IN-VITRO EVALUATION OF NOVEL FREEZE-DRIED PILOCARPI NE HYDROCHLORIDE BUCCAL TABLETS FOR THE TREATMENT OF R ADIATION-

INDUCED XEROSTOMIA

a Dissertation submitted by

M.Cristina Aller Garcia

in partial fulfilment of the requirements for the

MSc Degree in Pharmaceutical Analysis and Quality Control King’s College London University of London

Academic address

Department of Pharmacy King's College London

Franklin-Wilkins Building 150 Stamford Street

LONDON SE1 9NH

August, 2015

Acknowledgements Foremost, I would like to express my sincere gratitude to my supervisor Dr. Paul G. Royall for his

support, motivation, and immense knowledge. His guidance helped me at all times through the

learning process of this master dissertation.

My sincere thanks also goes to Abdulmalik Alqurshi PhD for introducing me to the topic as well

for the support on the way.

I would like to give a special thanks to my family: especially my parents Silvestre and Sabina and

my brother Xavi, for their faith in me and for teaching me that I should never surrender.

Last but not least, I would like to thank my companion, Peter Smith, who has supported me

throughout entire process, both by keeping me harmonious and helping me putting pieces

together. I will be grateful forever for your love.

Abstract

Introduction: Radiation-induced xerostomia is a highly debilitating complication for head and

neck patients receiving radiotherapy. Although pilocarpine hydrochloride 5 mg oral tablet is

currently used for its treatment, patient’s adherence is compromised by the large list of systemic

side effects that are associated with its administration. The aim of this study was to assess the

development of a freeze-dried buccal tablet of pilocarpine hydrochloride for local treatment to

overcome current limitations.

Methods: A blister, containing 1.5 g of the formulation solution in each well, was frozen at -20o C

overnight. Frozen tablets were loaded into vials in a freeze-drying chamber at ≤ -40o C and ≤ 0.01

mbar during 5 days. The quality control tests for each batch included appearance, uniformity of

dimension, weight, content, friability and moisture uptake. A conventional dissolution test and a

novel digital image assay (DIDA) were performed to study the drug release profile. The

morphological analysis was determined using Differential Scanning Calorimetry (DSC) and Hot

Stage Microscopy (HSM). Finally, a short-term stability study was also carried out at 4º C /71%

RH, 25º C /35% RH and 38º C/35% RH during three weeks.

Results: The tablets proved to be a stable unit with no signs of damage. All batches passed the

pertinent quality control tests. The average weight, width, length and content were 20.99 mg

±0.10, 17.15 mm ±0.60, 27.29 mm ±0.18 and 5.05 mg ±0.09, respectively (n=2). Tablets showed

high hygroscopicity which compromised their integrity. Significant differences in dissolution times

were spotted when using a volume of 0.7 mL as a dissolution medium compared to 0.1 and 0.05

mL (n=3). DSC showed an endothermic sharp peak at nearly 207º C for crystalline pilocarpine

HCl powder but none in the tablet thermogram. Stability studies resulted in the loss of dimension

uniformity for tablets stored at 38º C/35% RH.

Discussion: The integrity of the tablets is a result of the usage of a binder and a lyoprotectant

that offer structural strength and stability to the whole unit. Size and weight were found to be

appropriate for an easy buccal administration. The disappearance of the melting peak in the tablet

thermogram confirmed its amorphous state. Due to the unreliability of the conventional methods,

the necessity of a novel dissolution assay (DIDA) is proven. Finally, stability studies show no

significant differences in dissolution times, thermograms or drug content and none in dimension

except for tablets stored at 38º C.

Conclusion: A novel buccal pilocarpine HCL amorphous tablet was successfully developed by a

freeze-drying process.

Declaration

I declare that I have read the College Regulations on Cheating and Plagiarism and that this

research project / dissertation (delete as appropriate) is my own work and written in my own

words. Any experimental data obtained either by, or with the assistance of, others has been

acknowledged either in the Acknowledgements or at an appropriate position in the text. All

sources of information, including any quotations, are acknowledged by citation of an appropriate

source of reference.

Signed: Date:_______7th August 2015_________

Table of Contents

Title Page Acknowledgments

i

Abstract

ii

Declaration

iii

Table of Contents

iv

Literature Review 1

1. Introduction 1

2. Pathophysiology of radiation-induced xerostomia 1

3. Xerostomia management 3

4. Buccal delivery system 6

5. Summary 7

6. References 7

Research Paper 9

1. Introduction 9

2. Results 10

3. Discussion 12

4. Conclusions 14

5. Experimental Section 14

6. Acknowledgments 15

7. References 16

A-4 Poster

17

1

Literature Review

RADIATION-INDUCED XEROSTOMIA: IMPACT ON PATIENT QUA LITY LIFE AND MANAGEMENT

M. Cristina Aller Garcia[a], Dr. Paul G. Royall[a] Dry mouth or xerostomia is normally present in up to 100% of head and neck cancer patients during and after their treatment with

radiotherapy. Radiation causes damage to the salivary gland tissues, which leads to a decrease in saliva secretion and changes to

its composition. These patients not only suffer from oral distress but from other clinical complications such as malnutrition, dental

problems and depression thereby compromising their quality of life. The current management of radiation-induced xerostomia is the

symptomatic relief with salivary substitutes or saliva stimulation using sialogogues such as pilocarpine hydrochloride. However, many

studies have reported that its oral formulation is not totally accepted by the patients due to a large number of side effects. This review

argues for the buccal route as an alternative to the oral administration of pilocarpine hydrochloride and proposes a new delivery

system that will alleviate the problem of dry mouth.

1. Introduction Radiation-induced xerostomia is the highest occurring and

longest lasting side effect which patients suffering from neck

and head cancer have to face during and after conventional

radiotherapy treatment1. It is described as the subjective

sensation of dryness in the mouth as a result of the

hypofunction of the salivary glands. This complication arises

due to the proximity of the oral cavity and salivary glands to

the tumour, which locates the gland tissues within the

radiation field and therefore causes damage and loss of

salivary cells. Consequently patients experience a rapid

decline in salivary flow rate accompanied by qualitative

changes in the composition of the saliva and the decrease of

its production2. In healthy individuals, the normal average

whole stimulated saliva flow rate is around 4 to 5 mL/min in

contrast to a resting (unstimulated) rate of 0.3 mL/min;

whereas during night hours it decreases to 0.1 mL/min.

Hyposalivation is diagnosed when the unstimulated saliva

flow rate does not reach 0.1 mL/min3. The restoration of the

salivary function, and hence the disappearance of the

sensation of oral dryness after the radiotherapy treatment,

has been reported as partial or non-existent4,5 depending on

the patient characteristics, the administrated dose and the

duration of the treatment.

A wide range of clinical complications emerge as a

consequence of the lack of saliva which compromise the

quality of life of the patients during and after the treatment2.

It is well known that the production of saliva is crucial for the

maintenance of oral health as it offers lubrication, buffering

action and antibacterial activity therefore playing an

important role in taste perception, mastication and

swallowing and more importantly, in the prevention of oral

infections and tooth conditions6. Hence, the improvement of

xerostomia management is highly important, as the main

complaint that head and neck cancer patients have is the dry

mouth sensation which impacts on them physically and

emotionally thereby decreasing their quality of life7.

2. Pathophysiology of radiation-induced xerostomia The standard treatment plan for patients diagnosed with

head or neck cancer is radiation therapy usually combined

with surgery and chemotherapy2. Inevitably, the salivary

glands are more externally located than most tumours and

therefore are normally present in the fields of radiation when

treating the condition. As a result, the loss of function of the

salivary glands leads to oral complications impacting on

patient quality of life8.

Despite the fact that some studies have reported

that salivary gland cells have a low turnover rate and a low

mitotic index, it has been demonstrated that the alterations in

the gland tissues by ionizing radiation is a clear indicator of

their high radiosensitivity9. Alternatively, several studies

(Table 1) demonstrate that the extension of the salivary

dysfunction is related to the radiation field, dose of radiation

and the functionality of the salivary gland at the beginning of

the treatment.

[a] Dr. Paul G. Royall, Cristina Aller Garcia Institute of Pharmaceutical Science King’s College London, Franklin-Wilkins Building

150 Stamford Street, London, UK, SE1 9NH Fax: (+) 020 7848 4500 E-mail: paul.royall@kcl.ac.uk

2

Table 1. Studies reviewing different factors influencing salivary gland hypofunction.

