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Development

of

performance

matrix

for

generic product

equivalence

of acyclovir

topical

creams

Yellela S.R. Krishnaiah a, Xiaoming Xu a, Ziyaur Rahman a, Yang Yang a,Usha Katragadda a, Robert Lionbergerb, John R. Peters b, Kathleen Uhlb,Mansoor A. Khana,*aDivision of Product Quality Research, Of ce of Testing and Research, Of ce of Pharmaceutical Sciences, Center for Drug Evaluation and Research, Food and

Drug Administration, Silver Spring, MD, USAbOf ce of Generic Drugs, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA

A

R

T

I

C

L

E

I

N

F

O

Article history:

Received 14 May 2014Received in revised form 10 July 2014Accepted 24 July 2014Available

online

30

July

2014

Keywords:

AcyclovirTopical creamProcess variablesQuality

metricsSamenessQ1/Q2/Q3

A

B

S

T

R

A

C

T

The effect of process variability on physicochemical characteristics and in vitro performance of qualitatively (Q1)andquantitatively (Q2)equivalent generic acyclovir topical dermatological creamswasinvestigated todevelop amatrixof standards fordeterminingtheir invitrobioequivalencewith referencelisted drug (RLD) product (Zovirax1). A fractional factorial design of experiment (DOE) with triplicatecenterpointwas used to create11acyclovir cream formulationswithmanufacturingvariables suchas pHof aqueous phase, emulsication time, homogenization speed, and emulsication temperature. Threemore formulations (F-12–F-14) with drug particle size representing RLD were also prepared where thepH of the nal product was adjusted. The formulations were subjected to physicochemicalcharacterization (drug particle size, spreadability, viscosity, pH, and drug concentration in aqueousphase) and in vitro drug release studies against RLD. The results demonstrated that DOE formulationswere structurally and functionally (e.g., drug release) similar (Q3) to RLD. Moreover, in vitro drugpermeation studies showedthat extent of drug bioavailability/retention in human epidermis from F-12–F-14were similar to RLD, although differed in rate of permeation. The results suggestedgeneric acyclovir

creams can be manufactured to obtain identical performance as that of RLD with Q1/Q2/Q3.Published by Elsevier B.V.

1. Introduction

Zovirax1 cream

was

approved

by

US/FDA

in

2002

for

thetreatment

of

recurrent

herpes

labialis

(cold

sores)

in

adults

andadolescents.

It

is

a

topical

dermatological

product

containing

5%w/w of acyclovir in aqueous cream base formulated withcetostearyl alcohol, mineral oil, poloxamer 407, propylene glycol,sodium

lauryl

sulfate,

water,

and

white

petrolatum

as

inactiveingredients

(Zovirax,

2002).

Acyclovir

is

a

synthetic

purinenucleoside analog with in vitro and in vivo inhibitory activityagainst herpes simplex virus types 1 (HSV-1), 2 (HSV-2), andvaricella-zoster

virus

(VZV)

(Acosta

and

Flexner,

2011).

There

areno

generic

acyclovir

topical

dermatological

cream

productsavailable at this time in the market. The possible generic products

of

acyclovir

topical

cream

have

to

conform

to

the

same

standards

of quality as that of Zovirax1 cream (reference listed drug product,RLD)

and

demonstrate

clear

bioequivalence

(BE)

by

in

vivo

or

invitro

methodologies.

The

availability

of

product

quality

metrics

iscritical

to

demonstrate

that

generic

pharmaceutical

drug

productsare therapeutically equivalent and interchangeable with theirassociated innovator's product.A

list

of

in

vivo

and

in

vitro

methods

have

been

provided

toestablish

the

BE

under

regulation

21CFR320.24(b)

44 (21CFR320.1,2014; 21CFR320.24, 2014). In vivo studies in humans comparingdrug/metabolite concentrations in an accessible biological uid, invivo

testing

in

humans

of

an

acute

pharmacological

effect,

andcontrolled

clinical

BE

trials

in

humans

to

establish

ability

toachieve an equivalent clinical endpoint with no evidence of differing safety prole are to be chosen as the rst, second, andthird

approaches.

The

in

vitro

methods

are

to

be

chosen

as

the

nextavailable

choices.

The

sponsors

may

choose

any

other

rationalapproach and provide data to convince FDA on the use of suchapproach in demonstrating bioequivalence. One or more of theseapproaches

might

be

used

to

demonstrate

BE.

For example,

the

* Corresponding author at: FDA/CDER/OPS/OTR/DPQR, White Oak, LS Building 64,Room 1070, 10903, New Hampshire Ave, Silver Spring, MD 20993-002, USA.Tel.:

+1

301

796

0016.E-mail address: [email protected] (M.A. Khan).

http://dx.doi.org/10.1016/j.ijpharm.2014.07.0340378-5173/Published by Elsevier B.V.

International

Journal

of

Pharmaceutics

475

(2014)

110–122

Contents

lists

available

at

ScienceDirect

International

Journal

of

Pharmaceutics

journa l home page : www.e lsevier.com/loca te / i jpharm

mailto:[email protected]://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://www.sciencedirect.com/science/journal/03785173http://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.elsevier.com/locate/ijpharmhttp://www.sciencedirect.com/science/journal/03785173http://dx.doi.org/10.1016/j.ijpharm.2014.07.034http://dx.doi.org/10.1016/j.ijpharm.2014.07.034mailto:[email protected]://crossmark.dyndns.org/dialog/?doi=10.1016/j.ijpharm.2014.07.034&domain=pdf

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bioequivalence of solid oral dosage forms intended for systemicdelivery

is

established

by

in

vivo

pharmacokinetic

(PK)

studieswith

a

support

of

comparative

in

vitro

drug

release

data.

Thisapproach has been successfully applied to a large number of drugproducts (Kryscio et al., 2008). However, the conventional in vivoBE

study

with

PK

endpoints

such

as

C max and AUC is neitherappropriate

nor

feasible

for

establishing

BE

of

topically

applieddermatological products. Determination of topical bioequivalencefor locally acting drugs in skin is more complicated as local drugconcentrations

cannot

be

measured

directly.

