Transdermal Microneedle Lignocaine Delivery Versus EMLA Patch for Topical Analgesia Before Venepuncture Procedure To Adults in Clinical Setting
Phase: PHASE1
Conditions: Glaucoma, Cataract, Ophthalmological Disorder
Sponsor: Universiti Kebangsaan Malaysia Medical Centre

1 
 
 
 
 
Research Protocol 
Transdermal microneedle lignocaine delivery versus 
EMLA patch for topical analgesia before venepuncture 
procedure to adults in a clinic setting 
 
 
Date of Document: 12th December 2022 (Version 3) 
 
 
 
 
 
PRINCIPAL INVESTIGATOR: Professor Dr Cheah Fook Choe 
CO-INVESTIGATORS: Professor Dr Azrul Azlan Hamzah 
Professor Dr Mae-Lynn Catherine Bastion 
Dr Muhammad Irfan Abdul Jalal 
Dr Lam Chen Shen 
Ms. Chua Xin Yun 
COLLABORATORS: Dr Goh Chee Seong 

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CHAPTER 1 
INTRODUCTION 
 
1.1 Transdermal Drug Delivery 
Venepuncture, as one of the most common invasive medical procedures, is also one of the most 
feared and traumatic experiences that are frequently encountered by the hospitalised patients 
to perceive significant pain and stress. The anxiety and apprehension toward the experience 
with needle puncture may further be exacerbated in those patients experiencing chronic 
conditions which may require them to undergo frequent venous cannulation or venepuncture. 
In that sense, inadequate pain relief is such an unpleasant event and devastating for the patients 
which in long term may affect their emotional well-being and deter them from seeking medical 
aids in the future. 
With the current advances of medical science and technology, healthcare is continually 
improving with innovative approach to deliver comprehensive treatment to the patients. Local 
anaesthesia prior to venepuncture is regarded as a consistent approach that fit with Good 
Clinical Practice in providing a quality standard of care for the hospitalised patients. More 
recently, the emergence of transdermal drug delivery system has been valued for its inventions 
as a less invasive alternative in promoting effective drug administration. 
When it comes to transdermal drug delivery system (TDDS), the conventional drug-delivery 
through hypodermic needle injection and topical cream are the most common strategy to 
deliver the drugs systemically from the skin surface. However, both methods are having their 
inherent downsides. With the uses of hypodermic needle, perhaps, the greatest challenge for 
the patients is to overcome the elicits of pain, stress, and even needle phobia caused by its 
invasive nature applied through the skin, thus leading to its lower compliance and acceptance 
by the population.1,2 On the other hand, the topical drug delivery with formulated cream would 
be an alternative route that is much attractive compared to hypodermic needle injection in drugs 
administration with its valued advantages of being painless and ease of application by the 
patients themselves. Lignocaine cream is one of the examples as topical anaesthetics applied 
to administer anaesthesia. Nonetheless, as the drugs must passively diffuse across the skin, it 
would then lead to an issue on delayed onset of the drug action by its limited bioavailability to 
the effective site.3 Such delay in promoting the drug effects would be much infeasible and 
impractical for the face-paced environment within the clinical settings and practices. 
Besides with the above mentioned TDDS, there are also other developed fashions of 
transdermal delivery systems with the state-of-art technologies, such as iontophoresis 
(induction of electrical current to drive electrostatic diffusion of charged anaesthetic permeants 
through the skin layers), sonophoresis (generation of low-frequency ultrasound that facilitates 
the penetration of drugs), electroporation (formation of microchannels upon electric pulse to 
increase permeability), magnetophoresis (utilisation of magnetic field), thermophoresis (short 
thermal exposure to ablate the skin surface for better diffusion of drugs), and the jet injection 
(controlled compression of gas or spring to deliver anaesthetic drug into targeted skin 
layer).1,2,4–6 Collectively, both attributes developed from different types of TDDS are composed 
of the common objective to provide an efficacious mean in drug administration through the 
topical mean, which further underline the comprehension with the anatomy of human skin to 
understand with the fashion of drug delivery mechanism. 

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1.2 Skin Structure and Transdermal Drug Delivery 
Human skin is the most readily accessible and largest organ over the human body which 
extends over a coverage area of 1.5 - 2.0m2 and account for 16% of the total body weight of an 
adult.1,7 The primary role of human skin is to serve as a protective barrier that protects the 
human body against the relatively hostile external environment. However, such organised 
barrier function has also led to a tedious permeation of the applied topical drug to diffuse across 
the skin layer for its effective drug delivery and activation. 
The skin is histologically classified into 3 main compartments: (1) the outermost epidermis, (2) 
the middle dermis layer, and (3) the innermost hypodermis.1,8,9 The avascular layer of 
epidermis is predominantly consisting of keratinocytes (~95% of total cell in epidermis) which 
construct the non-viable constituent of the outermost stratum corneum (SC) and the viable 
sublayers beneath the SC. Several sublayers of the viable epidermis, i.e. stratum basale, 
stratum spinosum, stratum granulosum, and stratum lucidum collectively made up a thickness 
spanning from 50-100 μm that are adjoint together by tonofibrils. The whole layer of the skin 
epidermis is generally composed of 50-150 μ m in its thickness.10 The epidermis is 
subsequently connected to the deeper dermis layer with an undulating epidermal-dermal 
junction as a partition. Dermis is the thickest component of the skin with 2-3 mm thick and it 
is incorporated with mostly collagen and elastin fibres that confer to the strength and elasticity 
of the skin.11 The papillary and reticular layers of the dermis house the blood vessels, nerves, 
lymphatic vessels, skin appendages, and the connective tissues.8,9 Lying underneath is the 
innermost hypodermis or the subcutaneous layer, which is an elastic layer that mainly 
constituted of the adipose tissues along with the histological structures of blood vessels and 
nerve endings.9 As such, the implanted networks of the blood capillaries and nerves modules 
collectively made up a systemic platform for the route of transdermal drug administration. 
Figure 1 illustrated the structure and composition of human skin. 
 
 
 
Figure 1. Human skin structure and composition. The skin is mainly comprised of 3 layers, 
i.e. epidermis, dermis, and hypodermis with the different structural elements that reside at 
distinct anatomical positions. Figure courtesy: Tomáš Kebert &amp; umimeto.org - Own work, CC BY-SA 4.0 

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In the context of the influence of skin architecture on promoting effective transdermal drug 
delivery, the stratum corneum (SC) is the primary mechanical barrier that is rate-limiting the 
uptake of the topically applied drugs. The SC, made up of multilayered dead corneocytes 
scattered throughout a lipid-rich matrix, is principally harnessing a lipophilic nature by its 
barrier properties and which will exhibit a selective permeability for the penetration of 
relatively lipophilic molecules into deeper layers of the skin.12 As such, drugs that are rather 
lipophobic or high in molecular weight are relatively impeded for its effective systemic 
delivery. Apart from that, the underlying epidermal-dermal junction would be the secondary 
factor conferring the resistance on the molecular transportation of drug molecules across the 
epidermis layer and permeate to dermis.13 Only the drug molecules that managed to get through 
the SC and the papillary dermis will be effectively reaching the systemic circulation to exert 
its systemic effects.14 
The sophisticated structure of the skin has also implicated for a challenging issue of the TDDS 
to attain the optimal bioavailability at the systemic circulation. Evidence from previous study 
reported that the topical cream applied to the skin could only exhibit 10-20% of the total drugs 
permeated into the circulation. Whereas the hypodermic needle, despite with its high 
bioavailability distributed to the circulation (90-100% of the total drug), is intended to cause 
pain as its invasive nature may hit the nociceptors and elicit the pain stimulus.15 Combined 
with both limitations from different types of delivery systems, a new form of transdermal 
delivery method, named the microneedle, is established with the promising features to make 
up the flaws of conventional designs. 