Authors Test objectives Correlation found

Mira et

al.

(1982)10

Influence of the

pretreatment

salivary flow rates

and radiation in

the outcome of

xerostomia.

Statistically significant

(p<0.01) linear

correlation between

initial flow rate and

cumulative dose

resulting in salivary

hypofunction and

xerostomia.

D = 27Gy x Initial Flow

Rate + 3.6 Gy, “where

D is the dose required

to produce minimum

flow rate.”

Franzén

et al.

(1992)11

Impact of radiation

dosage on saliva

production.

<52 Gy treatment

group showed

secretion recovery but

patients treated with

doses >65 Gy showed

irreversible gland

hypofunction.

Roesink

et al.

(2001)12

Correlation

between the

volume of

radiation field and

salivary flow rate.

Negative correlation

between salivary flow

rate and volume of

gland irradiated with

doses between 35 and

45 Gy.

Common signs found in the salivary glands after

irradiation treatment are degranulation and necrosis of the

acinar cells due to their membrane damage, as well as

chronic inflammation and fibrosis of the gland lobules,

especially in the periductal and intralobular areas13,14. These

morphological changes can even be observed 6 to 8 months

after the end of the therapy and subsequently, the decrease

of the salivary function also occurs14.

The total radiation dose with treatment varies

between 50 and 70 Gy depending on the patient and the

extent of the tumour. However, the treatment is normally

fractionated during a period of 5 to 7 weeks with daily single

doses of 2.5 - 7.5 Gy15. This is characterised by a rapid

decrease of up to 50-60% in the salivary function in the initial

phase of treatment, if it involves the major salivary glands

(parotid, sublingual and submandibular) and by the

completion of the treatment, the saliva can reach its minimum

flow rate. The duration of the hyposalivation is proven to be

prolonged even after 12 months, with little recovery and in

the majority of the cases with irreversible damage16. It is

important to note, that both stimulated and unstimulated

salivary flow are affected. Dreizen et al.17 reported an 83.3%

reduction in flow rate. The average flow rate was reduced

from 1.3 mL/min to 0.22 mL/min after 6 weeks of

radiotheraphy.

Radiotherapy not only damages the salivary glands

with the consequent reduction of salivary flow but also

induces changes in the chemical contents of the saliva.

Studies have shown a statistically significant change in the

saliva electrolyte concentrations during the development of

xerostomia with concentration increases in sodium, calcium,

magnesium and chloride (Table 2), all independent of

salivary flow rate17, 18. In addition, radiation is also related to

the decrease in saliva bicarbonate concentration and

therefore its buffer capacity. These changes are related to

damage to the secretory units and tubules of the salivary

glands. As a result of these alterations and in addition to a

reduction in water content, the saliva becomes very viscous

and slightly more acidic with a change in pH from 7 to 517.

Table 2. Concentration of saliva components before and after radiation treatment. Table adapted from Dreizen et al (1976)17. Study done with samples of stimulated whole saliva of 30 patients with head or neck tumours.

Saliva electrolytes

Before Radiotherapy (mEq/L)

After Radiotherapy (mEq/L)

P value

Sodium 38.42 78.27 <0.001

Calcium 1.51 2.80 >0.05

Magnesium 0.37 0.99 < 0.001

Chloride 24.68 45.03 < 0.001

Bicarbonate 19.80 7.95 < 0.001

Proteins 0.48 1.01 < 0.001

P value= Probability level. P<.001 means statistically significant changes in the measurements.

The radiotherapy treatment also causes a small

increase in the concentration of antimicrobial proteins such

as actoferrin, lysozyme and salivary peroxidase and an

increase in the amount of immunoglobulins, such as IgG and

secretory IgA19 in order to provide protection against possible

infections. However, there is controversy on whether these

changes are due to the tumour itself or the oral complications

derived from this primary cause. Moreover, it is found that the

salivary levels of these proteins reach the normal

concentrations 6 months after the end of the radiation

3

therapy19. Additionally, the levels of salivary amylase, which

is synthesised in the acinar cells, tend to decrease as a

function of radiation dose and therefore it reflects directly the

radiation-induced damage caused to the glandular cells19.

The oral flora growth is also affected by the

changes in saliva production and composition. In general, the

noncariogenic population is shifted by cariogenic

microorganisms and, more specifically, an increase in the

number of Lactobacillus spp., Candida spp. and

Streptococcus mutans is clearly noted20. At the same time, a

decrease in Streptococcus sanguis, Neisserua and

Fusobacterium species was observed20. These microbial

changes are directly correlated with the alterations in the

salivary secretion rate, the acidification of the saliva and its

buffer capacity making the oral site more prone to the

invasion of these type of microorganisms. This bacterial

composition change can last for a long time, even years after

the treatment cessation20.

2.1 Impact on the patient’s quality of life Quality of life is defined as the assessment of an individual’s

well-being: the perception of the daily life quality of an

individual which includes all emotional, social and physical

aspects21. Xerostomia after radiotherapy for head and neck

cancer patients is very usual and significantly affects their

quality of life. Firstly, hyposalivation leads to oral distress

because the buccal mucosa gets sticky and dry with the

appearance of ulcers and tissue inflammation and this

discomfort is translated into difficulties in speech and in

eating because the mastication and swallowing is

compromised as the food cannot be moisturised2. Therefore,

patients have a depressed nutritional intake that is further

reduced by loss of appetite due to the radiotherapy itself and

therefore a significance loss of weight can be observed in the

majority of the subjects22. Moreover, a recent study related

the progression of gastro reflux disease with the reduction of

salivary flow rate and the decrease of oesophageal pH23.

Furthermore, the reduction of saliva and more importantly, its

chemical composition, results in the reduction in the number

of taste buds (chemoreceptors) which also results in loss of

taste or in the alteration of food choices24. As a consequence,

patients suffering from xerostomia are restrained in their

normal activities and most importantly in their social

interactions. By the end, patients tend to develop depression

and other related conditions20.

Most of the quality of life questionnaires, filled in by

neck and head cancer patients, highlight the presence of

dental complications, particularly the increase in the number

of caries7. This consequence is based on the change in pH

and electrolyte composition of saliva as well as its reduced

secretion that shifts normal oral microflora to cariogenic

microorganisms265. The risk of caries and other periodontal

diseases remains high and regular even after the end of the

treatment. Moreover, Brown et al.20 reported that the

characteristic increase in Candida albicans in these patients

commonly results in severe fungal infections. Patients

normally develop chronic moniliasis in the corners of the

mouth which increases the overall sensation of discomfort

and burning. By contrast, due to the lack of lubrication,

patients experience problems in the application of dental

prostheses that can irritate the epithelial layer even more8.

Finally, the sensation of extreme thirstiness can alter the

patients’ sleep patterns due to the need to moisturize the

mouth and as a consequence of the extensive liquid intake,

patients can also experience polyuria.

3. Xerostomia management

The management of radiation-induced xerostomia includes

the relief of the symptom of dryness by the application of

moistening agents and saliva substitutes or the

administration of sialogogues to increase the flow rate of

saliva2 for patients who are still able to produce it.

3.1 Artificial saliva, salivary substituents and moistening agents The lack of wetting medium in the oral mucosa can be

palliated by artificial saliva which is composed of

hydroxypropyl-, hydroxyethyl- or carboxymethylcellulose

(CMC). This is an aqueous solution whose formula is

completed by the addition of mineral salts in order to

resemble the actual saliva composition, as well as fluorides

to promote remineralisation, sweeteners with low cariogenic

potential and preservatives15. Second generation artificial

salivas include in their composition the addition of mucin

which is a normal component in saliva (Table 3)26. Numerous

studies concluded that mucin-based artificial salivas are

more effective and better tolerated than CMC-based ones.

However, some patients found in simply taking regular water

sips a much more effective alternative to relieve the dry

sensation2.

4

Table 3. Formulation comparison of CMC-based and mucin-based artificial saliva. Table adapted from Pal Singh et al. (2013)26.

