LeJ) were purchased from The Jackson Laboratory

5 6

(Bar Harbor, ME). The mice were 6-8 weeks old and weighed 18-22 g at the beginning of the

7

study. The animals were housed in groups of four in autoclaved individually ventilated cages at

8

an AAALAC-accredited facility. All animal handling was conducted at the Nelson animal

9

facility (Rutgers University). The mice were fed with PicoLab Rodent diet 20 (LabDiet, St ®

10

Louis, MO) and received autoclaved house water. The experimental protocol was reviewed and

11

approved by the IACUC at Rutgers-The State University of New Jersey.

12 13

2.8.2. Experimental design and treatments The total 15 rhino mice were used for this study and were divided in 3 groups: Differin

14 15

gel (0.1% w/w), adapalene-TyroSphere 15% MP407 gel-1 (0.05% w/w) and adapalene-

16

TyroSphere 15% MP407 gel-2 (0.03% w/w). The mice treated with similar formulations were

17

caged together.

®

18

The mice were anesthetized by inhalation of isoflurane (2-4% in 100% O2). The dorsal

19

skin of animals were treated topically with the following adapalene formulations: Differin (35

20

µg/cm ) or adapalene-TyroSphere MP407-1 (35 µg/cm ) and adapalene-TyroSphere MP407-2 (20

21

µg/cm ). The treated area was covered with Tegaderm™ dressing (3M Health Care, St. Paul,

22

MN) after dosing to prevent rub-off and oral ingestion of the formulations. After covering with

23

Tegaderm™ the gels were massaged on the skin for about 30 s using a cotton swap. The dosing

®

2

2

2


11

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1

was done once daily for 14 days. The mice were examined for signs of skin irritation every day

2

before receiving adapalene treatment.

3 4

2.8.3. Skin biopsies and epidermis mounting technique

5

On day 15 of the study the mice were euthanized by CO inhalation. Next, the skin was

6

cleaned and the treated dorsal skin was removed, cut into smaller fragments (about 1 cm ), and

7

stored in 10% neutral buffered formalin to be used for histological analyses. The untreated dorsal

8

skin areas were also collected and stored in formalin to be used as negative control.

9

2

2

In order to prepare the epidermal sheets, first the skin specimens were placed into 1%

10

acetic acid solution at 4°C overnight. The next day, the dermal layer was gently peeled using a

11

forceps and the epidermis pieces were dehydrated with ethanol (70, 95 and 100% v/v) and

12

cleared with xylene. The cleared epidermis was placed on a glass slide with the dermal side

13

facing up and subjected to microscopic evaluation.26, 27

14

2.8.4. Histological analyses of the mouse skin biopsies

15

Following fixation in formalin solution overnight, the tissues were dehydrated with

16

ethanol (70, 95 and 100% v/v) and xylene respectively following the routine procedure. The skin

17

samples were then embedded in paraffin. The samples were sectioned at 5 µm using a microtome

18

(Microm HM 3555). For each animal skin fragment, three sections were made 3-5 mm apart and

19

were collected on one glass slide. Next the samples were deparaffinized by xylene, rehydrated in

20

a gradient of ethanol solutions, and stained with haematoxylin and eosin (H&E).

21

2.8.5. Quantitative analysis of the utricles

22 23

The H&E stained samples were analyzed with Zeiss optical microscope equipped with Axiocam ERc 5s camera. The depth and diameter of the utricles were measured using


12


Page 14 of 35

1

AxioVision microscope software. Utricles were quantitatively assessed in 3 ways: i) density of

2

the utricles, which was done by counting number of utricles per 6 millimeters length of the skin

3

from vertical cross sections; ii) diameter of the utricle opening that was measured for all the

4

utricles in 6 millimeters length of skin from the vertical cross sections; and iii) the depth of the

5

utricles that was measured for 6 millimeters length of skin in the vertical cross sections (see

6

Figure 1).

7

8 9 10 11 12 13 14 15 16 17 18 19

Figure 1. Rhino mouse skin cross section stained with haematoxylin and eosin. The arrows are showing keratin-containing utricles in a 9-week old rhino mouse. The diameter of utricle opening and the utricle depth is shown by red and green line respectively.