The

guidance

onbioavailability

and

bioequivalence

drafted

by

Committee

of Proprietary Medical Products (CPMP) of the European regulatoryauthorities stated “for medicinal products not intended to bedelivered

into

the

general

circulation,

the

common

systemicbioavailability

approach

cannot

be

applied”

(EMA,

2000).

The

USFDA provided certain recommendations with respect to theestablishment of BE for such specic products (FDA, 2010). Draftguidance

documents

on

locally

acting

topical

drug

products

suchas

cyclosporine

ophthalmic

emulsion

and

acyclovir

ointment

havebeen developed by FDA to provide recommendations to sponsorsto meet statutory and regulatory requirements (FDA, 2012, 2013).Generally,

FDA

addresses

the

issue

on

a

case

by

case

basis

asoutlined

by

the

drug-specic

guidance.

Therefore,

it

is

necessary

to

identify the key scientic principles for consistent and ef cientidentication of bioequivalence methods for locally acting topicaldermatological

products.The

current

regulation

requires

conducting

clinical

endpointtrials for demonstrating BE between topical generic and RLDproducts when alternative methods, such as pharmacodynamicendpoint

measures

are

not

feasible

(21CFR320.1,

2014;21CFR320.24,

2014). Topical

glucocorticoids

(Chang

et

al.,

2013b)are an example of products where a clear pharmacodynamicendpoint (skin blanching) is possible. Clinical endpoint bioequiva-lence

studies

with

topical

drug

products

are

lengthy

and

expensive(Shah et al.,1998). These studies are subjected to greater variabilitythan other in vivo methods for determining bioequivalence. Thus,the

large

inter-subject

variability

and

dichotomous

nature

of

these

clinical endpoint bioequivalence studies demand the enrollment of several hundred subjects to achieve suf cient statistical power(Bhandari et al., 2002; Donner and Eliasziw, 1994). In order todetermine

BE

of

acyclovir

topical

cream

products

for

treatingherpes

simplex

labialis,

the

primary

endpoint

is

the

time

tocomplete healing of lesions. This is particularly challenging forthree reasons: (1) the severity of lesions is confounding; (2) lesionslast

a

short

period

of

time

and

heal

rapidly

regardless

of

treatment;and

(3)

the

effectiveness

of

therapy

is

related

to

the

rapidity

withwhich treatment is initiated. In two clinical studies conducted forZovirax1 cream, no signicant difference was observed betweensubjects

receiving

Zovirax1 cream

or

vehicle

(Zovirax,

2002). Themean

duration

of

the

recurrent

herpes

labialis

episode

wasapproximately half a day shorter in the subjects (n = 682) treated

with

Zovirax1

cream

(4.5

days)

compared

with

subjects

(n

=

702)treated

with

placebo

(5

days).

The

considerable

variability

inclinical

endpoints

is

common

and

renders

the

BE

clinical

designdif cult to detect the small difference in therapeutic responsebetween generic and RLD (Chang et al., 2013b; Yacobi et al., 2014).A

variety

of

surrogate

methods

such

as

skin

stripping/dermatopharmacokinetics

(DPK),

dermal

microdialysis

(DMD),in vitro permeation studies and near infrared (NIR) spectroscopyhave been explored to demonstrate the BE of topical dermatologi-cal

products

(Lionberger,

2008;

Narkar,

2010;

Yacobi

et

al.,

2014).Yet

these

surrogate

methods

are

even

more

prone

to

failures

indetecting low drug concentration in skin due to their limitedsensitivity, technical dif culty, and high variability. The scope andlimitations

associated

with

these

techniques

have

been

reviewed

(Herkenne

et

al.,

2008;

Mateus

et

al.,

2013;

Narkar,

2010;

Yacobi

et al., 2014). For example, skin stripping has been used for testingBE

of

topical

dermatological

products

acting

in

stratum

corneum(N'Dri-Stempfer

et

al.,

2008,

2009;

Navidi

et

al.,

2008;

Parry

et

al.,1992). But this is unsuitable for studying the BE of topicaldermatological products whose site of action is a compromisedskin

(e.g.,

cold

sores

due

to

herpes

labialis).The

in

vitro

drug

permeation

across

human

skin

and

in

vitrodrug release testing may be suitable to test the sameness (Q3) of Q1/Q2 equivalent topical dermatological products with respect totheir

performance.

Such

in

vitro

tests

have been

recommended

totest

the

product

sameness

under

certain

scale-up

and

post-approval changes (SUPAC) as it is believed to collectively reectanydifferences due to several physicochemical properties such assolubility,

particle

size

of

drug,

and

rheological

properties

of vehicle

(FDA,

1997).

The

present

study

was

carried

out

tounderstand and identify the appropriate in vitro quality metricsthat can discriminate the effect of process and formulationvariables

on

critical

quality

attributes

(CQA)

of

possible

genericacyclovir

topical

cream

formulations

having

the

same

Q1/Q2

tothat of Zovirax1.Quality by design (QbD) approach was used to study the effect

of

process

and formulation

variables on

CQA

of

acyclovir

topicalcream

formulations.

The

preparation

of

acyclovir

cream

typically

involves homogenization of oil-soluble and water-soluble com-ponents along with the drug to form oil-in-water cream at 70 C.Based

on

the

preliminary

process

understanding,

three

processparameters

(emulsication time,

homogenization speed, andtemperature of oil/water phases) were identied as criticalprocess parameters (CPP). Moreover, the HSV-1 infection andreplication

occurs

in

the

basal

cell

layer

of

the

epidermis

(Parryet

al.,

1992).

Therefore

effectiveness

of

acyclovir

topical derma-tological creams depends on drug permeation across skin anddrug retention in epidermis (DRE), which in general is a functionof

the

aqueous phase

drug concentration

(thermodynamicactivity). The equilibrium water solubility of acyclovir wasreported to be inuenced by pH with the highest solubilitybeing

at pH 9 (Shojaei et al., 1998). Thus pH of

acyclovir cream products was chosen as a formulation variable inaddition to the above three CPPs for studying their inuence onCQA. A fractional factorial design (24–1) with triplicate centerpoint

was chosen

to

study the

effects of

process and formulationvariability on

product

CQA.