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CHAPTER 2 
LITERATURE REVIEW 
 
2.1 Emergence of Microneedle and its Applications 
With the emergence on the microfabrication manufacturing technology, an intriguing solution 
of effective transdermal drug delivery has been introduced with the use of microneedle (MN), 
which also known as the microneedle patch. MN is a medical device that composed 
hypodermic needles mimicry with the micron-sized needles aligned in out-of-plane protruded 
arrays and encapsulated with the bioactive drug substances.16 These micron-sized needles 
function by the mechanism of creating multiple microchannels upon its puncture onto the SC 
of the skin and thus promoting a better permeability and faster onset of drug molecules onto 
the systemic circulation for its efficacious therapeutic effects.15 
MN offers several superior benefits that are more committed to the clinical compliance. 
Perhaps, the most significant advantage for the use of MN is to minimize the pain experienced 
by the patients by not stimulating the nociceptors or puncturing blood vessels, while the MN 
specification could also be tailored for the intended use of specific treatment.15 Moreover, MN 
is readily self-administered by the patients without the assistance by the healthcare 
professionals.15,16 Concerning with the complication of needle breakage by the conventional 
use of hypodermic needle, the design of MN has tackled with such issue by the utilisation of 
biodegradable materials which hold a strong promise for its safety of usage. Meanwhile, such 
materials would also minimise the biohazard wastage produced by its application in the clinical 
settings as compared to the materials that are conventionally applied, i.e. metal from needles.17 
Maltose, a natural carbohydrate with non-cytotoxic properties, is one of the most common 
sugars applied in the fabrication of MN due to its capability to promote good biodegradability 
and dissolution within minutes of administration.18,19 With a well-recognised safety standard, 
maltose is also widely used for various pharmaceutical formulations with its committed safety 
and efficacy upon clinical application.20 Among the types of MN, dissolving MN is a type of 
biodegradable microneedle patch encapsulated with formulated drugs with one-step approach 
for drug administration. Dissolving microneedle patch allows a continuous dissolution of drugs 
into the targeted skin layer upon its insertion onto the skin surface, whereby the microneedle 
and its drugs components will not be removed following its application.21 Such administration 
pattern could also be described as a ‘poke-and-release’ mechanism of drug delivery.21 
To date, there is a shortage of scientific investigation studying the effects of dissolving 
microneedles on delivering anaesthetics for the adult patients who requires regular blood 
transfusion by their clinical conditions. There are three previously published studies which 
evaluated the efficacy of microneedles for alleviating pain in patients. Rzhevskiy et al. (2022) 
demonstrated a clinical trial with the pretreatment of anaesthetics via a hollow microneedle 
(MicronJet600) prior to peripheral venous cannulation. Results from their study described a 
significant pain reduction (11-fold, p < 0.05) experienced by the study subjects with the 
administered anaesthetic, which the effectiveness of anaesthesic exhibited up to 2cm from the 
injection site and efficacy lasted for 30 minutes.22 On the other hand, Ornelas et al. (2016) 
conducted a randomised, single-blinded, parallel clinical trial to evaluate the effect of 
microneedles pre-treatment in accelerating the onset of topical cream anaesthesia. Results from 

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their study showed that the applied microneedle had shortened the incubation time from the 
typical period of one hour to 30 minutes and it significantly minimised (p < 0.05) the induction 
of pain (caused by needle lancet) after the anaesthetic administration at the particular time point 
(30 mins) as compared to another group which applied the sham patch (VAS score : 
microneedle: mean ± SD, 4 ± 1.3 mm; sham: 14.4 ± 3.8 mm).23 In a separate study, Gupta et 
al. (2012) compared the pain scales of anaesthetic injections administered by hollow 
microneedle and the conventional hypodermic needle to 15 healthy adults. They found out that 
both delivery systems exhibit a similar effects of local anaesthesia (p > 0.05) throughout the 
time of administration, but the study subjects reported significantly lower level of pain 
experienced by the intervention of microneedle which also suggested as their strong 
preference.24 
Based on these findings, the utility of microneedles is reportedly a promising approach for 
effective anaesthetics delivery and pain alleviation. However, the above published evidences 
may be restricted with some limitations that lead to an inconclusive result. For instance, clinical 
study done by Ornelas et al (2016) was limited to only men population, which requires further 
investigation with the whole population covered with both genders to generate a reflective 
result23. Gupta et al. (2012) stated some limitations with their studied prototype of using glass 
microneedle, which led to undesired leakage of drugs upon its administration in the pilot 
study24. Therefore, further investigations are necessary to illustrate a better improvement for 
the microneedle design and to elucidate the utility of microneedles for its potential application 
in anaesthetics delivery. 
Apart from that, the direct transdermal delivery of lignocaine through its embedment within 
microneedle matrix has not be studied so far. Hence, the safety, tolerability and 
pharmacodynamic property (preliminary efficacy) of lignocaine-embedded microneedle has 
not be investigated and reported thus far. Therefore, this trial is designed to provide the answers 
to such research questions. 
 
 
2.2 The general pharmacological properties of lignocaine 
Lignocaine, in its solid state, possesses a crystalline and colourless structure that is dissolvable 
in its hydrochloric salt form.25 It possesses anti-arrhythmic and anti-nociceptive properties 
mediated through voltage-gated sodium and potassium channel blockade.26 
It is primarily bound to α-acid glycoprotein (AAG) (approximately 50%) and to lesser extent, 
albumin (around 25%).27-28 Diseases such as myocardial infarction and cirrhosis and other 
factors such as smoking and age will influence the AAG concentration levels and this will in 
turn affect the fraction of free lignocaine concentration in the plasma.29-32 On the contrary, an 
increase in the AAG level will reduce the amount of unbound, hence active, lignocaine in the 
plasma, resulting in a decrease of its pharmacological effects at a specific plasma lignocaine 
concentration.33 The volume of distribution at steady state, Vss, in normal adults is 1.32 ± 0.27 
L/kg whilst Vss in those with chronic heart failure (CHF), chronic liver impairment and chronic 
renal failure CRF) are 0.88 L/kg, 2.31 L/kg and 1.2 L/kg, respectively.34-35 

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Lignocaine has a high degree of hepatic extraction ratio (62%-81%).36 Hence, factors affecting 
hepatic blood flow such as metoprolol-mediated reduction in hepatic blood flow, congestive 
heart failure and acute myocardial infarction will influence the rate of biotransformation of 
lignocaine.37-38 Apart from that, lignocaine is metabolized by CYP3A4, CYP3A5 and CYP1A2 
through consecutive deethylation process.39-40 Therefore, concurrent administration of 
CYP1A2 and CYP3A4 inhibitors such as amiodarone, fluvoxamine, erythromycin and others 
will cause mild to major elevations of serum lignocaine concentrations.41-43 Lignocaine dosage 
thus needs to be reduced by up to 60% when a drug such as fluxovamine, a CYP1A2 inhibitor, 
is simultaneously administered with lignocaine.44 
There are two major metabolites of lignocaine, monoethylglycinexylidide (MEGX) and 
glycinexylidide (GX).45 MEGX have a role of both therapeutic and toxic effects associated 
with lignocaine whilst GX only gives rise to toxic effect due to its insignificant therapeutic 
effects.46-47 Lignocaine is primarily eliminated via hepatic metabolism and the clearance rate 
ranges from 0.72 ± 0.15 L/hr/kg in healthy adults34 to 0.25 L/hr/kg in patients with Child-Pugh 
class C hepatic impairment.48 
The main adverse events (AEs) associated with toxic level of serum lignocaine can affect two 
major organ systems; the central nervous system (CNS) and cardiovascular system (CVS). In 
the former, the major AEs are confusion, slurring of speech, paraesthesia around the lips and 
tongue, double vision, tremor, seizures whilst sinus bradycardia, sinus arrest and disturbances 
in atrioventricular conduction are the major AEs in the latter.49 The toxic effects of lignocaine 
will appear when serum lignocaine concentration exceeds 5 mg/L and convulsion occurs when 
it exceeds 10 mg/L.50 Despite being associated with the development of nasal adenomas and 
tumours in murine models, lignocaine has not been associated with cancer development in 
humans.50-51 
In 1999, the US Food and Drug Administration (FDA) has approved the lidocaine 5% patch 
(Lidoderm®, Endo Pharmaceuticals Inc, Malvern, USA) for the treatment of postherpetic 
neuralgia.52 Since then, it has been diversely investigated for the treatment of other medical 
conditions such as low back pain, postoperative pain control, trauma patients experiencing rib 
fractures and for prevention of venepuncture or injection-related pain in paediatric patients.53- 
56 However, in a recent meta-analysis by Bai et al., the 5% topical lidocaine patch was found 
to be ineffective as an adjunct for the management of acute and postoperative pain since no 
significant differences were found with regard to the intensity of pain, duration of hospital stay 
and the postoperative consumption of opioid between the 5% topical lidocaine patch and 
placebo groups.57 Hence, a new transdermal drug delivery system for effective lignocaine 
administration is warranted, especially for optimal alleviation of pain associated with routine 
vein-puncturing procedures such as intravenous cannulation and venepuncture. 