CMC based Saliva Mucin-based Saliva

Sodium CMC 10 g Mucin 35 g

KCl 0.62 g KCl 1.20 g

MgCl 2 0.87 g NaCl 0.85 g

CaCl2 0.06 g K2HPO4 0.35 g

K2HPO4 0.17 g MgCl 2 0.05 g

KH2PO4 0.30

mg

CaCl2 0.20 g

NaFl 4.4

mg

Xylitol 20 g

Sorbitol 29.95

g

Methyl p-

hydroxybenzoate

1 g

Water to 1000

mL

Water to 1000 mL

The goal of salivary substituents is to provide

lubrication and moisturize the oral surfaces and therefore

relieve the sensation of dryness. They are available in

different forms such as solutions, sprays and lonzeges (Table

4)27 although the British National Formulary (BFN) does not

make clear their actual formulation details. They are targeted

at two types of populations: patients with decreased or no

salivary flow and for subjects with normal salivary flow that

want to enhance this function8.

Other methods employed are the use of sugar free

gums or candies which in general not only stimulate the

production of saliva but help to increase its pH and therefore

prevent the formation of caries15.

3.2 Pharmacologic Options

Drug therapy with sialogogues is an alternative treatment

which, instead of providing symptomatic relief of mouth

dryness, acts as a salivary gland stimulant28. In general,

sialagogues increase the flow rate of saliva and thus the

patients need to have a residual functional capacity of their

glands. Although many sialogogues (Table 5)29 have been

studied as possible drug candidates, pilocarpine has proven

to be the most effective. Pilocarpine hydrochloride is

approved for the treatment of radiation-induced xerostomia,

as an oral form, in some European countries and in the USA

Table 4. Saliva substitutes and preparations to treat xerostomia that are currently available and can be prescribed in the UK (British National Formulary)27 .

Pro

duct

nam

e (M

anuf

actu

rer)

For

mul

atio

n

Com

posi

tion

App

licat

ion

(whe

n re

quire

d)

AS

Sal

iva

Ort

hana

®

(AS

Pha

rma)

Ora

l spr

ay 5

0 m

L

Gas

tric

muc

in

3.5%

, xyl

itol

2%, s

odiu

m

fluor

ide

4.2

mg/

L, a

s w

ell a

s pr

eser

vativ

es

and

flavo

urin

g ag

ents

. pH

ne

utra

l. (A

ssum

ing

aque

ous

base

fo

rmul

atio

n)

Spr

ay o

nto

oral

an

d ph

aryn

geal

m

ucos

a 2-

3 tim

es.

30-lo

zeng

e pa

ck

Muc

in

65 m

g,

xylit

ol 5

9 m

g in

a s

orbi

tol

basi

s. p

H

neut

ral

Dis

solv

e 1

loze

nge

in

the

mou

th

Bio

tène

O

ralb

alan

ce®

(G

SK

) M

outh

Gel

50

g

Lact

oper

oxid

ase,

la

ctof

errin

, ly

sozy

me,

gl

ucos

e ox

idas

e an

d xy

litol

in a

gel

ba

sis.

A

lcoh

ol fr

ee

App

ly d

irect

ly

to g

ingi

vae

or

tong

ue

Bio

Xtr

a®

(R

IS

prod

ucts

)

Mou

th G

el 4

0 m

L

Lact

oper

oxid

ase

, lac

tofe

rrin

, ly

sozy

me,

w

hey

colo

stru

m,

xylit

ol a

nd

othe

r in

gred

ient

s.

Alc

ohol

free

. (A

ssum

ing

aque

ous

base

fo

rmul

atio

n)

Fol

low

med

ical

pr

escr

iptio

n

Gla

ndos

ane

®

(F

rese

nius

K

abi)

A

eros

ol s

pray

50

mL

Car

mel

lose

so

dium

50

0 m

g,

sorb

itol 1

.5 g

, K

Cl 6

0 m

g,

NaC

l 42.

2 m

g,

MgC

l 2 2.

6 m

g,

CaC

l 2 7.

3 m

g an

d K

2HP

O4

17.1

mg/

50 g

. pH

5.7

5.

(Ass

umin

g su

spen

sion

)

Fol

low

med

ical

pr

escr

iptio

n

Aqu

oral

®

(Sin

clai

r IS

)

Ora

l Spr

ay 4

0 m

L

Con

tain

s ox

idis

ed

glyc

erol

tr

iest

ers,

si

licon

dio

xide

an

d fla

vour

ing

agen

t. In

clud

es

aspa

rtam

e (A

ssum

ing

susp

ensi

on)

1 ap

plic

atio

n to

th

e bu

ccal

po

uch,

3-4

tim

es a

day

under the trade name of Salagen®2. It is a film-coated tablet

that contains 5 mg of pilocarpine hydrochloride,

microcrystalline cellulose as a binder, stearic acid as a

lubricant and acidifier and carnauba wax for polishing.

Pilocarpine hydrochloride is a direct acting cholinergic

parasympathicomimetic agent. Its mechanism of action is the

stimulation of muscarinic receptors present in the iris and in

the secretory glands30. These glands not only include the

salivary but also the sweat, lacrimal, intestinal and pancreatic

glands31. It is effective not only in radiation and drug-induced

xerostomia but also in diseases of the salivary glands such

as Sjögren’s syndrome31.

5

Table 5. Review of the systematic therapies studied for xerostomia based on the extended discussion of Grisius MM29.

Dru

g N

ame

Typ

e of

m

olec

ule

Mec

hani

sm

of a

ctio

n

Dis

adva

nta

ges

Pilo

carp

ine

Hyd

roch

lorid

e

Alk

aloi

d

Mus

carin

ic

agon

ist w

ith

med

ium

β-

adre

nerg

ic a

ctiv

ity

Incr

ease

in th

e

saliv

ary

flow

rat

e

but p

rono

unce

d

side

effe

cts:

swea

ting,

flus

h,

brad

i/tac

hyca

rdia

.

Bet

hane

chol

Chl

orid

e

Cho

line

carb

amat

e

Stim

ulat

ion

of th

e

para

sym

path

etic

nerv

ous

syst

em,

Lim

ited

stud

ies

as a

sial

ogog

ue. U

sed

for

med

icat

ion-

indu

ced

xero

stom

ia

patie

nts.

No

data

on

saliv

ary

flow

rat

e

impr

ovem

ent.

Car

bach

ol

Cho

line

carb

amat

e

M3

mus

carin

c

agon

ist

No

obje

ctiv

e

resu

lts a

chie

ved

(no

incr

ease

in

the

saliv

ary

flow

rate

)

Cev

imel

ine

Hyd

roch

lor

ide

Aza

spiro

deca

ne

deriv

ativ

es

M1&

M3

sele

ctiv

ity

Rap

id

inac

tivat

ion,

t 1/2

of 5

0 m

in.

Pos

sibl

e m

inor

side

effe

cts

but

no c

linic

al d

ata

to s

uppo

rt it

.

Ane

thol

e T

rithi

one

Ani

sole

Cho

liner

gic

effe

ct,

stim

ulat

ion

of th

e

saliv

ary

glan

ds

Con

trad

icto

ry r

esul

ts

in d

iffer

ent s

tudi

es,

ther

efor

e m

ore

inve

stig

atio

n ne

eds

to b

e do

ne to

dete

rmin

e its

effic

acy.

Bro

mhe

xine

Alk

aloi

d

Muc

olyt

ic A

gent

No

incr

ease

in

saliv

ary

flow

was

rep

orte

d.

Many studies have shown that the most effective

posology of pilocarpine is 5 to 10 mg three times per day for

up to 8 weeks and in even longer periods for some

patients32,33. Patients remarked upon improvements in

symptoms of xerostomia, which included speaking and

eating, in comparison to patients to whom a placebo was

given34. A double-blind, placebo-controlled study conducted

by Fox et al.35 showed not only subjective symptomatic relief

after a month of treatment with 5 mg pilocarpine three times

a day, but also an increase in the unstimulated salivary

secretion of 26 out of 39 patients after immediate

administration. Moreover, the salivary gland function of 9

patients receiving radiotherapy at the same time as

pilocarpine was studied36. This was a small double-blind

placebo-controlled study which showed that patients

receiving the placebo had a bigger loss in saliva secretion

and more dry mouth symptoms than the pilocarpine treated

ones, therefore confirming the possible benefits of

administering pilocarpine during irradiation therapy

treatment36. On the other hand, Le Veque et al.37 showed in

a multi-centre, randomised double-blind, placebo-controlled,

dose titration study with 162 patients with radiation-induced

xerostomia, a statistically significant improvement in the

subjective sensation of oral dryness and therefore in activities

such as swallowing, chewing and speaking, but no real

increase in the salivary flow rate was found.