The efficacy of TyroSphere gel treatment relative to Differin was evaluated by ®

comparing the % reduction in size of utricles, calculated as following: %reduction in size= (average utricle size in untreated skin-average utricle size after treatment)/average utricle size in untreated skin×100


13

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1 2


2.9. Statistical analysis The results of skin distribution studies, irritation assays, and animal studies are reported

3

as mean ± standard error (SE). Statistical analysis was performed using Prism Version 6

4

(GraphPad software, La Jolla California). Student t-test and one-way analysis of variance

5

ANOVA followed by Tukey’s post hoc or Wilcoxon signed-rank test (in case the normal

6

distribution assumption did not exist) were applied to determine the difference among the groups

7

in every study. For all analysis, a P value of less than 0.05 derived from a two-tailed test was

8

considered significant unless specified.

9 10 11

3. Results and Discussion

12

3.1. Adapalene-TyroSphere viscous formulations

13

DTO-SA copolymers are composed of hydrophilic PEG side chains and hydrophobic

14

desaminotyrosyl alkyl ester backbone. Because of its amphiphilic nature, this copolymer system

15

can self-assemble to nanomicelles (TyroSpheres) in aqueous media and encapsulate a wide range

16

of hydrophobic molecules like adapalene in its hydrophobic core. Gel formulations of adapalene-

17

TyroSphere were developed for a more convenient topical dosage form that was more viscous

18

and hence could be maintained more easily on the skin. The presence of nanoparticles results in

19

slight turbidity of the gel formulations. The MP407 TyroSpheres form a clearer gel than that

20

using HV HPMC TyroSpheres.


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Page 16 of 35

1 2

3.1.1. Particle size analysis TEM was used to study particle size and morphology of adapalene-TyroSphere viscous

3 4

formulations. The mean diameter size of adapalene-TyroSphere in the representative TEM

5

images (Figure 2) measured using Image J analysis are as follows: 32.2±5.2, 36.7±4.3, and

6

35.4±5.7 nm for adapalene-TyroSphere in liquid suspension, in liquid suspension, 2% HV

7

HPMC, and 15% MP407 respectively. TEM images of final adapalene-TyroSphere gel-like

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formulations (Figure 2) show particle size similar to what observed with adapalene-TyroSphere

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liquid dispersion, a narrow particle size distribution, and lack of particle agglomeration in the

10

presence of thickening agents. Increase or decrease in concentration of the thickening agent did

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not have any short term effects on the particle size and morphology of TyroSpheres (data not

12

shown)

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2 3 4 5 6

Figure 2. Transmission electron micrographs of adapalene-TyroSpheres from the top to bottom: in liquid suspension, 2% HV HPMC, and 15% MP407.

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3.1.2. Gel homogeneity

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Homogeneity of the gel-like adapalene-TyroSphere formulations (0.025% w/w) was studied by measuring drug content in the gel samples. The relative standard deviation (RSD) was

10

calculated as 4.5, and 2.9% for 2% HV HPMC and 15% MP 407 Adapalene-TyroSphere gels

11

respectively, which are in acceptable range for the pharmaceutical semi-solid dosage forms.

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Page 18 of 35

3.2. Rheological analysis The mechanical strength and viscoelastic properties of adapalene-TyroSphere viscous

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formulations were studied to analyze rheological behavior of these topical formulations and

4

compare them with Differin gel as a reference standard. ®

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3.2.1. Frequency sweep test

Rheological behavior of topical/transdermal formulations has a profound effect on their

8

spreadability, retention and contact time on the skin surface. Viscosity and spreadability are

9

regarded as critical quality attributes in the initial product development stage.

10

Oscillatory measurements are one of the most recommended methods for rheological

11

studies.28 The results of oscillation study are illustrated in Figures 3.C-3.D, where storage (G′)

12

and loss (G″) moduli were measured as a function of frequency.

13

Figure 3.A and 3.B depicts the frequency sweep test on adapalene-TyroSpheres in 2%

14

and 1.5% HV HPMC. At both concentrations, the HPMC formulations behaved as viscous

15

solutions and both G′ and G″ showed relative strong dependency on the frequency. The values

16

obtained for G′ were lower than G″ at similar oscillation rate, showing the system is primarily

17

viscous rather than elastic. This trend was observed at both 25 and 32 °C. Additionally, presence

18

of adapalene-TyroSpheres did not significantly affect the shear moduli.