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2.3 Justifications of study 
Based on our brief review of literature, we have then pointed out three problem statements in 
bridging the knowledge gaps from previous research with the highlighted significance of our 
current study. 
I) The safety profile of transdermally-delivered lignocaine that is directly embedded within the 
matrix of dissolvable microneedle in the adult patients requiring venepuncture during routine 
clinical settings have not been tested in previous studies. This study aims to evaluate the safety 
and tolerability of lignocaine-embedded microneedle in this cohort of patients. The microneedle 
patch was designed and used (without impregnated drugs) in a previous project approved by this 
committee among paediatric thalassaemia patients. This trial protocol has been published in 
clinicaltrials.org and also in Journal of Clinical Medicine 2022, Sep 8;11(18):5291. doi: 
10.3390/jcm11185291. The safety profile was good with side effects showing that out of 19 patients 
studied, none reported any adverse reactions to the microneedle, with only one case complained of 
mild itchiness at the patch site. 
II) The pharmacokinetic properties of lignocaine that is directly delivered transdermally 
through embedment within dissolvable microneedle’s matrix has not been investigated in the 
previous study. Therefore, a subset of this study patients will provide the preliminary 
pharmacokinetic data for lignocaine delivered via such administration route which will indicate 
the extent of absorption of lignocaine if any at all into the systemic circulation. Animal study 
in rats showed no scarring and complete healing after microneedle patching, and blood plasma 
lidocaine was subtherapeutic level, below 0.4ng/mL and diminished to almost nil after 150 
minutes. 
 

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III) A randomised clinical trial to compare the efficacy of pain relief at venepuncture of adult 
patients at Ophthalmology Clinic, HCTM who receive either the standard 5% EMLA patch 
versus those applied with lignocaine-embedded microneedle patch for 30 minutes prior to 
procedure. 
Hence, our current research is looking forward to providing the answers in response to the 
problem statement above as well as to highlight the potential of lignocaine-impregnated 
microneedle for its clinical application in topical anaesthetic administration and management. 
To summarize the foundations of our research, the conceptual framework of this research is 
shown in Section 2.4. 

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Adult patients who require venepuncture or 
venous cannulation for blood investigations 
Pharmacodynamic component 
Pharmacokinetic component 
Lignocaine is applied to promote 
anaesthetic effect for alleviating pain in 
venepuncture and venous cannulation 
Lignocaine applied to skin diffuse and 
penetrate through the skin layers 
Lignocaine-embedded MN speeds up 
the onset of anaesthesia with higher 
analgesic intensity 
Pharmacokinetic study of Lignocaine 
absorption into the systemic circulation 
from the administration site 
Lower VAS score and algesimeter 
readings in the group receiving 
lignocaine-impregnated MN after 30 
minutes of administration 
Interpret the pharmacokinetic data if 
the lignocaine is absorbed and 
distributed in the circulation 
Better treatment compliance and 
patient acceptance in healthcare 
2.4 
Conceptual Framework 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Establish a reliable platform of safety 
anaesthetic delivery with drug 
embedded microneedle 
Improved pain perception in the regular 
practice of venepuncture 
Microneedle (MN) aids in transdermal 
delivery of lignocaine drug 
Distribution of lignocaine under the skin 
promotes its anaesthetic effects for pain 
alleviation 

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3.1 General Objective 
CHAPTER 3 
OBJECTIVES 
To assess the safety and efficacy of lignocaine-embedded microneedles as a means of pain- 
reduction in adult patients requiring routine venepuncture procedure. 
 
 
3.2 Specific Objectives 
1. To evaluate the safety profile of lignocaine-embedded microneedle patch as a means of pain 
reduction in adult patients requiring routine vein-puncturing procedures 
2. To assess the pharmacokinetic (PK) parameters of lignocaine in the systemic circulation 
when the transdermal lignocaine delivery is enhanced through microneedle usage. 
3. To compare the efficacy of lignocaine-embedded microneedle patch with standard 5% 
EMLA dermal patch for pain reduction during venepuncture procedure based on mean changes 
in VAS scores and skin algesimeter index (pharmacodynamic (PD) study). 
 
 
3.3 Research Questions 
1. What is the safety profile of lignocaine-embedded microneedle patch used for pain reduction 
in adult patients requiring routine vein-puncturing procedures? 
2. What are the values of PK parameters (AUCinf, AUCt, Cmax, t1/2, volume of distributions (Vd) 
and clearance) for the lignocaine constituent in adult patients receiving lignocaine-embedded 
microneedles for routine vein-puncturing procedures? 
3. Are there differences between lignocaine-embedded microneedle patch and standard 5% 
EMLA dermal patch for pain reduction during venepuncture procedure based on mean changes 
in VAS scores and skin algesimeter index? 

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3.4 Research Hypotheses 
1. H0: The proportion of participants experiencing adverse event is not significantly different 
from 0. 
 
H1: The proportion of participants experiencing adverse event is not significantly different 
from 0. 
 
2. H0: The values of PK parameters for the lignocaine constituent in adult patients receiving 
lignocaine-embedded microneedles are not bioequivalent with the values of PK 
parameters for the lignocaine constituent in adult participants receiving lignocaine 
through the standard topical route. 
 
H1: The values of PK parameters for the lignocaine constituent in adult patients receiving 
lignocaine-embedded microneedles are bioequivalent with the values of PK parameters 
for the lignocaine constituent in adult participants receiving lignocaine through the 
standard topical route. 
 
3. H0: There are no differences in mean changes in VAS scores and skin algesimeter index 
between the trial participants receiving lignocaine-embedded microneedle patch and 
standard 5% EMLA dermal patch. 
H1: There are significant differences in mean changes in VAS scores and skin algesimeter 
index between the trial participants receiving lignocaine-embedded microneedle patch 
and standard 5% EMLA dermal patch. 

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4.1 Study Design 
CHAPTER 4 
METHODOLOGY 
This study can be divided into two distinct stages: 
a) Stage 1: Non-randomized single-centre open-label single group clinical trial to primarily 
assess the safety and tolerability of lignocaine-impregnated microneedle in adult patients 
undergoing routine vein-puncturing related procedures (pharmacokinetic (PK) study). 
b) Stage 2: A randomized single centre double blind two parallel group active controlled 
clinical trial to assess the efficacy of lignocaine-impregnated microneedle compared to 5% 
EMLA dermal patch (Pharmacodynamic (PD) study). 
 
 
4.2 Study Location 
The study will be carried out at the Ophthalmology Outpatient Clinic, Hospital Canselor 
Tuanku Muhriz (HCTM), UKM, Bandar Tun Razak. 
 
 
4.3 Study Period 
1st December 2022 – 30th November 2024 (24 months) 
 
 
4.4 Eligibility Criteria 
The inclusion and exclusion criteria of the study participants are as follows: 
 
 
4.4.1 Inclusion Criteria 
I) Patients aged 18 years old and above 
II) Patients requiring venous cannulation for blood investigations before eye surgery 
 
 
4.4.2 Exclusion Criteria 
I) Patient with a previous history of sensitization or allergy to lignocaine. 
II) Patient with a previous history of allergy to materials used in the study i.e., plaster, 
electrodes, maltose, Polyvinyl Alcohol (PVA), and Polyethylene Terephthalate (PET) 
III) Patient exposed to analgesic usage within 24 hours prior to the procedure 
IV) Generalized skin disorder/ rash 
V) Agitated/ fretful / uncooperative patient 

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VI) Uncommunicative/deaf/mute 
VII) Patients on hypnotics, or chronic pain relief medications 
VIII) Patients with psychiatric conditions or cognitive impairment 
IX) Patients with hepatic impairment 
X) Patients who are on CYP450 3A4, 3A5 or 1A2-inducing or inhibiting drugs (erythromycin, 
ciprofloxacin, amiodarone etc.) or pharmacotherapeutic agents that affect hepatic blood flow 
(metoprolol) since both may affect the metabolism of lignocaine. 
 
 
4.5 Reference Population 
All adult patients undergoing vein-puncturing procedures at the Ophthalmology Outpatient 
Clinic in Malaysia. 
 
 
4.6 Source Population 
All adult patients undergoing vein-puncturing procedures (intravenous cannulation, 
venepuncture) at the Ophthalmology Outpatient Clinic, Hospital Canselor Tuanku Muhriz 
(HCTM), UKM, Bandar Tun Razak. 
 
 
4.7 Sampling Frame 
All adult patients undergoing vein-puncturing procedures (intravenous cannulation, 
venepuncture) at the Ophthalmology Outpatient Clinic, Hospital Canselor Tuanku Muhriz 
(HCTM), UKM, Bandar Tun Razak and fulfil the eligibility criteria. 
 