3.2.1 Limitations of current delivery methods

The main disadvantages of saliva substitutes are

the short duration of their effect and therefore their

continuous application which can lead to high expenses for

the patients as they have to keep purchasing these

preparations30. Moreover, substitutes do not provide the

buffering capacity as the natural saliva does and thus they do

not replace its antibacterial protection.

Regarding pilocarpine, although it has been shown

as an effective treatment for radiation-induced xerostomia, its

cholinergic effects are a limitation in its administration.

Participants in different clinical trials reported side effects

after pilocarpine treatment. The most common were transient

sweating, flushing or warmth, increased urinary frequency,

nasal secretion, constant lacrimation and gastrointestinal

tract distress2,33. Patients also reported feeling nauseous and

dizzy and some described symptoms of blurred or altered

vision. As a parasympathomimetic agent, it could have

cardiovascular effects although no significant responses in

the heart rate or blood pressure have been noted38. However,

its administration is contraindicated in patients suffering from

hypertension or other cardiovascular or gastrointestinal

illnesses.

Due to the large collection of side effects, the

willingness of patients to take the medication decreases and

possible treatment cessation can occur. Moreover, due to its

oral administration, drug response variability can occur

because of drug losses during absorption as well as

6

degradation in the gastrointestinal tract. Most of these effects

are caused by the systemic delivery of the drug, which not

only stimulates the salivary glands but also the different

secretory glands15. Thus, in order to overcome these

limitations a more localised treatment, closer to the site of

action will be required.

Drug delivery within the oral cavity can be carried

out by buccal or sublingual formulations. The sublingual

mucosa is provided with more blood supply and is more

permeable than the buccal one, therefore the former route

offers a rapid absorption and good bioavailability hence it will

be excellent as a systemic delivery route39. However,

because the administration of 5 or 10 mg of pilocarpine has

a small elimination half-life time of 45 min or 1.35h

respectively2 and side effects are not wanted, the utilisation

of the buccal route, will allow the extension of time at the local

site of application of the delivery system, improving the flux

of the drug in that particular region.

Therefore, the development of a novel buccal

formulation of pilocarpine might allow prolonged localised

treatment for xerostomia with an effective stimulation of

saliva and at the same time limiting the side effects. Another

important advantage of the buccal route is the ease of

administration of the formulations40, especially for those

patients suffering from dysphagia or who have difficulties

taking conventional oral dosage forms. Consequently the

patients’ adherence with medication could be enhanced and

therefore their quality of life will be potentially improved

without compromising other aspects of their health40.

4. Buccal delivery system

Different buccal delivery systems have been developed

during the past few decades, although the most common

formulation for localised treatment is tablets37. However,

permeability is the main disadvantage in buccal delivery due

to its mucosa which is mostly immobile, presents low flux and

has a small absorption area resulting in poor bioavailability.

Thus, the development of a fast dissolution delivery system

is essential to improve buccal drug penetration because it will

increase the drug absorption on the site of application.40

Demand for fast disintegrating tablets has

increased over the past decade and the pharmaceutical

industry is focusing its attention on this particular formulation

field. These tablets are introduced into the mouth, between

the gingivae and buccal pouch and they dissolve or

disintegrate very rapidly without the need of water41. Good

disintegration times vary between a matter of seconds to

around a minute42. Thus, there is a need for a localised

treatment and a concomitant reformulation of pilocarpine

hydrochloride suitable for buccal delivery.

4.1 Ideal characteristics of a novel buccal formulation for the delivery of pilocarpine hydrochloride

The characteristics of the tablet formulation to fulfil these

requirements are summarised in Table 6 and critically

appraised below.

Table 6. Attributes for a good buccal delivery system in localised treatment

Attributes Essential Required Not

Required

Porous material

network �

Amorphous

structure �

Fast delivery

(<60 seconds) �

High moisture

content �

Dried storage

conditions �

Appropriate

dimensions �

Economic � To be

administered with

water

�

In order to achieve good bioavailability values of

pilocarpine within the buccal area, the drug must dissolve

quickly and therefore its absorption will be enhanced. This

requires that tablets contain highly porous material as well as

highly water-absorbent excipients in order to allow saliva into

the tablets. A glassy amorphous structure of the components

will also be essential because, thanks to their instability, they

will disintegrate very easily. The combination of properties

will allow the achievement a fast releasing system. Moreover,

appropriate packaging and storage is required to avoid

components coming into contact with high levels of humidity.

Adapted width and length dimensions of the tablet for the

buccal zone, in combined with the lack of need for water, will

7

make its administration easier for patients thus improving

their adherence to the medication.

Today’s market only provides oral radiation-

induced xerostomia treatment, therefore a fast released

buccal delivery system is required. These rapid dissolving

properties are normally achieved through a special

manufacturing process named lyophilisation or freeze

drying42. A freeze-dried product will have the desired large

surface area and a high porosity network that, in combination

with the possible amorphous structure of the excipients and

the drug, will enhance the dissolution rate and therefore its

absorption in the buccal area.

5. Summary

As said above, the current management of radiation-induced

xerostomia consists of salivary substitutes and

pharmacologic options given orally. Although they have

shown to be effective in most patients, they also present

limitations such as adverse reactions and difficulties in

administration which results in reduced patient compliance

with the treatment. This is why the use of freeze drying in the

development of a novel buccal pilocarpine formulation can

improve not only the drug absorption but also the patient

adherence to the treatment thanks to its local administration.

6. References 1. Wijers O.B, PC Levendag, Braaksma M.M, Boonzaaijer M, Visch L.L, Schmitz P.I. (2002) Patients with head and neck cancer cured by radiation therapy: a survey of the dry mouth syndrome in long-term survivors, Head Neck, 24:737–747. 2. Guchelaar H.J, Vermes A, Meerwaldt J.H (1997) Radiation-induced xerostomia: pathophysiology, clinical course and supportive treatment, Support Care Cancer 5:281-288. 3. Bergdahl M, Bergdahl J (2000) Low unstimulated salivary flow and subjetive oral dryness: association with medication, anxiety, depression, and stress. J Dent Res 79: 1652-8. 4. Dobbs J, Barrett A, Ash D (1999) Practical radiotherapy planning. London:Arnold. 5. Liu R.P, Fleming TJ, Toth B.B, Keene HJ. (1990) Salivary flow rates in patients with head and neck cancer 0.5 to 25 years after radiotherapy. Oral Surg Oral Med Oral Pathol. 70:724–729. 6. Humphrey S.P, Williamson R.T (2001) A review of saliva: normal composition, flow, and function J Prosthet. Dent., 85:162–169. 7. Epstein J.B, Emerton S, Kolbinson D.A, et al. (1999) Quality of life and oral function following radiotherapy for head and neck cancer. Head Neck Surg 21:1–11.

8. Beumer J, Curtis T, Harrison R.E (1979) Radiation therapy of the oral cavity: sequelae and management, part 1. Head Neck Surg 1: 301–312. 9. Shannon I.L, Trodahl J.N, Starcke E.N (1978) Radiosensitivity of the human parotid gland. Proc Soc Exp Biol Med 157:50–53. 10. Mira J.G, Fullerton G.D, Wescott W.B (1982) Correlation between initial salivary flow rate and radiation dose in production of xerostomia. Acta Radiol Oncol 21:151–154. 11. Franzen L, Funegard U, Ericson T, Henriksson R(1992) Parotid gland function during and following radiotherapy of malignancies in the head and neck: A consecutive study of salivary flow and patient discomfort Eur J Cancer, 28: 457–462.

12. Roesink J.M, Moerland M.A, Battermann J.J, Hordijk G.J, Terhaard C.H (2001) Quantitative dose-volume response analysis of changes in parotid gland function after radiotherapy in the head-and-neck region. Int J Radiat Oncol Biol Phys 51:938–946. 13. Konings A.W, Coppes R.P, Vissin A (2005) On the mechanism of salivary gland radiosensitivity Int J Radiat Oncol Biol Phys,62:1187–1194. 14. Frank R.M, Herdly J, Philippe E (1965) Acquired dental defects and salivary gland lesions after irradiation for carcinoma. J AM Dent Association 70:868-883. 15. Chambers M.S, Garden A.S, Kies M., et al. (2004) Radiation-induced xerostomia in patients with head and neck cancer: Pathogenesis, impact on quality of life, and management, Head Neck, 26: 796–806. 16. Pinna R et al. (2015) Xerostomia induced by radiotherapy: an overview of the physiopathology, clinical evidence, and management of the oral damage Ther Clin Risk Manag. 11: 171–188. 17. Dreizen S, Brown L.R, Handler S., Levy B.M (1976) Radiation-induced xerostomia in cancer patients. Effect on salivary and serum electrolytes, Cancer, 38: 273–278.