19

In Figure 3.C and 3.D the G′ and G″ of MP407 gels are plotted logarithmically against

20

frequency. At both 15 and 20 wt% of MP407, the oscillation rate did not affect G′ and G″ and the

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G′ was much higher than G″ indicating the system is elastic. Similar trend was observed with the

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gels at 32°C with higher values for both of the shear moduli The results of frequency sweep test

23

showed that the gels made with MP407 are well-structured system and sedimentation of particles


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are unlikely to occur. Presence of TyroSpheres slightly increased the viscosity but did not change

2

the behavior of the system.

3 4

The gelling agent in Differin is Carbomer 940, which is a cross-linked polyacrylate

5

polymer capable of providing high viscosity and forming clear aqueous and hydroalcoholic

6

gels.29 The results of frequency sweep test for Differin gel is demonstrated in Figure 3.G.

7

Rheological measurements of G′ and G″ for Differin showed independency of both shear moduli

8

from oscillation rate. At both temperatures the G′ is much more than G″ indicating the system is

9

elastic. Both shear moduli were not affected by change in the temperature.

10

®

®

®

The results of the frequency sweep tests on the viscous formulations revealed that the

11

gels made with MP407 behaved similar to Differin gel. In both gels, G′ and G″ are independent

12

of the frequency. In both systems, elastic modulus dominates the complex modulus meaning the

13

system is very elastic. In the gels made with HV HPMC, the loss and storage moduli are both

14

enhanced with increase in the frequency. Presence of TyroSpheres in the gels slightly increased

15

the shear viscosity but did not change the rheological behavior and strength of the gels.

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Page 20 of 35

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Figure 3. A) The influence of oscillation rate on shear moduli of 2% and 1.5 wt% HV HPMC at 25°C. B) The influence of oscillation on elastic modulus (G′) in HV HPMC formulations at 25 and 32°C with and without adapalene-TyroSpheres (Ada-Tyr). C) The influence of oscillation rate on shear moduli of 20 and 15 wt% MP407 gels at 25°C. D) The influence of oscillation rate on elastic modulus (G′) in MP407 formulations at 25 and 32 °C with and without adapaleneTyroSpheres (Ada-Tyr). Strain sweep test on E) adapalene-TyroSpheres in 2% HV HPMC, F) adapalene-TyroSpheres in 15% MP407, and H) Differin® to establish the linear viscoelasticity regime of the gels. G) Oscillatory viscometry of Differin® gel recorded at 25 and 32°C.


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3.2.2. Strain sweep test The strain sweep test (Figure 3.E, 3.F and 3.H) was performed on TyroSphere viscous

3

formulations and Differin in order to establish the regime of linear viscoelasticity (LVE) and to

4

study mechanical strength of the gels. In the cellulose-based system (HPMC) the loss modulus

5

(G″) dominates and relative to MP407 and Differin gel, this formulation showed the highest

6

resistance to structural change following increase in shear strain. But in MP407 and Differin

7

storage modulus (G′) was higher than loss modulus, confirming both systems are elastic. Also

8

the structure of both gels break down at nearly 5% shear strain.

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®

®

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Viscosity of a semi-solid formulation may impact skin retention of the dosage form and drug

11

delivery/penetration via the skin. Based on the results we obtained from the rheological

12

characteristics of TyroSphere viscous formulations, we found 15% MP407 the best gel system to

13

use for TyroSpheres with closest rheological properties to Differin . ®

14

15 16

3.3. Adapalene-TyroSphere MP407 microscopy characteristics In the commercial formulation (Differin ) adapalene exists in form of microcrystals ®

17

dispersed in the vehicle and these microcrystals can be visualized under fluorescent microscope

18

(Figure 4.E). However, in fluorescent images taken from adapalene-TyroSphere MP407 gels no

19

microcrystal was observed even after one month storage at room temperature (Figure 4.F). This

20

confirms drug encapsulation by TyroSpheres and lack of drug leakage from nanoparticles in

21

short-term storage.

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FE SEM images taken from adapalene-TyroSphere MP407 gel demonstrate homogenous dispersion of nanoparticles in the gel structure and lack of particle agglomeration (Figure 4.A-


20


1

D). The nanoparticles are seen as white (average 32 nm) size spheres in the gel system. The

2

particle size of TyroSpheres found to be similar to what was observed with TEM.