 
4.8 Study Subjects 
All adult patients undergoing vein-puncturing procedures (intravenous cannulation, 
venepuncture) at the Ophthalmology Outpatient Clinic, Hospital Canselor Tuanku Muhriz 
(HCTM), UKM, Bandar Tun Razak, fulfil the eligibility criteria and consent to trial 
participation. 
 
 
4.9 Sampling Method 
Stratified random sampling, a type of probability sampling method, will be used for both stages 
of the trial since the sample size required (section 4.9) is smaller than the whole pool of 
participants. Since differential pain experience has been shown in females and male adults58, 
the stratification variable is the gender of the participants to ensure that our study sample 
contains an equal number of female and male adults and is representative of patient population. 

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4.10 Sample Size Calculation 
4.10.1 Pharmacokinetic (PK) study 
Due to the paucity of prior information, formal sample size calculation for stage 1 
(pharmacokinetic (PK)) study based on power analysis cannot be carried out. Based on 
recommendations by Ogungbenro and Aarons (2010) and Julious (2012), the sample size is set 
at 20 subjects each for the PK study59-60. Even though, it is recommended that the sample size 
can be minimally set at 12 subjects for a single-group pilot pharmacodynamic trial (Julious, 
2012), the sample size is increased to 20 subjects since based on Julious 2012’s results (Figure 
3 of Julious 2012), the statistical asymptote is reached when the sample size is at least 20 
subjects59. Hence, the addition of another subject will result in only non-substantial gain in the 
precision of the parameter estimates when the sample size of 20 participants is reached. 
 
 
4.10.2 Pharmacodynamic (PD) Study 
For stage II of the trial, the sample size was calculated using Power and Sample Size (PS) 
Program version 3.1.6 (Vanderbilt University, Nashville, Tennessee, USA; 2018). The 
standard deviation (SD) of the VAS score of 2.1 were obtained from a prior study and this is 
for patients who received 5% EMLA.61 We consider a 1-point VAS difference between the 
intervention group as the minimum detectable difference (MTD) and type I error and study 
power (1- β) were fixed at 0.05 and 0.80, respectively. The ratio of controls to cases is fixed at 
1:1 ratio. Based on these parameter values, the calculated sample size is 70 participants per 
group. 
After accounting for a 10% drop-out rate, the final sample size is 77 participants per group 
(ntotal= 154 participants) for this stage of trial. 
 
 
4.11 Physical Description of Lignocaine-Embedded Microneedle 
Currently, we are able to manually produce some microneedle array patches (MAP) in a lab 
scale quantity. Different designed parameters MAP has been used for initial test on animal skin 
and also human skin. In this project, two major parts will be carried out. First, we will design 
a semi-auto machine to fabricate MAP. Refine parameters will be adapted from previous 
designs for the fabricate of the drug impregnated microneedles. Second, we will carry out a 
clinical trial for testing on the real implementation and application of the fabricated MAP. The 
details for each session are discussed as below 
 
 
Part I: Preparation of Biodegradable Drug Impregnated Microneedle Array Patch 
In a previous project, few parameters such as base plate size, number of microneedles per unit 
area, height etc. that have been determined and optimized. They will be used as a guideline for 
the design of new microneedle fabrication machine for better microneedle fabrication. With a 
new fabrication machine, we will be able to have a small-scale production to achieve a pre- 
commercialization technology readiness. 

16 
 
In this part, we will focus on the fabrication and implementation of biodegradable microneedle 
array patch. Sugar compounds such as sucrose, trehalose, and maltose have been experimented 
as biodegradable matrix materials for microneedles. In particular, maltose itself is a 
carbohydrate that is widely-acknowledged as a generally recognized safe excipient material for 
drug delivery. MAP fabricated from maltose generally demonstrate strong mechanical 
properties, and as such can facilitate perforation of skin and formation of micro-channels for 
transdermal drug delivery. Besides, for enabling the function as dissolvable MAP, maltose can 
rapidly dissolve in the dermal regions within minutes under body temperature, and is thus able 
to deliver therapeutic compounds such as proteins and peptides rapidly, safely and in an 
environmentally-friendly manner. The MAP that we propose as a prototype will consist of two 
basic but essential structures, i.e. the microneedles and the substrate (baseplate). This special 
design allows the microneedles to be separated from the substrate once the MAP is applied 
onto the skin. While the substrates can be readily disposed of after being peeled off from the 
skin, the microneedles and the therapeutic loads that they carry would stay on the skin and 
continue to dissolve and release drugs in a predetermined and sustained manner. Furthermore, 
as the microneedle is only 150 µm in length, thus, maximum penetration would only reach 
epidermis-dermis intersection. There is no possibility of the microneedles reaching the blood 
vessels, due to the limited length of the microneedles. Therefore, the drug distribution will 
follow a topical mode of distribution. No systemic distribution of drug is expected. 
A. Preparation of microneedle matrix mix for the dissolving microneedle 
 
I. 
To prepare the calcium ion cross-linked alginate/sugar (Ca2+/Alg-sugar) composites, 
sodium alginate powder is firstly dissolved in DI water at a weight ratio of 1:4 with the stir 
in a water bath at 60 °C until to gain the homogeneous solution. 
II. 
And then, the 15% (w/w) CaCl2 solution is added slowly with rapid mixing to cross-link 
alginate (CaCl2/Cross-linked alginate weight ratio = 1:10). 
III. 
To enhance the mechanical properties of composite microneedles, 15% (w/w) maltose 
monohydrate or any carbohydrate equivalents is added simultaneously into the sodium 
alginate solution to form precursor for preparation of paste for the fabrication of drug- 
loaded microneedles and baseplate (substrate): 
 
 
 
 
 
Di-ionized water 
 
 
Sodium alginate 
der 
 
CaCl2 solution 
 
 
 
 
 
 
 
 
Sodium alginate powder 
was measured 
 
 
Alginate and maltose in the ratio of 
1:4 maintained at an elevated 
temperature of 60C for forming an ion 
cross-linked composite 
ss-linked alginate 
15% (w/w) CaCl2 solution is 
added slowly with rapid mixing 
to cross-link alginate 
(CaCl2/Cross-linked alginate 
weight ratio = 1:10). 
 
Figure 1: The microneedle composite will be prepared based on the above-mentioned steps 

17 
 
A(i) 
A(ii) 
A(iii) 
A(iv) 
B. Fabrication of mould as a microneedle stamp used to fabricate negative mould 
with polydimethylsiloxane (PDMS) via the moulding technique 
 
A mould will be fabricated based in this fabrication process. The moulds for different sizes, 
shape and physical scales of microneedles will be designed. 
 
 
I. 
In the previous lab-scale fabrication, the pyramidal microneedle patch is fabricated 
using polymethyl methacrylate (PMMA) and used as the male mould. In this project, 
we will use a more solid and hard materials such as metal for the male mould. 
II. 
Subsequently, reversed mould (which is the female mould for the microneedle 
fabrication) is fabricated. To do so, a PDMS mixture is blended and added to a dust- 
free master structure, followed by centrifugation (4000 rpm, 30 min, 25°C, centrifuge 
and application of vacuum (200 mbar, 30 min, 25°C, vacuum drying oven) to remove 
air bubbles. 
III. 
After curing at 100°C for 5 h, the female mould is obtained, which can be used to 
produce microneedles made of sugar compounds (such as maltose). 
IV. 
The above processes are to prepare reverse mould with different physical parameters to 
test for the suitability and conformity of the mould for fixing onto the semi-auto 
fabrication machine. After that, the designs have the highest aptness will be used. The 
parameters will be adapted into a 3D printer for small scale produce of these female 
moulds. 
 
 
 
 
 
Female mould with the best parameters will be used as standard for small –scale production of the 
mould with different design and physical parameters used for microneedle fabrication 
 
Figure 2: Moulds with different sizes, shapes and physical parameters will be designed and 
prepared 

18 
 
C. Semi-auto machine for the fabication of dissolving microneedle array patch 
 
With the use of MAP-fabrication machine, 
I. 
A two-step casting process will be used to fabricate Ca2+/Alg-sugar composite 
microneedles. Firstly, the microneedle matrix loaded with therapeutic protein or drug 
is poured onto the centrifuged by a refrigerated centrifuge at 10000 rpm in 20 °C for 10 
min to fill up the porous container of microneedle mould to form a thicker pre-casting 
microneedle baseplate. 
II. 
Then, the preformed drug (lignocaine)-loaded microneedle preforming plate will be 
loaded to the machine and go through stamping, forming hardening to become a final 
microneedle array patch as shown in a schematic diagram in Figure 3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sugar compounds such as sucrose, trehalose, and 
maltose will be used to mixed to the dedicated 
drug/s and the mixture will go through a centrifuge 
process in a perforated container to form a thicker 
pre-casting microneedle baseplate 
Preformed drug loaded microneedle 
preforming plate will be loaded to the 
machine and go through stamping, forming 
hardening to become a final microneedle 
array patch 
 
 
Figure 3: The microneedle matrix composite will have filled into a porous container to form 
a pre-casted microneedle plate, the plate will be used to form microneedle tips and baseplate 
after a series of stamping, forming, hardening processes. 
 