This

is

a

Resolution

IV

design

whereestimation of the main effects is not confounded by two-factorinteractions (Chang et al., 2013a). Accordingly 11 formulationswere

prepared

and subjected

to

physicochemical

characterizationand

in

vitro

performance

testing

to

test

the

sameness (Q3)

of Q1/Q2 equivalent acyclovir creams.

2.

Materials

and

methods

2.1. Materials

Zovirax1 cream

was

obtained

from

Bradley

Drugs,

Bethesda,MD,

USA.

Acyclovir

(>99%)

was

purchased

from

RIA

InternationalLLC, East Hanover, NJ, USA. Propylene glycol USP, white petrolatumUSP, mineral oil USP, glacial acetic acid USP and sodium laurylsulfate

(SLS)

NF

were

purchased

from

Fischer

Scientic,

Norcross,GA,

USA.

Poloxamer

407

NF

and

cetostearyl

alcohol

NF

werepurchased from Spectrum Chemical Manufacturing Co., NewBrunswick, NJ, USA.

2.2.

Preparation

of

acyclovir

cream

formulations

Four process/formulation variables (pH of aqueous phase,emulsication

time,

homogenization

speed

and

emulsication

temperature)

were

studied

using

a

fractional

factorial

design

with

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triplicate

center

points.

Based

on

this

Design

of

Experiment

(DOE),11

acyclovir

formulations

(DOE

1

to

11)

were

prepared

(Table

1).Briey, aqueous phase and oil phase were mixed and homoge-nized. Aqueous phase was prepared by dissolving poloxamer 407in

water

by

mechanical

stirrer

(RW-20

digital,

IKA,

WilmingtonUSA)

at

500

rpm

for

30

min,

adding

propylene

glycol

and

adjustingits pH (5.5, 6.75, or 8.5). Acyclovir was dispersed in the aqueous

phase by mechanical stirring at 500 rpm at 70, 80, or 90 C for10

min.

Similarly,

oil

phase

was

prepared

by

melting

whitepetrolatum,

cetostearyl

alcohol,

and

mineral

oil

at

70,

80,

or90 C. Sodium lauryl sulphate was dispersed in the oil phase bySilverson

homogenizer

(L5M-A,

Silverson,

Baltimore,

MD)

at2000

rpm

for

2

min.

Aqueous

phase

was

added

to

oil

phase

andhomogenized

by

Silverson

Homogenizer

for

15,

22.5,

or

30

min

at2000, 3750, or 5000 rpm. The cream was allowed to cool at roomtemperature

while

being

homogenized

at

1000

rpm

for

2

h.Placebo

cream

was

also

prepared

in

the

same

way

but

withoutdrug

and

pH

adjustment.

Drug

was

physically

added

to

the

placebocream

to

make

its

composition

equal

to

DOE

formulations.

Threemore

formulations

(F-12–F-14)

were

prepared

with

a

slightmodication

in

the

preparation

of

aqueous

and

oil

phases

using

acyclovir drug particles representing Zovirax1 cream. The aqueousphase

pH

was

adjusted

after

dissolving

SLS

and

dispersingacyclovir,

and

the

oil

phase

was

without

SLS.

The

aqueous

andoil

phases

were

homogenized

at

5000

rpm

and

70 C

for

15

min(process

conditions

representing

as

those

of

DOE-9).

The

pH

of acyclovir

cream

formulations

was

measured

using

a

pH

meter.

Allthe

acyclovir

cream

formulations

were

packed

(5

g

in

quantity)

inmultiple-dose

aluminum

tubes,

and

stored

in

a

chamber

at25 C/60%

RH

until

used

for

further

studies.

2.3.

Drug

content

uniformity

Topically

applied

semisolid

drug

products

such

as

acyclovircream

may

show

physical

separation

during

manufacturing

process

and

during

their

shelf

life.

To

ensure

their

integrity,

it

isessential

to

evaluate

the

uniformity

of

the

nished

product

withrespect

to

visual

uniformity

and

uniformity

of

active

ingredients.This

was

carried

out

as

per

the

procedure

described

in

USP

(USP36-NF31, 2013a). The bottom tube seal was cut off and a vertical cutwas

made

from

the

bottom

to

the

top

of

the

tube.

The

tube

aroundthe

upper

rim

was

cut

to

open

the

two aps and aps laid open toexpose the product. The product was inspected for the presence of phase separation, and change in physical appearance and texture(e.g.,

color

change).

An

appropriate

amount

of

accurately

weighedproduct

(100

mg)

was

removed

from

the

top,

middle,

and

bottomportions of the tube, and transferred to a ask containing 400 mlsolvent (pH 9.2 borate buffer). The contents were homogenized at7600

rpm

for

15

min

(25 C), ltered, diluted suitably, and injected

into

HPLC

column

for

determining

acyclovir

concentration.

This

was

used

to

calculate

the

amount

of

acyclovir

in

the

samplesobtained

from

the

tube.

2.4. Drug concentration in aqueous phase

The

acyclovir

cream

formulations

including

Zovirax1

were lled into Eppendorf tubes (capacity 5 ml) without air gaps.

These tubes were centrifuged at 14,000 rpm for 5 h (sampletemperature

set

to

25 C).

The

top

layer

of

oil

phase

separated

fromthe

cream

was

scooped

out

carefully,

and

remaining

aqueous

phasecentrifuged at 14,000 rpm for 1.5 h to clearly separate the traces of oil

phase.

The

resultant

aqueous

phase

was

suitably

diluted,ltered

through

0.45-mm

syringe

lter

and

injected

into

HPLC

fordetermining

acyclovir

concentration.

2.5.