18. Ben-Aryeh H, Gutman D, Szargel R, Laufer D (1975). Effects of irradiation on saliva in cancer patients. Int J Oral Surg 14:205–210. 19. Makkonen T.A, Tenuvuo J, Vilja P, Heimdahl A (1986) Changes in the protein composition of whole saliva during radiotherapy in patients with oral cancer, Oral Surg Oral Med Oral Pathol, 62: 270. 20. Brown LR, Driezen S, Handler S, et al. (1975), The effect of radiation-induced xerostomia on human oral microflora. J Dent Res 54:740-750. 21. Dirix P, Nuyts S, Vander Poorten V, Delaere P, Van den Bogaert W (2008) The influence of xerostomia after radiotherapy on quality of life: results of a questionnaire in head and neck cancer. Support Care Cancer 16:171–179. 22. Chencharick J.D, Mossman K.L (1983) Nutritional consequences of the radiotherapy of head and neck cancer. Cancer 51:811–815. 23. Helm J.F (1989) Role of saliva in esophageal health and disease. Dysphagia 4:76-84.

8

24. Henkin R.I, Tala1 N, Larson AL, et al. (1972), Abnormalities of taste and smell in Sjijgren’s syndrome. Ann Intern Med 76:375-383. 25. Beumer J, Curtis T, Harrison R (1979a). Radiation therapy of the oral cavity: sequelae and management. Part 2. Head Neck Surg 1:301–312. 26. Pal Singh O. et al. (2013) How to manage xerostomia in prosthodontics. Dental Journal of Advance Studies 3:144-151. 27. British National Formulary: 12 Ear, nose and oropharunx, 12.3 Drugs acting on the oropharynx; 12.3.5 Treatment of dry mouth [https://www.medicinescomplete.com/mc/bnf/current/PHP7432-treatment-of-dry-mouth.htm] Access date: June 2015. 28. Vissink A, Panders AK,’s-Gravenmade EJ, Vermey A (1988) The causes and consequences of hyposalivation. Ear Nose Throat J 67: 166–176. 29. Grisius M.M (2001). Salivary gland dysfunction: a review of systemic therapies. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 92:156-162. 30. Holmes S. (1998) Xerostomia: aetiology and management in cancer patients, Support Care Cancer 6:348-355. 31. Wiseman L.S, Faulds D (1995) Oral pilocarpine: a review of its pharmacological properties and clinical potential in xerostomia. Drugs 49: 143–155. 32. Johnson J.T, Ferretti C.A, Nethery J.W, et al (1993) Oral pilocarpine for postirradiation xerostomia in patients with head and neck cancer. N Engl J Med 329:390–395. 33. Schuller DE, Stevens P, Calusen KP, et al (1989) Treatment of radiation side effects with oral pilocarpine. J Surg Oncol 42:272–276. 34. Fox P.C, Ven P.F van der, Baum B.J, Mandel D (1986) Pilocarpine for the treatment of xerostomia associated with salivary gland dysfunction. Oral Surg Oral Med Oral Pathol 61:243–248. 35. Fox P.C, Atkinson J.C, Macynski A.A, Wolf A, Kung D.S, Valdez I.H, Jackson W, Delapenha R.A, Shiroky J, Baum B.J (1991) Pilocarpine treatment of salivary gland hypofunction and dry mouth (xerostomia). Arch Intern Med 151:1149–1152. 36. Valdez H.I, Wolff A, Atkinson J.C, Macynski A.A, Fox P.C (1993) Use of pilocarpine during head and neck radiation therapy to reduce xerostomia and salivary dysfunction. Cancer 71:1848–1851. 37. LeVeque F.G, Montgomery M, Potter D, Zimmer M.B, Rieke J.W, Steiger B.W, Gallagher SC, Muscoplat SC (1993) A multicentre, randomized, double-blind, placebo-controlled, dose-titration study of oral pilocarpine for treatment of radiation-induced xerostomia in head and neck cancer patients. J Clin Oncol 11:1124–1131. 38. Kusler D.L, Rambur B.A (1992) Treatment for radiation-induced xerostomia. Cancer Nurs 15: 191–195. 39. Rossi S, Sandri G, Caramella C.M (2005) Buccal drug delivery: a challenge already won? Drug Discov. Today: Technol., 2: 59–65.

40. Smart J.D (2005) Buccal Drug Delivery, Expert Opin. Drug Deliv. 2(3):507-517. 41. Badgujar B, Mundada A, (2011) The technologies used for developing orally disintegrating tablets: A review, Acta Pharma. 61: 117-139. 42. Fu, Y., Yang, S., Jeong, S. H., Kimura, S., & Park, K. (2004). Orally fast disintegrating tablets: Developments, technologies, taste-masking and clinical studies. Crit. Rev. Ther. Drug Carrier. Syst., 21(6): 433–476.

9

Research Paper

IN-VITRO EVALUATION OF NOVEL FREEZE-DRIED PILOCARPI NE HYDROCHLORIDE BUCCAL TABLETS FOR THE TREATMENT OF R ADIATION-

INDUCED XEROSTOMIA

M. Cristina Aller Garcia[a], Abdulmalik Alqurshi[a], Paul G. Royall[a]

The treatment of radiation-induced xerostomia by pilocarpine hydrochloride oral tablets presents several limitations due to its

large list of systemic side effects. Therefore, the present study is aimed at the development and characterisation of pilocarpine

hydrochloride buccal tablets for a more localised delivery with a fast onset of action, prepared by freeze drying technology.

The mean average tablet length, weight and content for all batches was 27.29 ±0.18 mm, 20.99 ±0.10 mg and 5.05±0.09 mg,

respectively. Furthermore, a novel dissolution assay was developed to imitate buccal mucosa conditions. Dissolution times

resulted in less than 40 seconds thanks to the tablet porosity and its amorphous nature confirmed by Differential Scanning

Calorimetry and Hot Stage Microscopy. However, size and shape of tablets were negatively affected by their high

hygroscopicity and a short-term stability study showed the same results for tablets stored at 38o C/35% RH. In conclusion,

fast dissolving tablets were successfully manufactured by a freeze drying process although future studies might be conducted

in order to improve tablet integrity and stability.

1. Introduction

Nearly 100% of neck and head cancer patients treated with

radiotherapy experience continuous sensation of mouth

dryness or xerostomia1. Because of the proximity of the

tumour to the salivary glands, they, inevitably, fall within the

radiation field and as a consequence their cells are damaged.

Thus the function of salivary glands is decreased leading to

a reduction of saliva levels which causes oral distress and

other clinical conditions2. Various studies have shown that

the radiation dose given, the extension of the radiation field

and the salivary flow rate at the beginning of the treatment

directly influence the salivary hypofunction3,4,5. However, the

majority of the patients suffer between a 50 and 60%

decrease of salivary flow rate after the first radiotherapy

session and by the completion of the treatment most of them

reach the minimum flow rate which can be prolonged after 1

year with little or no recovery6. Additionally, changes in the

saliva composition are also noticeable. Studies have

demonstrated an increase in different electrolytes such as

sodium, chloride and magnesium and a decrease on

bicarbonate and water content7. These changes are

translated into an acidification of the saliva from a pH 7 to 57.

Radiation-induced xerostomia is a disabling condition and

highly unpleasant for patients. The lack of saliva produces

tissue injuries and discomfort due to the dryness of the buccal

mucosa, which therefore compromises the ability to speak

and eat, and as a consequence, patients find difficulties in

social interaction and in the development of daily activities8.

Moreover, dental complications such as cavities arise due to

the change in pH and electrolyte levels of saliva9. Thanks to

the new buccal conditions cariogenic microorganisms are

more prone to growth and consequently the normal flora

population is shifted and thus an increase of caries and other

periodontal diseases is commonly reported by most of the

patients9.