 
The microneedle patch is made in a prototype lab in Alnair Incorporated, Tokyo, Japan, which 
is the collaborator in this study. The microneedles were individually heat treated at 120°C for 
sanitation before packaging. 

19 
 
4.12 Trial Conduct 
4.12.1 Pharmacokinetic (PK) Study Conducts 
Therefore, for this project, we will conduct a subproject as Phase 1 trial to assess the safety and 
tolerability of 12.5mg lignocaine-embedded microneedle on a small number of adult patients 
(20 patients; 10 males and 10 females) without hepatic or renal dysfunction. All participants of 
this phase I will be cataract patients who will be recruited from the HCTM ophthalmology 
outpatient clinic. Pre-treatment fasting is not required for all participants. 
On the day of the study, each potential participant will be screened for study eligibility based 
on our pre-specified inclusion and exclusion criteria. An interim abridged medical history will 
be taken from each participant and their list of medications will be reviewed. Vital signs 
(systolic and diastolic blood pressures, oral temperature, pulse and respiratory rates) will be 
taken and targeted clinical examinations will be performed by the medical officers to assess 
the overall health of the participants. 
After the 12.5mg lignocaine-impregnated microneedles have been applied to the designated 
sites, intravenous cannula will be placed on the dorsal aspect of the participant’s right hand. 
Approximately 3.0 ml venous blood samples will then be withdrawn from the antecubital fossa 
at each t=0, 30, 60, 90, 120, 180 minutes and collected into separate 3.5-ml of plastic blood 
collection tube with accelerator & separator gel (BD™, New Jersey, USA). Heparinized saline 
will be periodically infused to ensure that the cannula lumen remains patent throughout the 
sampling periods. The blood samples will then be sent to Jabatan Kimia Malaysia for 
lignocaine concentration measurements using the validated methodology of Gas 
Chromatography Nitrogen Phosphorus Detector (GC-NPD). 
 
 
a) Determining Serum Lignocaine Concentration using Gas Chromatography-Nitrogen 
Phosphorus Detector (GC-NPD): A Brief Protocol 
One (1) mL of the blood will be taken from the collection tube and alkalinized with NaOH 
solution of pH 12. The internal standard, Methaqualone and the organic solvent, Chlorobutane 
will then be added to the mixture. The mixture will be subsequently mixed using a roller mixer 
and centrifuged to extract the organic the layer which will be then concentrated. from the 
partition. A clean-up solution, hexane-ethanol, will be then added to the sample mixture which 
will be again vortexed and centrifuged. The bottom organic layer will be then moved into 
another tube and it will be evaporated to complete dryness under nitrogen gas flow in room 
temperature. The residue will be subsequently reconstituted using absolute ethanol prior to 
loading into the GC-NPD system. 
Lignocaine in the blood matrix will be spiked based on the level below or within the range in 
the therapeutic level. Besides, the response of the drug in the gas chromatography (i.e. the 
resolution and the peak of in GC) will be taken into consideration. In normal practice, 
lignocaine at the amount of 0.5 parts per million (ppm) will be spiked and a lower amount of 
lignocaine (0.3 ppm) will be used for quality control, which are based on previous 
recommendations by Winek et al (Lignocaine: Therapeutic: 1.5-5.0 ppm; Toxic: 7-20 ppm; 
Lethal:>25 ppm)62. For calibration, a 1-point calibration to estimate serum lignocaine 

20 
 
concentration will be used. A series of 1-point calibration will also be carried out whenever 
serum lignocaine concentration exceeds the therapeutic range. 
 
 
b) Post-intervention Monitoring and Pharmacokinetics Data Analysis 
The participants will be allowed to return home after the last blood sample is taken at t=180. 
The participants will be further monitored for any adverse events (AEs) such as redness, pain, 
itchiness, blistering, etc (local reactions) and light-headedness, euphoria, tinnitus, diplopia etc. 
(systemic reactions), serious adverse events (SAEs) and suspected unexpected serious adverse 
reactions (SUSARs) for up to 48 hours via telephone calls. 
The pharmacokinetic data will be first summarized in mean / standard deviation or median / 
interquartile range for continuous data and count and percentage for categorical data. The 
pharmacokinetic parameters (AUCinf, AUCt, Cmax, Cmin, tmax, t1/2, volume of distribution (Vd), 
Clearance (Cl)) of lignocaine will be evaluated using blood samples obtained at times t=0, 30, 
60, 90, 120, and 180 minutes after the application of lignocaine-impregnated MN patch. The 
intraindividual and interindividual variations of the pharmacokinetic parameters will be 
evaluated using coefficient of variation (CV) and these will be classified as low (CV≤10%), 
moderate (CV ≈ 25%) and high (CV>40%).63 The pharmacokinetic data will be analysed using 
the non-linear mixed effect models based on two-compartmental model which will be 
implemented on NONMEM® version VI (Icon Development Solutions, Ellicott City, 
Maryland, USA). The influence of clinically relevant covariates such as participant’s age, 
gender, BMI and others on pharmacokinetic parameters will be evaluated in a stepwise fashion. 
First-order conditional likelihood (FOCE INTER on NONMEM) will be used to fit the data 
and model selection will be dependent upon the likelihood ratio test, the estimates of 
pharmacokinetic parameters and their 95% confidence intervals and goodness-of fit measures. 
 
 
4.12.2 Pharmacodynamic (PD) Study Conduct 
a) Randomization Procedure and Blinding (Masking) of Trial Participants 
For random allocation, block randomization procedure with varying block size (permuted 
block) will be utilized to guarantee that both intervention groups will have an equal number of 
trial participants. This will be carried out by the trial statistician using the R package, blockrand 
version 1.50 which will be implemented on R platform.64 The list of generated random numbers 
will be used to allocate the study participants to either intervention or control branch. The 
allocation sequence generated will be kept in a password-protected document that is only 
accessible to the statistician to maintain allocation concealment. To further ensure the adequacy 
of allocation concealment, randomisation code will not be revealed until the potential trial 
participants have been definitively enrolled into the trial, which will be after all baseline 
measurements are made and all eligibility criteria are deemed fulfilled by the study recruiters. 
In addition, allocation concealment is further safeguarded by ensuring identity of the allotted 
treatment is only revealed to the interventionist (i.e. the person who will be administering the 
intervention) via secured telephone calls (central randomization). Consecutive recruitments 
will be made until the final intended sample size is achieved. 

21 
 
For this study, the outcome assessors and care providers (may be the same individual) will be 
masked to the identity of interventions (single blinding / single masking). Only the statistician 
and interventionist/procedurist will be unmasked to the study interventions. Furthermore, 
unique ID code to indicate each treatment sequence assignment will be generated and utilised 
to ensure that the unintentional / intentional unmasking of one trial participant will not 
compromise the integrity of blinding for the rest of study participants. The primary unblinded 
trial persons (subjects, the statistician and the procedurist/interventionist) are instructed not to 
divulge the identity of the allotted treatments to other blinded trial personnel. The success of 
blinding will be determined by asking the blinded trial persons to guess the identity of 
interventions received and then compare the results obtained with what would be anticipated 
by chance. Blinding indices such as James’ Blinding Index or Bang’s blinding Index could also 
be calculated to objectively assess whether blinding has been successfully achieved in this 
trial.65,66 
 