X-ray

powder

diffraction

(XRPD)

Acyclovir

physical

forms

in

the

cream

were

conrmed by XRPD.Diffractogram

were

collected

using

a

Bruker

D8

Advance

withDaVinci

design

(Bruker

AXS,

Madison,

Wisconsin)

equipped

withthe

LYNXEYE

scintillation

detector

and

Cu

Ka radiation

(l= 1.5405 Å) at a voltage 40 kV and current 40 mA. About500

mg

sample

were

placed

in

the

sample

holder

and

diffracto-gram

was

collected

over

2u

range

of

4–40 with

an

increment

of 0.0114

at

1

s

per

step

(3000

total

steps).

Sample

holder

was

rotatedduring

run

to

get

the

average

diffractogram

of

the

sample.

TheXRPD

operation,

data

collection,

and

data

analysis

were

achievedthrough

Diffrac.SuiteTM (V2.2).

2.6. Particle size analysis

Cream

samples

wereapplied

onto a

glass

slide

and

spread

evenlyusing

a

cover

slip.

Images

were acquired

using an

Olympus

BX51polarized

light

microscopy

(Olympus

America

Inc.

Melville,

NewYork).

On

average about

10

microscopy

images

(about

200–500

particles)

were

acquired

for

each

sample

(200

magni-cation).

In

each

image,

particles

were manually

counted

andmeasured

using

Olympus

cellSens

software

(Olympus

America

IncMelville, New

York)

to obtain

particle

size

and

elongation

informa-tion which were then imported into Excel to generate statisticalinformation

(i.e.,

D10, D50and D90) with a percentile function.Particlesize

of

acyclovir

API

powder

was

determined

using a

HALEOS

laserdiffraction instrument (Sympatec, Clausthal-Zellerfeld, Germany),equipped with a R5 lens (0.5–875mm). For each measurement,approximately

100mg

of

dry

sample

was

dispersed

at

a

feed

rate

of 50%

by a

controlled

feeder

(VIBRI/L),

with

dry

dispersal

(RODOS)attachments set at a main pressure of 1.0bar. The triggeringcondition was set at 0.2% optical concentration. Data were analyzedusing

Fraunhofer

theory

with

WINDOX

5

software

(Sympatec,

Clausthal-Zellerfeld,

Germany).

Table 1

Fractional factorial design (24–1) to assess the process variables of acyclovir cream formulations.

ID Emulsication time(min)

Homogenization speed (rpm) Temperature(C)

pH of aqueous phase

DOE-1 15 2500 90 8.5DOE-2 15 5000 90 5DOE-3 30 2500 90 5DOE-4 30 5000 90 8.5DOE-5 22.5 3750 80 6.75DOE-6 22.5 3750 80 6.75DOE-7 22.5 3750 80 6.75DOE-8 30 2500 70 8.5DOE-9

15

5000

70

8.5DOE-10

15

2500

70

5DOE-11 30 5000 70 5

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2.11. HPLC analysis of acyclovir

An

Agilent

1260

Series

high-performance

liquid

chromatogra-phy (HPLC) system equipped with binary solvent pump, autosam-pler, photodiode array detector, thermostated columncompartment

and

Chemstation

chromatographic

software

wasused

for

estimating

concentration

of

acyclovir.

The

methoddescribed elsewhere in the literature was used with a fewmodications (Parry et al., 1992). Waters SunFireTM C18 column(5

mm, 4.6 150 mm) maintained at 25 C was used to eluteacyclovir.

The

mobile

phase

used

was

an

isocratic

mixture

of

glacialacetic acid and water (0.5% v/v). The ow rate was 1.2ml/min.Standard solutions (5 or 50 ml) containing 0.05 to 10 mg/ml of acyclovir

were

injected

into

the

HPLC

column,

and

the

elutingacyclovir

solutions

were

detected

at

254

nm.

The

peak

areas

wereobtained and subjected to regression analysis. A good linearrelationship was observed between the peak area of acyclovirstandard

solutions

and

their

concentration

with

a

high

correlationcoef cient

(r 2 =

1.0).

The

HPLC

analytical

method

was

validatedaccording to USP Validation of Compendial Methods (USP36-NF31,2013c). The method was precise (intra- and inter-day variation was

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3.1. Acyclovir physical form

X-ray

powder

diffraction

of

acyclovir

drug

powder,

placebocream, physically mixed drug in the placebo cream, and DOEformulation are shown in Fig. 1. The drug was crystalline as itshowed

characteristics

diffraction

peaks

at

6.28,

6.85,

10.46,

13,

16,21,

23.90,

26.15

and

29.2.

On

the

other

hand,

placebo

creamshowed broad and low intensity peaks at 6.35, 21.24, and 21.58

suggesting its partly crystalline nature. Physically mixed drug inthe

placebo

cream

showed

peaks

of

drug

and

placebo

cream.However,

intensity

of

drug

peaks

in

the

placebo

cream

was

muchlower when compared with raw API peaks. This was probably dueto dilution of the drug substance with the placebo cream. Similarly,DOE

formulations

showed

identical

diffraction

pattern

as

that

of physically

mixed

drug

in

the

placebo

cream.

This

suggested

thatprocessing of drug during cream manufacturing has not changedits polymorphic/physical forms.

3.2.

PH,

drug

content

uniformity,

and

drug

concentration

of

aqueous

phase

In

the

nal

dosage

form

of

acyclovir

cream

drug

product,acyclovir

may

exist

in

both

oil

and

aqueous

phases

as

soluble

form

(aqueous and oil) and suspended form (aqueous and oil). Becausethe amount of drug present in the formulation (5% w/w) greatlyexceeds

the

equilibrium

solubility

of

the

drug

(2–4

mg/ml

betweenpH

2–9),

majority

of

the

drug

is

expected

to

be

in

the

aqueousphase as suspended form (Shojaei et al., 1998). However, withrespect to the product performance and its therapeutic outcome,solubilized

drug

in

aqueous

phase

is

the

most

relevant

parameter.