Page 22 of 35

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Figure 4. A-C) FE SEM micrograph of adapalene-TyroSpheres in 15% MP407 gel, (the scale bars are 200 nm, 20µm and 200nm respectively) and D) TyroSphere MP407 gel without drug (scale bar = 200 nm). TyroSpheres are the white spherical particles in the images. E) Fluorescent images of Differin® gel and F) adapalene-TyroSphere MP407 gel. Adapalene microcrystals (in blue) are observed in Differin® sample, but not in TyroSphere gel formulation.

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3.4. In vitro Irritation study on MatTek epidermal skin model (EpiDerm ) TM

The most frequent side effect of topical retinoids is known as the “retinoid reaction”, which is characterized by pruritus, peeling, burning, and redness in the site of application and

10

peaks within the first few weeks of the treatment. This phenomenon is due to the free carboxylic

11

acid in the structure of retinoids and occurs more with application of tretinoin and tazarotene

12

than with adapalene. Release of pro-inflammatory cytokines such as IL-1α, IL-6, TNF-α, and IL-

13

8 in is known to initiate this retinoid reaction.4, 30

14

Commercially available artificial skin models such as EPISKIN and EpiDerm™ are

15

widely used nowadays to assess acute irritation potential of the topically applied compounds.

16

They have proved to be a reliable alternative to the in vivo irritation test that is conducted on

17

albino rabbits.31 We used EpiDerm™ skin model from MatTek corp. to compare irritation

18

potential of adapalene-TyroSphere MP407 gel with the commercial adapalene gel, Differin .

19

Based on MTT tissue viability assay performed on EpiDerm™ treated with the adapalene

20

formulations, both Differin and TyroSphere formulations were found to be non-irritant since the

21

% tissue viability was observed at over 50% at all time points (Figure 5.A). No significant

22

difference was found with the tissue viability of the skin treated with both formulation during 3,

23

24 and 40 h application.

24

®

®

Release of proinflammatory cytokines (a well-established biomarker of acute irritation)

25

was used to quantify the amount of irritation in EpiDerm™ skin model. ELISA assays were used

26

to measure concentrations of IL-1α and IL-8 that were secreted from EpiDerm™ treated with


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Page 24 of 35

1

adapalene formulations. At some of the time points (especially with shorter exposure time)

2

application of adapalene-TyroSpheres MP407 resulted in less secretion of IL-1α and IL-8

3

compared to treatment with Differin (Figures 5.B-C). There was no significant difference in

4

release of pro-inflammatory cytokines from EpiDerm™ treated with adapalene-TyroSphere and

5

negative control (PBS) except the IL-1α secretion during 40 h application of the formulations.

6

We assume that encapsulation of adapalene in the nanoparticles and the sustained release manner

7

that was observed with TyroSphere gel formulation prevent the non-physiological overload of

8

adapalene in the skin; and therefore, reduce irritation potential of the retinoid formulation

9

compared to the commercial product. With Differin since the drug crystals are dispersed in the

®

®

10

vehicle they directly contact the skin, inducing release of inflammatory cytokines. Similar

11

observations have been reported in the literature, where irritant drugs like retinoic acid were

12

encapsulated in particulate systems. Castro et al. 6 reported significant decrease in skin irritation

13

of retinoic acid in solid lipid nanoparticle formulations without reduction in efficacy in vivo.

14

Moreover, the absence of other potentially irritant ingredients such as chemical penetration

15

enhancers in the TyroSphere MP407 gel could be another reason for decreased skin irritation that

16

was observed from our formulation relative to the commercial gel.

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.


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Figure 5. A) Percentage (%) tissue viability of EpiDerm™, treated with adapaleneTyroSpheres MP407 gel (Ada-Tyr Mp407) and Differin® for 3, 24, and 40 h. Release of B) IL1α and C) IL-8 from EpiDerm™ treated with adapalene-TyroSpheres in 15% MP407 (Ada-Tyr MP407), Differin®, and phosphate buffered saline (PBS) for 3 and 24, and 40 h. Data are shown as mean ± SE, * P

Development and characterization of a topical gel formulation of

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