msc paqc_dissertation_cristina aller
TRANSCRIPT
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: [email protected]
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: [email protected]
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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