As shown in Fig. 2, the amount of drug dissolved in aqueous phase(total

aqueous

drug)

is

determined

by

two

equilibriums:

solubili-zation

and

partitioning

(log P = 1.56) (Kristl et al., 1993), which

should remain relatively constant considering that both solid drugamount and oil phase concentration remain unchanged. Depend-ing

on

the

actual

pH

of

aqueous

phase,

the

total

dissolved

drug

inaqueous

drug

phase

may

present

as

three

different

species:cationic, zwitterionic, and anionic, each of which may have slightdifferent skin permeation potential (Shojaei et al., 1998). For thisreason,

in

the

current

study,

pH

of

aqueous

phase

was

identied

asone

risk

factor

that

may

impact

the

product

performance

(i.e.,

drugretention in epidermis) and was incorporated into the experimen-tal design.The

measured

pH

of

acyclovir

cream

DOE

formulationsremained

relatively

constant

ranging

from

7.92

to

8.46

despitethe intentional change in pH of the aqueous phase (to 5.00, 6.75, or8.50). The statistical analysis showed no effect of investigatedprocessing

variables

on

the

nal

pH

of

DOE

formulations.

The

pH

of Zovirax1was

also

almost

at

similar

level

(7.92).

The

lack

of

changein the pH of DOE formulations is most likely due to the pH alteringeffect of formulation ingredients, particularly the SLS (pH > 9) andthe

drug

addition

method.

To

investigate

such

a

possibility,

threeadditional

acyclovir

cream

formulations

(F-12–F-14)

were

pre-

pared wherein both the drug and SLS were added to aqueousphase, and then pH adjusted to a varying degree (5.00, 6.75, or8.50).

As

expected,

when

pH

was

adjusted

in

the

presence

of

SLS,the

pH

matched

the

design

values

(pH

of

F-12–F-14

were

5.11,

6.75,and 8.43, respectively).The top, middle, and bottom portions of acyclovir cream in the

tubes

showed

no

evidence

of

phase

separation

or

change

in

physical

Fig.

2.

Role

of

drug

partitioning/solublization

and

pH

on

skin

permeation

and

retention

in

epidermis

from

acyclovir

cream

formulations.


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appearance

and

structure.

When

tested

for

drug

content

uniformity,all

the

formulations

including

Zovirax1 complied

with

USPspecications (USP36-NF31, 2013c). The drug concentration inaqueous phase of cream formulations was almost the same rangingfrom

3.4

to 4.0

mg/ml,

which

was

slightly

higher

than

the

aqueoussolubility

of

acyclovir

(2.5

mg/ml

at

37 C)

(Zovirax,

2002).

This

wasattributed

to the

solubilizing

effect

of

formulation

ingredients

suchas Poloxamer 407, SLS (surfactants), and propylene glycol (cosol-vent).

Absence

of

statistically

signicant

difference

( p

>

0.05)

inaqueous

phase

drug

concentration

of

the

investigated

acyclovircream

formulations

conrms that processing variables have nosignicant impact on the drug concentration in the aqueous phase.

3.3. Effect of various factors on particle size determined by polarized

light microscopy

Under polarized light microscope, acyclovir crystal exhibited arectangular/square

shape

as

can

be

seen

in

Fig.

3.

All

eleven

DOEformulations

exhibited

very

similar

particle

size

values

(Table

2),and no processing factors showed a signicant effect on the drugparticle size. ANOVA conrmed that the model was not signicantand

independent

factors

had

no

relationship

with

the

response( p > 0.05). However, it is clear that DOE formulations showedsignicantly ( p

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rheological properties: viscoelastic behavior, yield stress, effect of shear rate on cream viscosity (viscosity at low, medium, and high

shear rate), etc.Zovirax1 exhibited

a

typical

viscoelastic

behavior

whensubjecting to increasing strain. As shown in Fig. 4A, the storagemodulus of Zovirax1 cream remained constant up to approxi-mately

1%

strain,

after

which

it

started

to

decline.

This

region

isgenerally

identied

as

the

linear

viscoelastic

region

(LVR)

withinwhich any disturbance to the microstructure is instantaneouslyrecovered (reversible process). Note that for Zovirax1 cream, thestorage

modulus

(G0)

is

signicantly

higher

than

the

loss

modulus(G00),

suggesting

that

the

microstructure

of

the

cream

is

highlyorganized and dominated by cohesive forces. Overall materialbehaviors like a solid. As the shear strain increases, both G0 and G00

decrease

and

the

material

becomes

progressively

more

uid-likeand

eventually

G00 exceeds

G0.

As

discussed

earlier,

when

plotting

G0

as

a

function

of

shear

stress

exerted

on

the

material

during

theoscillation strain sweep, the onset of G0 curve indicates anirreversible

plastic

ow

of

the

material

and

corresponds

thematerial's dynamic yield stress (s 0). With respect to theviscoelastic behavior, all DOE formulations exhibited similar LVR as

the

Zovirax1 (data

not

shown).Experimentally,

yield

stress

can

be

measured

via

severaldifferent ways. One commonly used technique is to determine

the stress at the viscosity maximum during a stress rampexperiment (Kryscio et al., 2008). In this type of test, the viscosity

maximum occurs as a result of two competing effect: (1) timedependent

viscosity

build-up

of

the

viscoelastic

material;

and

(2)viscosity decrease due to structure break-down with increasingstress (Barnes, 1999; Franck, 2014). Though fairly reproducible, themeasurement

result

of

this

technique

is

sensitive

to

the

selection

of ramp

rate.

For

this

reason,

in

this

study

another

method

waschosen, by determining the onset of storage modulus (G0) versusshear stress curve during an oscillation stress/strain sweepexperiment

(Fig.

4B)

(Pal

1999).

3.4.1. Effect of various factors on product's yield stress

All DOE formulations exhibited similar yield stress values(ranging

from

45.9

to

91.6

Pa)

to

both

blank

(57.6 1.9Pa) and

Zovirax1 (73.0 13.7 Pa) as shown in Fig. 5. Statistical analysis

conrmed

that

the

investigated

processing

parameters

showed

nosignicant ( p > 0.05) impact on the product yield stress. Though theyield

stress

might

be

affected

by

formulation

variables,

it

is

notaffected by the process variables investigated in the present study.The formulation variables were kept constant for all the DOEformulations

(Q1/Q2

equivalent).