Radiation-induced xerostomia management can be

divided into symptomatic relief treatment or stimulation of

saliva production using pharmacological options1. A wide

variety of saliva substitutes are available in the UK and

described in the British National Formulary (BNF)10. Their

purpose is to provide lubrication and dry mouth relief,

although their short duration of action, the necessity of

continuous application and their inability to offer buffer

capacity are noticeable limitations for their use. Besides,

sialagogues have been studied for gland stimulation in order

to increase the production of saliva and its flow rate although

[a] Dr. Paul G. Royall, Cristina Aller Garcia, Abdulmal ik Alqurshi Institute of Pharmaceutical Science King’s College London, Franklin-Wilkins Building

150 Stamford Street, London, UK, SE1 9NH Fax: (+) 020 7848 4500 E-mail: paul.royall@kcl.ac.uk

10

Figure 1 . Molecular structure of pilocarpine hydrochloride Image taken from www.sigmaaldrich.com

they have been focused on medication-induced xerostomia

or for the treatment of xerostomia in patients with Sjögren’s

syndrome11. In 1995, pilocarpine hydrochloride (Fig. 1) oral

tablet (Salagen®, Novartis) was approved as a muscarinic

agonist for the treatment of radiation-induced xerostomia.

Many studies have shown its efficacy and establish its

posology as 5 to 10 mg three times per day and it was

concluded that improvements in subjective sensation of

dryness but also a statistically significant increase in

unstimulated salivary flow rate were achieved11. However,

due to its systemic delivery and therefore the stimulation of

muscarinic receptors in all secretory glands, a wide range of

side effects have been described in different clinical trials12.

The most common adverse reactions included excessive

sweating, increased urinary urgency, flushing, nasal and

lacrimal secretion, and gastrointestinal tract discomfort.

Additionally, symptoms of blurred or altered vision,

taquicardia and hypertension have been less frequently

reported12. Consequently, patient compliance is

compromised, leaving the patients only with a palliative

alternative.

Local delivery is a potential solution to overcome

these limitations as it reduces the amount of drug absorbed

systematically, and therefore the delivery within the oral

cavity by a buccal formulation should be an alternative to the

current normal release oral tablet. The buccal mucosa is less

irrigated than the mucosal one allowing the drug to prolong

its time on the specific site and to be absorbed there, avoiding

first pass metabolism and systemic side effects. However,

the main disadvantages of this type of route are the limited

absorption area and difficult permeation which can result in

poor bioavailability13. Thus, the development of

orodispersible tablets (ODT) or fast disintegrating tablets

(FDT) which are defined by the European Pharmacopoeia

(EP) as ‘‘uncovered tablet for buccal cavity, where it

disperses before ingestion,‘’ is essential14. FDTs fast

dissolution process allows a rapid onset of action thanks to

their absorption enhancement and therefore tablets

bioavailability is increased. Moreover, their ease of

administration inside the buccal pouch without the necessity

of water is very convenient for the improvement in patient‘s

compliance14.

The techniques used for the production of

orodispersable tablets include freeze-drying, direct

compressing, spray drying, mass extrusion or moulding15.

Freeze-drying process or lyophilisation is a process where

the water is sublimed from the drug solution, previously

frozen, when subjected to vacuum and is the most suitable

technique as it provides drug stability and ensures a glassy

amorphous porous structure in order to achieve rapid

disintegration and then absorption16. However, no standard

dissolution test is described in the pharmacopoeias for FDT

buccal tablets and the development of a novel assay is highly

required.

Thus, the aim of this study was to produce

pilocarpine hydrochloride freeze-dried buccal tablets and

characterized them by means of different techniques as well

as to develop a novel dissolution assay suitable for buccal

freeze-dried tablets.

2. Results

A novel formulation of pilocarpine hydrochloride FDT was

prepared using a freeze drying method developed by

Alqurshi A. et al.17 In that study, different combinations of

gelatine, mannitol and sodium bicarbonate were studied in

order to provide the best mechanical properties to the tablet

as well as faster release rates. It was concluded that the

optimum ratio of excipients was 2:6:1, respectively.

Therefore, this ratio was used in this study and the successful

manufacturing of 4 different batches of 5 mg pilocarpine

hydrochloride buccal tablets was achieved (Fig. 2).

All tablets showed no signs of damage and

exhibited a uniform white colour, a smooth surface and a

defined shape from the blister-well as shown in Figure 2.

Moreover, they met the quality specifications for dimensions,

weight and drug content, results that are summarised in

Table 1. However, after one day of storage, at room

temperature, batch number 4 suffered the collapse of 90% of

the tablets.

Figure 2. Appearance of Pilocarpine HCl tablets

11

Friability studies showed no damaged or broken

tablets after rolling. Interestingly, the calculated mean weight

loss % was a negative value, -0.76% w/w ±0.23 (n=3),

indicating an unexpected increase of weight. These results

can be correlated to the moisture uptake study which

illustrated an average increase in weight, shown in Figure 3.

Figure 3. Moisture uptake study (n=3) at room T o and average of 49% RH. Error bars calculated using SD

In order to validate the HPLC method for the

pilocarpine HCl assay, a number of parameters were

evaluated. The linearity range of the HPLC method was

found to be over the concentration range of 10-200 μg/mL

(Fig. 4). A good linear relationship was confirmed by the

regression equation, y= 11,7864 + 13,2874x, which exhibited

a correlation coefficient (R2) of 0.9999. The limits of detection

(μg/mL) and quantification (μg/mL) were 1.317 and 3.99,

respectively. The precision was high, with a value of 0.076,

expressed as relative standard deviation (RSD%). The

accuracy was determined by the mean % recovery, which

resulted in 101.33 ±2.30, thus indicating a satisfactory

accuracy of the method. Moreover, the method was able to

detect degradation products after subjecting a standard

solution to different stress factors. Although, light, heat and

storage time didn’t affect the retention time of pilocarpine

HCl, around 2.2 min, the method was able to detect a second

peak at 1.8 min after preparing a standard solution in acidic

and in basic conditions.

Figure 4. Calibration curve of Pilocarpine HCl (n=3). The interval for the error bars is visually negligible

The in-vitro dissolution test that was applied in the

study reported here was not a suitable method for drug

release study of freeze-dried tablets due to their fast release,

as shown in Figure 5. Therefore, in order to record the

dissolution process in the second range, it was necessary to

develop a novel digital image dissolution assay (DIDA).

Using DIDA, the mean dissolution time (95% dissolved) for

the 4 batches was found to be 17.2 s ±2.49 which met the

specifications (< 40s). In order to study the dissolution profile

of representative volumes in dry mouth, DIDA was again

performed. In this case, 0.05 and 0.1 mL of artificial saliva

represented volumes of saliva found in xerostomic patients,

while 0.7 mL was the control volume18. Although all tablets

dissolved within 40 seconds some differences were noted as

shown in Figure 6. When looking at the logarithm of the

0.7mL concentration plotted against the time taken, the

values show linear tendencies with a gradient of -2.23

suggesting that the equation of the dissolution is ���.���. In

order to find the t50 (time required to dissolve 50%), it is

necessary to solve the equation ���.��� = 0.5 which gives the

solution t = 0.31 seconds. When applying the same method

to the other two concentrations, the 0.1 case results in the

need to solve ���.��� = 0.5 giving a solution of t = 0.55

seconds and the 0.05 case gives out ���.��� = 0.5 leading to

a t50 value of 0.61 seconds. These solutions agree with

solutions obtained using linear interpolation of the graphs

plotted (Fig 6) and represent a statistically significant

difference on dissolution times for the 0.7 case as opposed

to 0.1 or 0.05.

The DSC thermogram of pilocarpine hydrochloride

powder (Fig. 7) showed a sharp endothermic peak at 207o C

corresponding to its melting peak temperature. However, its

disappearance on the thermogram of the freeze-dried tablets

for each batch (Fig. 7) indicated their amorphous nature. A

Table 1. Results of tablet size, uniformity of weight and content. Mean values ± SD n=2.