b) Administration of Lignocaine-embedded Microneedle (intervention) and EMLA 
(control) patches 
Prior to the administration of intervention/control, relevant clinic-demographic profiles (age, 
gender, ethnicity, anthropometric measurements, presence of comorbidities,) will be recorded 
and entered in the case report forms (CRFs) that are specifically designed for this study. This 
research study uses lignocaine embedded microneedles. The comparison of pharmacodynamic 
properties (i.e. efficacy) between 12.5 mg lignocaine delivered through direct embedment 
within the microneedle matrix and standard 5% EMLA dermal patch containing 1 finger-tip- 
unit (1 FTU = 0.5g) of 12.5mg lignocaine and 12.5mg prilocaine will be assessed via VAS 
score and skin algesimeter index for the pain induced by venepuncture. 
The window period given to lignocaine for it to be effective will be based on the usual clinical 
practice observation where it is usually applied for 30 minutes prior to venepuncture. The 
rationale behind it is due to logistical issues and for the day care's operational convenience. 
Nevertheless, in a busy clinical setting, the application time is sometimes shortened to 15 
minutes for a slight anaesthetic effect. Thus, we postulate that, with the aid of microneedle, 
the time to onset of action for lignocaine could be greatly reduced resulting in a much more 
reduced pain sensation when the clinical assessment is carried out 30 minutes after treatment 
application. 
The administrator of interventions (procedurist) will identify and draw a grid of 1cm × 1cm at 
the dorsum hand, which will serve as an ideal site for cannulation. The procedurist will then 
apply the lignocaine-impregnated microneedle patch. After a 30-minute application time, the 
attending medical officer will perform venepuncture line using a 21-gauge (G) hypodermic 
needle inserted into the vein in the dorsal aspect of the participant’s hand. 
For the participants allotted to the standard 5% EMLA dermal patch, 1 FTU of 5% EMLA 
cream will be applied and covered with a piece of adhesive to form a dermal patch. This will 
be applied for 30 minutes on the dorsum of the hand which will be cleaned and dried first. This 
application is equivalent to 12.5mg lignocaine and 12.5mg prilocaine. The EMLA patch will 
be firmly applied to the designated area (dorsal aspect of the hand). 

22 
 
During the trial day, the participants will not be allowed to take any analgesic medications 
(NSAID, Opioids, Paracetamol) since they will modulate the level of pain experienced by the 
participants due to the received interventions. Other medications and concomitant care will be 
permitted during the trial. 
 
 
c) Pain Assessment 
The study participants will first be guided on the operating manual for a 10-points, 100mm 
VAS pain score by an outcome assessor. The participants will be presented with a ruler that 
contains 100-mm slots with “No Pain” written on the left side and “Worst Pain” on the opposite 
right side. The study participants will then be asked to move and place the slider in the slot that 
accurately describes his/her pain at the following time points: 1) within 5 minutes after 
application of lignocaine-impregnated MN patch and before venepuncture/IV cannulation 
(baseline VAS score); 2) within 5 minutes after venepuncture/IV cannulation. The 
investigator/outcome assessor, who is blinded to the subject intervention arm will record the 
location of the slot where the slider is placed in millimetres (mm), clearly printed on the ruler’s 
reverse side and this will be the participant’s VAS score. Throughout the process, there will be 
a trained investigator standing by to assist the verification of the pain scale and to aid the 
participants who require additional assistance. 
For a subset of randomly selected patients, before applying MN patch and EMLA Cream, the 
patients will be attached with the PainMonitor™ (Med-Storm Innovation AS, Oslo, Norway) 
device whereby the electrodes will be attached to the hypothenar eminence of the opposite 
hand not receiving the vein cannulation. The procedurist will set up this machine and 
application before the interventions are commenced. The skin conductance peaks (in 
microSiemens (μS) and the skin algesimeter index (in microSiemens per second (μS/s)) will 
be recorded by the outcome assessor who will be blinded to the subject intervention arm. 
Those parameters indicate the skin’s sympathetic nerve block induced by the topical 
anaesthetic. The measurement time points start from the point of intervention and the 
recordings continued for at least 15 seconds. 
 
 
4.12.3 Clinical Data Collection 
All study personnel (i.e. interventionist/procedurist, investigator/outcome assessors, healthcare 
providers) will receive training (e.g. use of open ended questions when assessing VAS pain 
score and adverse events related to interventions) in VAS measurement, administration of 
lignocaine-embedded MN patch prior to trial commencement to standardise data collection, 
enhance data quality and reduce data inconsistency and measurement variability. 
All data collected will be checked for data quality using double data entry practice and checking 
for sensible data range and format (e.g. integer for the number of adverse events experienced 
within 24 hour of intervention). All paper-based CRFs will be stored in locked cabinets that 
are only accessible to the principal investigators, data manager and statistician and these will 
be maintained for 5 years after the trial ends. For quality control, periodic random check on a 
subset of CRFs will be carried out. 

23 
 
All data recorded on paper-based CRFs which will then be transcribed into an SPSS 
spreadsheet in an .sav extension at the central site conducted by statistician-trained data entry 
personnel. This will then be converted into a Stata-friendly file format (dta extension) to aid 
statistical analysis. The dataset will be password-protected and is only accessible to the 
principal study researchers and statistician to prevent any intended or unintended breach of 
patient confidentiality. Backup datasets will be stored in a password-protected thumb drives 
and cloud storage (GoogleDrive) that are again only accessible to the principal study 
investigators and statistician. The password for all datasets will be regularly changed to ensure 
maximum prevention against any data breach. 
 
 
4.12.4 Interventional Safety Assessment 
We define adverse events (AE) as "an abnormal sign, symptom, laboratory test, syndromic 
combination of such abnormalities, untoward or unplanned occurrence (e.g. accident), or any 
unexpected deterioration of concurrent illness".67 For serious AE (SAE), this is defined as 
"adverse events that result in the following outcomes: 1) death; 2) life-threatening AEs; 3) 
inpatient hospitalization or prolongation of existing hospitalization; 4) a persistence of 
significant incapacity or substantial disruption of the ability to conduct normal life functions, 
or a congenital anomaly or birth defect.68 
We classify the likelihood of AEs / SAEs (unrelated, possible, probable, definite) based on 
Naranjo et al. classification.69 All AE/SAE will be recorded and graded based on the Common 
Terminology Criteria for Adverse Events (CTCAE) Version 5 and US FDA's Toxicity Grading 
Scale Healthy Adults and Adolescents Volunteers Enrolled in Preventive Vaccine Clinical 
Trials. All AEs or SAEs can be classified into local skin reaction (pain, erythema, ecchymosis, 
swelling, itchiness, tenderness) or systemic reaction (fever, irritability, tiredness, anorexia, 
vomiting, tachycardia, seizure, hypotension). 
All AEs will be recorded on the CRFs. The detailed characteristics, the time and dates of onset 
and disappearance, and severity of AEs will be included in the CRFs. The study investigators 
will assess each participant experiencing AEs and they will receive appropriate treatments 
accordingly. The relationships between AEs and lignocaine-embedded microneedles will be 
evaluated by the investigators and classified as either unrelated, possible, probable or definite 
based on Naranjo et al. classification. AEs are considered as unexpected AEs when the AEs 
are not previously observed and not reported in the Investigator’s Brochure or standard 
lignocaine package insert. Any incidence of AEs or SAEs classified as possibly, probably and 
definitely linked to lignocaine-embedded microneedles will be monitored until the AEs/SAEs 
resolution is complete or the Investigator deem that the AEs or SAEs have become stable or 
irrevocable. 
All AEs of grade 3 and above will be reported to the JEPUKM within 5 business days. All 
SAEs (including Serious Unexpected Suspected Adverse Events (SUSARs)) will be reported 
within 24 hours of occurrence (expedited reporting) to the JEPUKM. If AEs / SAEs occur or 
are still ongoing by the end of the study period, the study participants will still be continuously 
followed up until complete resolution of AEs / SAEs which will take the following form: 1) 
additional participant visit to the trial centre / hospital; 2) telephone calls to the subjects; 3) 
additional reporting in the form of letters from the treating physicians. 

24 
 
Participant enrolment, intervention allocation and administration will be stopped if one of the 
following occurs (study halting criteria): 
a) Death related to lignocaine-impregnated MN patch 
b) Any participant experiences bronchospasm, laryngospasms or anaphylaxis within 24 hours 
post lignocaine-impregnated MN patch 
c) Any SAE related to lignocaine-impregnated MN patch 
d) Any AE of grade 3 and above or any SAE that cannot obviously be implicated on other 
causes 
e) Any study participant who develops abscess, ulceration or erosion at the site(s) of lignocaine- 
impregnated MN patch 
To ensure the independence of safety monitoring, all recorded safety data will be reviewed by 
JEPUKM which functions as an independent Data Safety Monitoring Board (DSMB) for our 
trial. 
4.13 Operational Definitions of Study Variables 
a) Independent variables 
 
i) Age: The age of a study participant at the first study visit. The variable will be measured in 
years and month and modelled as a continuous numerical variable and will not be categorised 
into separate age groups. 
 
ii) Gender: A categorical variable that will be recorded in the SPSS data frame as 0 = female 
(base category) and 1 = male. Missing data shall be recorded as 999 
 
iii) Ethnicity: A categorical variable that will be recorded in the SPSS data frame as 0 = Malay 
(base category); 1 = Chinese; 2 = Indian; and 3 = Other ethnicity. Missing data shall be recorded 
as 999. 
 
iv) Body mass index (BMI): A continuous numerical variable that is calculated using the 
standard body mass index formula; BMI = kg / m2. This variable will be categorised according 
to a widely used BMI classification70-71: 
<18.5= underweight (SPSS code: -1) 
18.5-24.9= normal BMI (SPSS code: 0; base category) 
25.0-29.9= Overweight (SPSS code: +1) 
≥30 = Obese (SPSS code: +2) 
 
The categorized variable will then be used as a predictor variable for statistical modelling 
purposes. Missing data will be recorded as 999 in the SPSS spreadsheet. 