Three

additional

formulations(F12-F14)

exhibited

similar

yield

stress

values

compared

toZovirax1, blank, and DOE formulations, suggesting that all samples

Fig. 4. Strain sweep test (0.05–50% strain at 1 Hz) for a Zovirax1 cream sample: (A) assessment of linear viscoelastic region; (B) determination of yield stress using onset of

storage

modulus.

Table 2

Particle size of acyclovir in various samples, determined using polarized light microscopy.

Sample Particle count Length Min.(mm)

Length Max.(mm)

D10(mm)

D50(mm)

D90(mm)

Zovirax1 200 2.5 43.4 4.3 0.4 9.3 1.7 21.8 2.0DOE-1 279 4.0 210.8 9.4 0.3 18.4 1.0 60.5 11.9DOE-2 283 4.4 211.4 8.7 1.6 17.2 2.3 68.7 17.9DOE-3 261 3.1 147.2 8.7 2.1 17.5 2.2 61.6 11.5DOE-4 433 5.1 255.1 8.6 0.7 18.9 1.4 55.6 8.4DOE-5 372 3.0 197.5 8.0

0.4 26.7

3.5 59.4

7.6

DOE-6 450 3.6 165.4 13.3 2.1 33.8 1.2 65.9 1.6DOE-7 408 3.5 234.4 13.6 1.4 37.3 3.0 78.6 14.1DOE-8

433

2.3

197.2

9.5 1.2 23.9 2.3 58.3 2.8

DOE-9

338

7.0

168.5

14.3 0.4 27.9 1.6 50.9 1.7

DOE-10 417 2.8 181.9 10.6 3.3 25.7 3.6 54.6 3.7DOE-11

417

3.1

176.5

10.5 0.5 25.4 2.0 53.0 3.7

API-raw 206 4.1 199.4 11.9 3.9 23.3 2.6 44.7 8.1API-milled 217 4.5 52.8 9.2 0.6 14.3 0.4 23.7 2.4

F-12 403 3.2 54.4 7.1 1.0 14.1 0.4 27.5 0.4F-13

401

4.0

60.0

7.6 0.3 14.1 0.1 27.8 1.7

F-14

410

2.9

61.3

6.6 0.9 13.0 1.8 26.0 5.4


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should have similar spreading behavior over the skin. A yield stressvalue

between

10

and

100

Pa

requires

very

minimal

force

to

initiatethe

ow

(1–10mN

force

over

1

cm2 area),

and

hence

the

cream

isexpected to be very easily applied onto and spread over the skin.

3.4.2.

Effect

of

shear

rate

on

sample

viscosity

All

tested

formulations

exhibited

similar

plastic

ow

behavioras compared to Zovirax1 (Fig. 6 and 7). Under the low shearcondition, the cream displays very high viscosity (10,000 Pa s),

giving

the

rmness

feel

to

the

product.

As

the

shear

increases,product

viscosity

quickly

reduces

(to 0.05). However, one formulation (DOE-3) showedslightly

higher

viscosity

at

low

(0.001

s1)

and

medium

(1

s1)shear

rate

conditions

but

slightly

lower

viscosity

at

high

(>20

s1)shear rate, suggesting that this particular formulation would havehigher rmness feel to it under the low shear but easier to ow

during

spreading

than

Zovirax

1

.

Three

additional

formulations(F12-F14)

exhibited

similar

viscosity

proles

compared

to

all

theDOE formulations.

Fig.

6.

Viscosity

pro

le

of

acyclovir

cream

formulations

as

a

function

of

shear

rate

(n

=

5

for

Zovirax

1

and

n

=

3

for

other

samples).

Fig. 5. Yield stress of various formulations (n = 5 for Zovirax1 and n = 3 for all the other samples).

118 Y.S.R. Krishnaiah et al. / International Journal of Pharmaceutics 475 (2014) 110–122

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3.5. Cream stability: rheological considerations

It

was

noticed

that

during

storage

all

of

the

DOE

formulations(DOE-1

to

DOE-11)

were

accidentally

exposed

to

elevatedtemperature (32–38 C) for an unknown period of time due tothe air-conditioner malfunctioning at the facility. After such anexposure,

the

yield

stress

and

viscosity

of

DOE

samples

weresignicantly lower (data not shown). This suggests a destabiliza-tion of the cream structure. To understand the effect of temperature

on

cream

stability,

temperature

ramping

tests

wereperformed

on

a

series

of

new

samples

(Zovirax1 and

three

newformulations F-12–F-14). As shown in Fig. 8, three new formula-tions behaved similar to the Zovirax1: the storage modulesremained

relatively

constant

until

temperature

reached

approx.32 C,

above

which

sample

behaved

more

like

a

liquid.

Storage

under this elevated temperature conditions may accelerate phase

separation under static environment, causing irreversible changein

the

rheological

prole.

On

another

interesting

note,

the

cream'sstorage

modulus

was

signicantly

lower

at

temperature

above32 C (skin temperature). This makes it even easier to spread thecream over the skin (in addition to the shear rate effect).

3.6. In vitro drug release study

The

in

vitro

drug

release

studies

were

carried

out

to

detectthe

effect

of

minor

changes

in

process

variables

involved

in

themanufacturing of acyclovir cream formulations (FDA, 1997). Thepercent of drug transported from acyclovir cream formulationsinto

the

dissolution

medium

ranged

from

1.74 0.23 to 2.10 0.16based

on

the

total

drug

amount

(dissolved

and

undissolved),

or

31.26

3.37

to

41.69

3.18

based

on

aqueous

drug

amount(assuming acyclovir concentration remains constant in aqueousphase).

At

the

end

of

the

test,

there

was

still

a

large

portion

of

drugavailable

for

release.

In

order

to

enhance

the

passive

transport

of drug across the skin, the thermodynamic activity of drug(s) ismaintained high with large quantity of drug in topical andtransdermal

products

(Allen

Jr

et

al.,

2005;

Davis

and

Hadgraft,1991;

Iervolino

et

al.,

2000;

Pellett

et

al.,

1994).