Parameter Limits Batch1 Batch2 Batch3 Batch4

Length (mm)

26 - 30 26.69 ±0.014

27.95 ±0.35

27.22 ±0.18

28.20 ±0.03

Width (mm)

14 - 18 16.95 ±0.64

17.35 ±0.64

17.16 ±0.52

18.00 ±0.14

Weight (mg)

20.6 - 25.2

20.82 ±0.11

21.36 ±0.085

20.79 ±0.12

21.22 ±0.14

Content (mg)

4.5-5.50 5.10 ±0.08

4.97 ±0.012

4.85 ±0.20

5.29 ±0.06

y = 13.287x + 11.786

R² = 0.9999

0

500

1000

1500

2000

2500

3000

0 50 100 150 200 250

Mea

n P

eak

Are

a

Concentration (µg/mL)

0.00%

0.50%

1.00%

1.50%

2.00%

2.50%

3.00%

3.50%

4.00%

0 2 4 6 8 10 12

wei

ght i

ncre

ase

(%)

exposure time (min)

12

Figure 5. Drug release profile of Pilocarpine HCl tablet (n=3). Errors bars calculated using SD

Figure 6. % of undissolved tablet in the first 5 seconds (n=3). Error bars calculated using SD

broad endothermic peak is also spotted which is by the

residual solvent evaporation and confirmed by the

mean %loss of weight of the pan after the cycle in the four

batches (8% w/w ±0.022). The HSM offers a clear image of

the porosity of the lyophilised tablets (Fig.8). Moreover, it

indicates the glass transition temperature (Tg) of the system

by observing the conversion of the glassy formulation to a

more rubbery state. The phenomenon appeared to start at

approximately 37.5o C and therefore could not be determined

using DSC as the evaporation process was masking this

transition.

The short term stability study showed that only

tablets exposed at 38o C during 3 weeks suffered loss in

dimensions, although they passed the quality control test of

weight (Table 2). All tablets met the specifications for content

(average of 4.89 ±0.11 mg) and dissolved in an average time

of 21.06 ±5.55 sec. DSC thermograms of tablets in the above

temperatures showed no melting peak and confirmed its

amorphous stability once again.

Figure 7 . Representative DSC thermogram of Pilocarpine HCl powder as received and Pilocarpine HCl tablet.

Figure 8. HSM images of Pilocarpine HCl tablets. A) 25o C; B) 37. 5o C; C) 47. 5o C; D) 117. 5o C; E) 142. 5o C; F) 187. 5o C

3. Discussion

Gelatine, which is a glassy amorphous polymer with a Tg

between 50-90o C, was used in the formulation of these

freeze-dried tablets as a binder because it gives the required

structural strength to the tablets thanks to the formation of an

extensive highly porous gelatine matrix structure within the

dosage form as a consequence of water sublimation during

the freeze-drying process19. The formation of more polymer

cross-links, which at the same time extends the porosity of

the system, is enhanced by increasing amounts of gelatine.

However, beyond a certain concentration level, it facilitates

the formation of gels which are difficult to disintegrate and

can negatively affect the porous network19. Moreover, the

Table 2. Results of tablet size and uniformity of weight for the stability study.

Parameter Limits 4o C 25o C 38o C

Length (mm) 26 - 30 27.3

27.95

16.3

Width (mm)

14 - 18 17.2 17.35

9.5

Weight (mg) 20.6 - 25.2 20.908 21.36

21.828

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 5 10 15

Dru

g R

elea

sed

(mg)

Time (min)

0%

20%

40%

60%

80%

100%

120%

0 1 2 3 4 5

% u

nd

isso

lve

d t

ab

let

Time (sec)

DIDA 0.7 mL

0.1 mL

0.05 mL

13

main disadvantage of using gelatine as an excipient is in its

animal origin, which can cause problems in medication

adherence due to religious or dietary convictions of the

patients, such as vegetarian beliefs20. Besides, mannitol as a

crystalline compound is used to give elegance and integrity

to the amorphous material in order to avoid the collapse of

the structure. Interestingly, its application in this study is

different as it acts as a lyoprotectant giving protection and

stabilisation to the gelatine. Hence, it is essential that

mannitol remains amorphous during the freeze drying

process and afterwards, as it will ensure its validity as a

stabilizer21. However, its high tendency to crystallise and its

low Tg makes it difficult to be maintained in an amorphous

state. Mannitol is present in 3 different anhydrous

polymorphs (α, β and δ) and also exists as mannitol

hemihydrate, which is known to be formed during the

lyophilisation process and stay during the shelf life of the

product20, 21. However, as an unstable hydrate, it can have

negative impact in the stability of the tablets as the water

retained might be released, leading to a degradation of the

API by means of hydrolysis or other chemical changes22.

Various studies have reported that the addition of salts could

inhibit mannitol crystallisation during several steps of the

freeze-drying process23. Telang et al.22 described in a study

the high melt miscibility of sodium chloride and mannitol

which implies their thermodynamic compatibility. Hence the

interactions between the salt and the sugar perturb the

mannitol molecules’ rearrangement and therefore stops its

crystallisation. Thus, the use of sodium bicarbonate in the

formulation of this study is explained. Its presence offers as

well the reduction of gelatin cross-links and inter-chain

bonding and therefore an increase of the freeze-dried tablet

matrix porosity can be seen24. Moreover, the presence of

water in the freeze concentrated solution is also a requisite

to avoid crystallisation, thanks to the formation of hydrogen-

bonding which again reduces the mobility of mannitol

molecules22. Therefore, in order to avoid the complete loss of

water within the tablet, the second drying stage of the freeze-

drying process was removed.

The size and shape of the tablets were established

to provide the best contact within the buccal pouch and were

appropriate for an easy administration. The failure on

specifications of the 4th batch is explained due to a

manufacturing misstep. It was suggested that an early

finalisation of the freeze drying process by 1 day led to an

increase in water content of the tablets which then melted

gradually.

Regarding the HPLC method, it was concluded as

an accurate, precise and linear HPLC assay for the

quantification of pilocarpine hydrochloride content of the

freeze-dried tablets. The degradation peaks appeared on the

stress study were due to either the epimerisation of

isopilocarpine or hydrolysis in acid or alkaline conditions to

pilocarpic acid25 making the assay also specific.

The European Pharmacopoeia26 states that FDT

should be dissolved in 1min or less, and although no

standard in-vitro dissolution tests are described for this type

of tablets, a conventional dissolution test was performed. The

method was not fit for purpose due to the rapid disintegration

of the tablets and the large volume of medium employed,

which did not imitate the conditions found in the buccal

mucosa where the unstimulated salivary flow rate does not

exceed 2 mL/min in healthy individuals and is found to be

around 0.05- 0.1 mL/min in patients suffering from dry

mouth18. The rapid disintegration and therefore dissolution

was due to the porosity of the tablets. Thus, saliva could

penetrate easily through the whole tablet, wetting the active

ingredient and dispersing it. Many studies have

demonstrated that the increase in tablet porosity leads to an

increase in water uptake and therefore a better dispersibility

of the drug which then shows shorter dissolving times.

However, mechanical properties such as hardness or tensile

strength might be compromised making the tablet to fragile

and unstable24. Interestingly, friability and moisture uptake

studies showed the ability of the tablets to absorb amounts of

water when exposed to the environment resulting in a fast

tablet deformation. Thus, hardness must be improved in

order to maintain tablet integrity while manipulating it.

Generally, the hardness is related to the intermolecular

bonding force between excipients which at the same time

depends on the porosity extension27 which again plays an

important role in the disintegration times. Thus, an

improvement on the formulation is suggested in order to

achieve good mechanical strength.

The amorphous nature of the tablets showed in the

DSC in all batches and in the stability study explain their rapid

dissolution times. Crystalline compounds require higher

energies to break their intermolecular forces in comparison

with amorphous materials which are more unstable and

therefore possess a higher dissolution rate28,29.

Finally, the physical integrity of the freeze-dried

tablets during their storage is highly important and its related

to the Tg. Tablets should be stored below their Tg in order to

maintain the stability of the product30. If the tablets are

14

exposed to temperatures above the Tg, they lose the porous

matrix due to an increase in the mobility of the molecules

within the sample, thus experiencing a reduction in viscosity,

as seen in Fig. 9, which induces the contraction and

shrinkage of the tablet. This is why tablets stored at 38o C

collapsed and why the tablets at 4 and 25o C conditions

maintained their properties within the time.

4. Conclusions

In the present study, pilocarpine HCl buccal tablets were

succesfully manufactured using freeze drying technique.

Tablets offered an appropiate size, shape and content and

possesed an amorphous porosity matrix, assuring a very

rapid dissolution and therefore a very fast onset of action.