25 
 
v) Intervention groups: A categorical variable representing the types of interventions 
received by the study participants in each study visit. This variable will be recorded in the 
SPSS data frame as 0 = 5% EMLA dermal patch only group (control, base category, 30 
minutes); 1 = Lignocaine-Embedded Microneedle Patch (30 minutes) 
 
vi) Baseline VAS score: A continuous numerical variable that will be measured before the 
administration of intervention. This will be used as a predictor variable to control the 
confounding effect of heterogenous baseline VAS scores among study participants. Missing 
data will be recorded as 999 in the SPSS spreadsheet. 
 
vii) Baseline pain score obtained via PainMonitor™ device: A continuous numerical 
variable that will be measured before the administration of intervention. This will be used as 
a predictor variable to control the confounding effect of heterogenous baseline VAS scores 
among study participants. Missing data will be recorded as 999 in the SPSS spreadsheet. 
 
 
b) Dependent (outcome variables) 
 
i) VAS score (30-minutes post intervention application): A continuous numerical variable 
that will be measured during each visit; 30 minutes after lignocaine-embedded microneedle or 
5% EMLA application. Missing data will be recorded as 999 in the SPSS spreadsheet. 
 
ii) Pain score from PainMonitor™ device (30-minutes post intervention application): A 
continuous numerical variable that will be measured during each visit: 30 minutes after either 
lignocaine-embedded microneedle or 5% EMLA application. Missing data will be recorded as 
999 in the SPPS spreadsheet. 

26 
 
4.14 Ethical Issues 
Voluntary written informed consent will be obtained from each study participant. This study 
will be conducted in accordance with the principles of ethics in human research as stipulated 
by the Declaration of Helsinki (18th World Medical Association General Assembly, 1964), the 
Good Clinical Practice (GCP) guidelines, and ISO14155:2020 Clinical Investigation for 
Medical Devices for Human Subjects. Ethical approval will be obtained from the UKM 
Research Ethics Committee (Human) (JEPUKM). 
All research participants will sign informed consent forms prior to their study participation. 
The participants will be made aware that their participation is completely voluntary and they 
can withdraw from the study at any time point. The research participants will also be notified 
that their decisions to withdraw from the study will not jeopardise their current or subsequent 
treatments and healthcare services received. To ensure the confidentiality of patient 
information, each participant will be assigned an anonymous research ID code that will be used 
for data storage and analysis. The data will solely be made available to the research team 
members and access to the storage may only be granted by the principal investigators. 
To aid the transparency of reporting, the trial will be registered at the Clinical Trials Registry 
(https://clinicaltrials.gov/) and the Malaysian National Medical Research Registry (NMRR) 
(https://nmrr.gov.my/). The full trial protocol will be made available in the same trial registries 
and prepared according to the Consolidated Standards of Reporting Trials (CONSORT) 
guideline for parallel design and the 2013 Standard Protocol Items: Recommendations for 
Interventional Trials (SPIRIT) statement. Any future protocol modifications will be submitted 
first to the JEPUKM for approval and the list of protocol changes will be made available to the 
public via both the Clinical Trials and NMRR registries. 
The trial data with study participant identification numbers removed (Hospital RN, Identity 
Card (IC) numbers, subject’s identification number (SIDNO) will be made available to the 
public via Harvard Dataverse repository for research data (https://dataverse.harvard.edu) to 
ensure the transparent dissemination of study findings and adherence to recommendations 
made by leading medical journal editors for the future publication of this research. 

27 
 
4.15 Statistical Analysis 
Data analysis will be performed using Statistical Package for Social Science (SPSS™) (IBM 
Corp. Released 2020. IBM Statistics for Windows, Version 27.0, Armonk, NY: IB Corp) and 
STATA™ version 15 (StatCorp. 2017. Stata Statistical Software: Release 15. College Station, 
Texas: StataCorp LP). Our primary analysis will be based on the intention-to-treat (ITT) 
principle by which all trial participants will be analysed according to their original intended 
treatment assignment. For missing observations, we will use the multiple imputation method 
to fill in the missing data, assuming the missing at random (MAR) mechanism. To check the 
robustness of the results, we will also conduct sensitivity analysis by comparing the results 
obtained using complete case analysis (i.e. including patients with complete observations) with 
full case (i.e. patients with complete and imputed observations for missing data) analysis. 
The differences in terms of the outcomes measures between the groups will be statistically 
assessed using an independent t-test if the parametric assumption is met (i.e. the data are 
normally distributed) or the Mann-Whitney test if the data are non-normally distributed. To 
control and adjust the effects of confounding variables such as baseline pain scores, body mass 
index (BMI), gender of the patients, multiple linear regression analysis will be used. Variable 
selection will be based on a mixture of strategies: 1) stepwise regression based on Akaike 
Information Criterion (AIC), Bayesian Information Criterion (BIC); 2) purposeful selection of 
covariates; 3) clinical domain knowledge. Effect modification will be assessed by creating and 
assessing the significance of the statistical interaction terms. The goodness of fit of the model 
will be evaluated using the coefficient of multiple determination, R2. 
Model assumptions (linearity, independence, normality, and homoscedasticity of residuals) 
will be assessed using a studentised residual vs predicted values scatter plots and Durbin 
Watson statistics. Box-Cox transformation with carefully selected exponent (lambda) will be 
employed in the presence of skewness in the outcome variables. The presence of influential 
observations will be suspected in the presence of large leverage (extreme value in the x space) 
and residual values (extreme value in the y space) for any outlying observations. This will be 
further confirmed using influential diagnostic measures such as Cook's distance72, dfFITS and 
dfBeta.73 The significance threshold will be set at 0.05 and 95% confidence intervals will be 
presented for each effect estimate. 

28 
 
TRIAL WORKFLOW 
 
Participant recruitment and screening based on the trial’s 
eligibility criteria from ophthalmology clinic (n =) 
 
Participant excluded for not 
meeting eligibility criteria (n=) 
Stratified sampling applied. Consent will be obtained from 
each eligible trial participant 
 
 
 
 
Study Subjects (n=) 
Participant excluded for not 
consenting to trial participation (n=) 
 
 
 
Pharmacokinetic Study 
(n=) 
Pharmacodynamic Study 
(n=) 
 
 
 
 
Blood sampling at t=0,30,60, 
Lignocaine- 
embedded MN 
(n=) 
5% EMLA cream 
(n=) 
90, 120, 180 minutes. 
Briefing of participants, Baseline assessment for 
VAS score and skin algesimeter index 
 
Serum lignocaine concentration 
quantification using GC-NPD to be 
conducted at Jabatan Kimia Malaysia 
Lignocaine-embedded 
MN application and 
wait for 30 minutes 
(window period) 
5% EMLA dermal 
patch application and 
wait for 30 minutes 
(window period) 
 
Analysis of pharmacokinetic parameters (AUC, 
Cmax, Tmax,T1/2, Vd, Clearance) using NONMEM 
Venipuncture procedure. Then, post-intervention 
VAS score and skin algesimeter index assessments. 
 