When

the

amountof drug diffused per cm2 of membrane was plotted against squareroot of time (Higuchi diffusion kinetics), there was a linearrelationship

with

high

correlation

coef cient

(0.9687

to

0.9987)(USP36-NF31,

2013b).

The

drug

release

rate

for

the

acyclovir

creamformulations was ranging from 0.32 0.03 to 0.38 0.02 mg/cm2 h0.5. The RLD also showed almost the same drug release rate

of

0.35

0.04

mg/cm

2

h

0.5

.

Statistical

analysis

of

the

DOE

modelFig.

8.

Temperature

effect

on

storage

modulus

of

acyclovir

cream

formulations.

Fig. 7. Viscosity of acyclovir cream formulations at three different shear rates (n = 5 for Zovirax1 and n = 3 for other samples).


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showed that none of the processing variables or pH of aqueousphase affected drug release rate of acyclovir from the topical creamformulations.

Even

direct

comparison

of

drug

release

rates

between individual DOE cream formulations and RLD showedno signicant difference (ANOVA with Holm–Sidak test).Because outliers are expected to occur on occasion with in vitro

drug

release testing

(for

example,

due

to an

air

bubble

between

theproduct

sample

and

the

membrane),

a

nonparametric statisticaltechnique was used as describedin USP and FDA guidance document(FDA, 1997; USP36-NF31, 2013b). Since the investigated acyclovircream

formulations

(DOE-1–DOE-11,

F-12–F-14)

are

Q1/Q2

equiva-lent,

the

sameness

of

these

formulations

with

respect

to theirperformance (in vitro drug release) may be considered as level 1change (due to possible minor variation in processing parameters).The

in

vitro drug

release

rate

of

DOE

formulations,

F-12–F-14

(testformulations)

were compared

against

Zovirax1 (Reference

formu-lation) as a two-stage test. If the 90% condence interval for the ratio

of

test

toreference

release

rates

does

not

fall

within

0.75

and

0.13333(or75–133.33%)

in rststage (6 cells each for test and reference), four

additionaltests(12

cellseachfortest

and

reference)werecarriedout.All the acyclovir cream formulations except DOE-1, DOE-2, and F-12complied with the sameness of drug release rate in rst stage.However,

the

DOE-1,

DOE-2,

and

F-12

formulations

also

compliedwith

the

sameness

of

drug

release

rate

at

the

second

stage

of

testing(Table 3). These results showed that that none of the processingvariables

affected

the

drug

release

performance

of

acyclovirformulations

indicating

that

Q1/Q2

equivalent

generic

acyclovircream formulations can be prepared to match the performance of Zovirax1 (RLD).

3.7. Drug deposition (or retained) in human epidermis

The in vitro drug permeation studies across human skin candetect

the

difference

in

topical

delivery

of

generic

acyclovir

topical

creams which vary in formulation composition, and therefore usedin this study for detecting the effect of process variables on Q1/Q2equivalent

acyclovir

cream

formulations

(Trottet

et

al.,

2005).

The

functional ability of acyclovir topical dermatological productsdepends on the ability of the formulation to retain the drug at thesite of action in basal layers of epidermis. Drug retained inepidermal

layers

after

24

h

of

in

vitro

skin

permeation

study

wasable

to

detect

the

functionality

difference

between

1%

penciclovircream and 5% acyclovir cream (Hasler-Nguyen et al., 2009). Hence,the amount of acyclovir retained at the end of 24 of in vitro drugpermeation

study

(DRE)

was

determined

in

the

present

study.Since

none

of

the

investigated

process

variables

or

pH

(aformulation variable) affected the structural characteristics(viscosity, yield stress, particle size, drug concentration of aqueousphase)

and

performance

(in

vitro

drug

release

rate)

of

acyclovircream

formulations,

DRE

studies

were

carried

out

only

with

F-12–F-14 cream formulations against Zovirax1 cream (RLD). All the

three

formulations

(F-12–F-14)

showed

similar

structural

charac-teristics

(viscosity,

yield

stress,

particle

size,

drug

concentration

of aqueous

phase)

and

drug

release

rate

prole as that of RLD, butdiffer only with respect to pH of aqueous phase. The observed pH of F-12–F-14 acyclovir cream formulations were 5.11, 6.95, and 8.43,respectively

whereas

the

pH

of

Zovirax1 was

7.92.

As

discussedabove,

the

pH

of

acyclovir

cream

formulations

play

a

critical

role

intheir functional ability to retain the drug in the targeted layers of human

epidermis

(Shojaei

et

al.,

1998).The

mean

(S.E.M.) in vitro permeation parameters of Zovirax1, F-12–F-14 acyclovir cream formulations across humanepidermis (n = 9) are shown in Table 4. There was a steady statedrug

ux

from

acyclovir

topical

cream

formulations

(F12,

F-13,

F-14

and

Zovirax1)

across

human

epidermis.

The

quantity

of

drug

permeated

in

24

h

from

F-12

and

F-13

were

signi

cantly

( p

<

0.05)higher when compared to Zovirax1 (about 50% higher). Accord-ingly

the

steady-state

ux

of

acyclovir

from

F-12

and

F-13

Table 4

Mean (S.E.M.) in vitro permeation parameters of Zovirax1, F-12–F-14 acyclovir cream formulations across human epidermis (n = 9).

Formulation Quantity of drug permeated at 24 h (mg/cm2) Flux (mg/cm2 h) Drug retained in epidermis, DRE (mg/cm2) after 24 h of in vitro permeation

Zovirax1 12.04 1.10 0.78 0.06 2.20 0.34F-12

19.41 1.53** 1.22 0.12** 2.91 0.43

F-13

17.89 1.99* 1.14 0.13* 3.42 0.49

F-14 16.25 2.24 1.07 0.15 2.16 0.41

* p

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formulations

was

signicantly

higher

than

Zovirax1 (Fig.

9).

However,

neither

the

ux

nor

the

amount

of

drug

permeated

at24 h from F-14 was signicantly different to Zovirax1.The

signicantly

higher

skin

permeation

from

F-12

and

F-13compared

to

Zovirax1 as

shown

in

Fig.