Thus, the aim of this study is fulfilled as it confirms the

plausibility of developing a commercially buccal FDT

formulation of pilocarpine HCl.

5. Experimental Section

Formulation of FDT of pilocarpine hydrochloride

The drug solution was prepared by the addition of 0.780 g of

gelatine powder EP (Fragon Ltd., lot RM148/14), 0.132 g of

sodium bicarbonate powder EP (Fragon Ltd., lot RM151/14)

and 2.931 g of mannitol 10% (Fresenius Kabi) in water for

injection heated to 40o C. Finally, 0.33 g of pilocarpine

hydrochloride (Sigma Aldrich, lot #MKBS0848V, ≥98%) were

added and the solution was brought to volume in a 100mL

volumetric flask and was cooled to room temperature.

Furthermore, 1.5 g of the solution were weighed in each

pocket of two empty aluminium blisters (Zhejang Xinfei

Machinery Ltd.) specially designed for this study. The freeze-

drying protocol started by freezing the blisters over 24h at -

20o C. The frozen tablets were loaded into vials (1 oz Clear

Glass Universal Type 1) pre-cooled to -40o C and placed in a

freeze-drying chamber (Lyotrap freeze dryer, LTE Scientific

Ltd.). The freeze drying cycle lasted 5 days at ≤ -40o C under

a pressure of ≤ 0.01 mbar. Once the cycle ended, the vials

were sealed with rubber stoppers and screw lids under

nitrogen gas inside the chamber. The prepared FDTs were

left to reach room temperature and stored in appropriate

conditions until further use. In total, 4 batches of 20 tablets

each were produced.

Appearance

A visual test was performed in order to check the overall

appearance of the tablet dosage form. This included the

description of colour and shape and the inspection of any

signs of damage.

Dimension uniformity

2 tablets from each of the 4 batches were measured in width

and length using a digital calliper.

Weight uniformity

2 tablets from each of the 4 batches were weighed using an

electronic balance (Micro balance: Sartorius UK Ldt) and the

average weight was calculated.

Friability

An adapted E.P method is used to determine tablet strength

by measuring its % loss. Three tablets, previously weighed,

were placed in a roller mixer (STR2, Stuart Scientific) that

rotated with a speed of 33 rpm for 3.1min. The tablets were

dropped continuously within the vial and after 100

revolutions, they were weighted. The % loss was determined

using this formula:

% loss =

� � �� �� ����� ��� �� ���

� � �� �� ��� x 100 Eq. 1

Moisture uptake study

3 tablets were taken from the vials and exposed at room

temperature and room humidity for 10 min, while they were

weighed. The percentage increase in weight was calculated.

Humidity was measured at 49% using an EL-USB-LCD-2

humidity data logger (Lascar Ltd.)

Pilocarpine HPLC assay

The assay test was carried out by high-performance liquid

chromatography (HPLC). HPLC testing was performed on an

Agilent® 1100 system with Agilent® diode array detector

(DAD). The HPLC method consisted of a C-18 Gemini-NX 5

µm x 4,6 x 250 mm column, a mobile phase of 50% v/v

methanol HPLC grade and 50% 0.1 ammonium acetate (pH

5.8) with isocratic gradient, a flow rate of 1 mL/min, a column

temperature of 37o C and an injection volume of 20 µL in

triplicate. Peak responses were measured at 229 nm.

- Content uniformity

A standard preparation was prepared by dissolving 5 mg of

pilocarpine hydrochloride in a 100 mL volumetric flask with

water HPLC grade to obtain a concentration of 0.05 mg/mL.

The test solution was prepared by completely dissolving 1

tablet in a 100 mL volumetric flask with water HPLC grade to

obtain a nominal concentration of 0.05 mg/mL, based on the

content claim. Both solutions were injected separately in

triplicate. The quantity in mg of pilocarpine hydrochloride can

be calculated using this formula:

mg of pilocarpine HCl = ����

�� Eq. 2

15

Where C is the concentration in mg/mL of the standard

preparation, V is the volume in mL used for the test solution,

and ru and rs are the peak area responses obtained from the

test solution and standard preparation, respectively. The test

was performed in duplicate for each of the four batches.

- System suitability

To check the linearity of the method three sets of working

standards were carried out in 3 different days. The standards

were prepared by accurately weighing 25 mg of pilocarpine

hydrochloride and dissolving them in 50 mL water HPLC

grade to make a stock solution of concentration 500 μg/mL.

Serial dilutions were carried out with HPLC-water to get

concentrations of 10, 20, 30, 50, 100, 150 and 200 μg/mL. A

duplicate injection of each concentration was done and the

plot of peak area Vs concentration was subjected to linear

regression analysis. The precision was determined using the

relative standard deviation (%RSD) of the peak area values

of a standard solution of pilocarpine hydrochloride injected 6

times. The accuracy was calculated using the %recovery of

drug after the HPLC analysis of three different standard

solutions. Finally a stress study was conducted by subjecting

a standard solution of pilocarpine to various factors such as

time, heat and light as well as in acidic and alkaline

conditions. The possible degradation products were detected

by the HPLC assay.

In-vitro drug release study

The study was carried out using a USP XXIII Dissolution

Apparatus I (paddle type) at 40 rpm. The drug release profile

was studied in 900 mL of phosphate buffer at pH 7.3 at a 35o

C. Aliquots of 5mL were withdrawn at intervals of 1, 2, 4, 5,

10 and 15 minutes. The amount of drug was determined by

HPLC assay. Dissolution of three tablets (n=3) was

determined and mean amount of drug release was

calculated.

Digital Image Dissolution Assay (DIDA)

Novel dissolution test17 using artificial saliva as the

dissolution medium. The synthetic saliva consisted of 2g of

NaCL, 0.0475 g of KH2PO4, 0.595 g of Na2HPO4 and 0.54 g

of mucin from porcine stomach, all dissolved in 250 mL of

distilled water. The saliva was kept in a water bath in order to

reach the targeted temperature (35o C). A black-painted well

of a thermal-jacketed blister was filled with appropriate

volume of dissolution medium. Using GeneSnap gel imager

software version 6.07.3, a reference image was taken. Then,

the well was thoughtfully dried and a freeze-dried tablet was

placed on it. Again, another image was taken, this one

corresponding to time 0 sec. Then, 100 consecutive images

were taken at 0.4 sec intervals after the addition of an

appropriated volume of dissolution medium at a controlled

temperature of 35o C which permitted to trace the

disappearance or dissolution of the tablet. The image

analysis was carried out using Image J analysis software

which determined the mean grey value (MGV) of the images

taken. The background of each value was corrected and

were plotted against time for the construction of a dissolution

curve. The volumes employed were 0.7 mL for the quality

control of each batch and 0.7, 0.1 and 0.05 mL to study the

effect of different quantities. The temperature was controlled

using thermocouples connected to a data logger

thermometer (YC-747UD data logger thermometer, YCT

Ldt.)

Differential Scanning Calorimetry (DSC)

The analysis was performed in a DSC Q20 (TA instrument,

New Castle, USE) with a refrigerated cooling accessory

under a nitrogen atmosphere. The set up parameters

included an isotherm at 25º C for 5 min, a ramp to 235º C,

followed by a cooling cycle to 25º C and a final heating ramp

to 235º C. The heating rate was 10º C/min and the samples

which weighed between 2 and 10 mg where placed in pin-

holed hermetic pans. All analysis were done in triplicate and

an empty pin-holed pan was used as a reference pan. Pans

were accurately weighed before and after the completion of

the cycle with an electronic balance (Micro balance: Sartorius

UK Ldt.).

Hot Stage Microscopy (HSM)

A portion of the tablet was placed on a microscopic slide and

heated at a rate 10 °C/min to 200°C on a Linkam Hot Stage.

Microscopic examinations were carried out using a Leitz

Dialux 22EB microscope.

Stability study

Tablets of the first batch were stored in sealed vials for 3

weeks at 4º C/71% RH, 25 ºC/35% RH and 38 ºC/35% RH in

triplicate for each temperature. Samples were tested for drug

content, dissolution test (DIDA) and DSC.

6. Acknowledgements

This research was made possible by King’s College London

to whom I am extremely grateful for providing me with the

necessary equipment and knowledge.

Keywords: pilocarpine HCl · freeze drying technology · fast

disintegrating tablet · in-vitro disintegration time · crystallisation ·

hot stage microscopy

16

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