10

can

be

explained

on

thebasis of the observed differences in their pH values. The pK a valuesof acyclovir are 2.27 and 9.25, giving its isoelectric point at pH 5.76(Shojaei

et

al.,

1998). The

pH

of

F-12

(5.11)

is

closest

to

the

drug'sisoelectric point, resulting in acyclovir less charged than in F-13(pH 6.75), F-14 (pH 8.43), and Zovirax1 (pH 7.92). The polarity of acyclovir

in

these

four

cream

formulations

increases

with

anincrease

in

the

pH

of

the

product.

As

the

non-polar

small

moleculesare more skin permeable than the polar counterparts (Subedi et al.,2010), the less charged acyclovir in F-12 achieved the highest skinpermeation,

followed

by

F-13

and

F-14,

as

compared

to

Zovirax1.The

pH

of

F-14

is

the

closest

to

Zovirax1,

demonstrating

similarskin permeation proles (Table 4).Therapeutic performance of topically applied dermatological

creams

depends

on

their

ability

to

act

locally

in

epidermal/dermallayers

of

skin.

In

case

of

acyclovir

topical

dermatological

creams,the site of therapeutic action is basal epidermal layers (Parry et al.,1992). Hence, it is necessary to determine the amount of drugretained/deposited

in

epidermis

(DRE)

at

the

end

of

24

h

of

in

vitro

permeation study. This represents the best quality metrics toassess

the

in

vitro

performance

of

acyclovir

topical

dermatologicalcreams.

The

mean

(S.E.M.) amount of drug retained in epidermis(DRE) with Zovirax1was 2.20 0.34 mg/cm2. The DRE values withF-12–F-14 were 2.91 0.43, 3.42 0.49, and 2.16 0.41 mg/cm2,respectively.

However,

there

was

no

statistical

signicant

differ-ence

in

DRE

values

of

F-12–F-14

formulations

when

compared

tothat observed with Zovirax1 (Table 4). Although not statisticallysignicant, F-12 and F-13 exhibited greater DRE and skinpermeation

ux

than

Zovirax1,

also

indicating

the

pH

effect

ontheir

in

vitro

performance.

The

ability

of

epidermis

to

retain

thedrug in its layers is a saturation process. Since acyclovir creamformulations are in contact with the epidermis for 24 h, it ispossible

that

their

functional

ability

reached

to

saturation

levels,and

thus

the

DRE

values

are

not

signicantly

different.

The

DREvalues were also expressed as the amount (mg) of acyclovirretained per cm3 (ml) of epidermis. The volume of exposedepidermis

was

calculated

by

multiplying

the

average

thickness

of human

epidermis

(0.007127

cm)

with

exposed

area

(1.717

cm2).The mean (S.E.M.) DRE with Zovirax1 was 308.4 47.9 mg/mlwhereas the DRE values with F-12–F-14 were 408.4 60.8,497.7

68.3 and 303.7 57.2 mg/ml, respectively. These drugconcentrations

available

in

epidermal

layers

are

in

far

more

than

the desired IC50 values of acyclovir (0.02–13.5mg/ml) to producetherapeutic effect (Zovirax, 2002). The results of in vitro drugpermeation

studies

across

human

epidermis

suggest

that

Q1/Q2equivalent

generic

acyclovir

topical

cream

products

show

same-ness in terms of functional ability to provide the desiredtherapeutic drug concentrations at the targeted site of action inepidermal

layers.It

is

well

known

that

change

in

formulation

composition

of acyclovir topical creams affects their therapeutic ef cacy. Forexample, it was reported that generic acyclovir topical creamscontaining

varying

levels

of

propylene

glycol

exhibited

differencesin therapeutic ef cacy (Trottet et al., 2005). However, in thepresent study formulation excipients and their composition werekept

the

same

(Q1/Q2).

The

results

suggested

generic

acyclovir

creams can be manufactured to obtain identical performance asthat of RLD with Q1/Q2/Q3.

4.

Conclusions

Quality by Design approach was used to identify the effect of process variability on structural and functional sameness (Q3) of qualitatively

(Q1)

and

quantitatively

(Q2)

equivalent

genericacyclovir

topical

dermatological

cream

products

in

comparisonwith RLD (Zovirax1). The investigated critical process variables(homogenization speed, homogenization time, emulsicationtemperature)

did

not

affect

the

structural

and

functional

character-istics

with

respect

to

content

uniformity,

particle

size,

spreadabili-ty on skin (yield stress), viscosity (at low, medium, and high shear

rates),

drug

concentration

in

aqueous

phase,

and

in

vitro

drugrelease.

Although

not

statistically

signicant,

acyclovir

creamswith

a

pH

of

5.11

(F-12)

and

6.75

(F-13)

exhibited

greater

DRE

andskin permeation ux than Zovirax1 indicating the pH effect ontheir in vitro performance. The thermo-rheological characteriza-tion

suggested

that

storage

conditions

above

30 C

should

beavoided

as

it

may

bring

irreversible

changes

in

storage

modulusaffecting product storage stability. The study concluded thatgeneric Q1/Q2 equivalent acyclovir topical creams can bemanufactured

with

similar

Q3

matching

to

RLD.

Conict of interest

This

scientic

contribution

is

intended

to

support

regulatory

policy

development.

The

views

presented

in

this

article

have

not

Fig. 10. Mean (S.E.M.) amount of drug permeated from Zovirax1, F-12–F-14

acyclovir

cream

formulations

across

human

epidermis

(n =

9).

Fig. 9. Mean (S.D.) drug release rate of acyclovir from acyclovir topical creamformulations (n = 12 to18).


8/21/2019 1-s2.0-S037851731400533X-main (1)

13/13

been adopted as regulatory policies by the Food and DrugAdministration

at

this

time.

Acknowledgements

Of ce

of

Generic

Drugs

is

gratefully

acknowledged

for

providingfunding

to

carry

out

this

research.

The

authors

would

like

to

thankRobert Hunt for his help with the in vitro drug release studies. TheNational Disease Research Interchange (NDRI, Philadelphia, PA,USA)

is

also

acknowledged

for

providing

the

human

cadaver

skin.

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