 
 
 
 
 
Final number of participants 
Participant excluded due to: 
Missing data (n=) 
Study withdrawal (n=) 
Serum lignocaine assay 
error (n=) 
 
 
 
 
 
Final number of 
 
 
 
 
 
Final number of 
included for data analysis (n=) 
participants included for 
data analysis (n=) 
participants included for 
data analysis (n=) 
 
Statistical analysis using SPSS 
 
 
Publication of trial results and end of project report submission to JEPEM, UKM 
 
Figure 1: Anticipated trial workflow based on the 2010 CONSORT statement flowchart 

 
29 
 
GANTT CHART 
 
 
 
Project activities 
Timeline 
2022 
2023 
2024 
11 12 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
Research 
application for 
ethics approval 
Trial subject 
recruitment 
Serum assays for 
lignocaine levels 
Data analyses and 
interpretation 
Data presentation, 
Manuscript 
preparation, Final 
project report, 
and Publication 
End of project 

30 
 
BUDGET 
 
No. 
Description 
Unit price (RM) 
Quantity 
Price (RM) 
1 
PROFESSIONAL SERVICE 
1.1 
Lignocaine quantitation assay with GC-NPD 
125 sample 
120 
15000 00 
Total 
15000 

31 
 
REFERENCES 
1. 
Alkilani AZ, McCrudden MT, Donnelly RF. Transdermal Drug Delivery: Innovative Pharmaceutical 
Developments Based on Disruption of the Barrier Properties of the stratum corneum. Pharmaceutics. 
2015;7(4):438-70. doi: 10.3390/pharmaceutics7040 438. 
2. 
Jeong WY, Kwon M, Choi HE, Kim KS. Recent advances in transdermal drug delivery systems: a 
review. Biomater Res. 2021;25(1):24. doi: 10.1186/s40824-021-00226-6. 
3. 
Lee BM, Lee C, Lahiji SF, Jung UW, Chung G, Jung H. Dissolving Microneedles for Rapid and 
Painless Local Anesthesia. Pharmaceutics. 2020;12(4):366. doi: 10.3390/pharmaceutics12040366. 
4. 
Murthy SN, Sammeta SM, Bowers C. Magnetophoresis for enhancing transdermal drug delivery: 
Mechanistic 
studies 
and 
patch 
design. 
J 
Control 
Release. 
2010;148(2):197-203. 
doi: 
10.1016/j.jconrel.2010.08.015. 
5. 
Zempsky WT, Sullivan J, Paulson DM, Hoath SB. Evaluation of a low-dose lidocaine iontophoresis 
system for topical anesthesia in adults and children: a randomized, controlled trial. Clin Ther. 
2004;26(7):1110-9. doi: 10.1016/s0149-2918(04)90183-x. 
6. 
Kim TY, Jung DI, Kim YI, Yang JH, Shin SC. Anesthetic effects of lidocaine hydrochloride gel using 
low frequency ultrasound of 0.5 MHz. J Pharm Pharm Sci. 2007;10(1):1-8. 
7. 
Benson HAE, Grice JE, Mohammed Y, Namjoshi S, Roberts MS. Topical and Transdermal Drug 
Delivery: From Simple Potions to Smart Technologies. Curr Drug Deliv. 2019;16(5):444-460. doi: 
10.2174/1567201816666190201143457. 
8. 
Yousef H, Alhajj M, Sharma S. Anatomy, Skin (Integument), Epidermis. [Updated 2021 Nov 19]. In: 
StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: 
https://www.ncbi.nlm.nih.gov/books/NBK470464/ 
9. 
Ali S, Shabbir M, Shahid N. The structure of skin and transdermal drug delivery system - A review. 
Research Journal of Pharmacy and Technology. 2015; 8(2):103-109. doi: 0.5958/0974- 
360X.2015.00019.0 
10. 
Duarah S, Sharma M, Wen J. Recent advances in microneedle-based drug delivery: Special emphasis 
on its use in 
paediatric population. 
Eur J Pharm Biopharm. 
2019;136:48-69. doi: 
10.1016/j.ejpb.2019.01.005. 
11. 
Liu X, Kruger P, Maibach H, Colditz PB, Roberts MS. Using skin for drug delivery and diagnosis in 
the critically ill. Adv Drug Deliv Rev. 2014 ;77:40-9. doi: 10.1016/j.addr.2014.10.004. 
12. 
Brown MB, Martin GP, Jones SA, Akomeah FK. Dermal and transdermal drug delivery systems: 
current and future prospects. Drug Deliv. 2006;13(3):175-87. doi: 10.1080/10717540500455975. 
13. 
Andrews SN, Jeong E, Prausnitz MR. Transdermal delivery of molecules is limited by full epidermis, 
not just stratum corneum. Pharm Res. 2013;30(4):1099-109. doi: 10.1007/s11095-012-0946-7. 
14. 
Ita K. Transdermal Delivery of Drugs with Microneedles-Potential and Challenges. Pharmaceutics. 
2015;7(3):90-105. doi: 10.3390/pharmaceutics7030090. 
15. 
Waghule T, Singhvi G, Dubey SK, Pandey MM, Gupta G, Singh M, Dua K. Microneedles: A smart 
approach and increasing potential for transdermal drug delivery system. Biomed Pharmacother. 
2019;109:1249-1258. doi: 10.1016/j.biopha.2018.10. 078. 
16. 
Li J, Zeng M, Shan H, Tong C. Microneedle Patches as Drug and Vaccine Delivery Platform. Curr Med 
Chem. 2017;24(22):2413-2422. doi: 10.2174/092986732466617 0526124053. 
17. 
Xu J, Xu D, Xuan X, He H. Advances of Microneedles in Biomedical Applications. Molecules. 
2021;26(19):5912. doi: 10.3390/molecules26195912. 

32 
 
18. 
Zhang Y, Jiang G, Yu W, Liu D, Xu B. Microneedles fabricated from alginate and maltose for 
transdermal delivery of insulin on diabetic rats. Mater Sci Eng C Mater Biol Appl. 2018;85:18-26. doi: 
10.1016/j.msec.2017.12.006. 
19. 
Larrañeta E, Lutton REM, Woolfson AD, Donnelly RF. Microneedle arrays as transdermal and 
intradermal drug delivery systems: Materials science, manufacture and commercial development. 
Materials 
Science 
and 
Engineering 
R: 
Reports. 
2016;104:1-32. 
https://doi.org/10.1016/j.mser.2016.03.001. 
20. 
Kolli CS, Banga AK. Characterization of solid maltose microneedles and their use for transdermal 
delivery. Pharm Res. 2008;25(1):104-13. doi: 10.1007/s11095-007-9350-0. 
21. 
Sartawi Z, Blackshields C, Faisal W. Dissolving microneedles: Applications and growing therapeutic 
potential. J Control Release. 2022;348:186-205. doi: 10.1016/j.jconrel.2022.05.045. 
22. 
Rzhevskiy A, Popov A, Pavlov C, Anissimov Y, Zvyagin A, Levin Y, Kochba E. Intradermal injection 
of lidocaine with a microneedle device to provide rapid local anaesthesia for peripheral intravenous 
cannulation: 
A 
randomised 
open-label 
placebo-controlled 
clinical 
trial. 
PLoS 
One. 
2022;17(1):e0261641. doi: 10.1371/journal.pone.0261641. 
23. 
Ornelas J, Foolad N, Shi V, Burney W, Sivamani RK. Effect of Microneedle Pretreatment on Topical 
Anesthesia: 
A 
Randomized 
Clinical 
Trial. 
JAMA 
Dermatol. 
2016;152(4):476-7. 
doi: 
10.1001/jamadermatol.2015.5544. 
24. 
Gupta J, Denson DD, Felner EI, Prausnitz MR. Rapid local anesthesia in humans using minimally 
invasive microneedles. Clin J Pain. 2012;28(2):129-35. doi: 10.1097/AJP.0b013e318225dbe9. 
25. 
Lidocaine Hydrochloride and Epinephrine Injection USP [Package Insert]. Quebec, Canada: Pfizer 
Canada ULC; 2006. 
26. 
Olschewski A, Hempelmann G, Vogel W, Safronov BV. Blockade of Na+ and K+ currents by local 
anesthetics in the dorsal horn neurons of the spinal cord. Anesthesiology. 1998;88(1):172-9. doi: 
10.1097/00000542-199801000-00025. 
27. 
Edwards DJ, Lalka D, Cerra F, Slaughter RL. Alpha1-acid glycoprotein concentration and protein 
binding in trauma. Clin Pharmacol Ther. 1982;31(1):62-7. doi: 10.1038/clpt.1982.10. 

33 
 
28. 
Tesseromatis C, Kotsiou A, Tsagataki M, Tigka E, Vovou J, Alevizou A, Perisanidis C, Saranteas T, 
Karakitsos D, Karabinis A, Kostopanagiotou G. In vitro binding of lidocaine to liver tissue under the 
influence of propranolol: another mechanism of interaction? Eur J Drug Metab Pharmacokinet. 
2007;32(4):213-7. doi: 10.1007/BF03191006. 
29. 
Routledge PA, Stargel WW, Wagner GS, Shand DG. Increased alpha-1-acid glycoprotein and lidocaine 
disposition in myocardial infarction. Ann Intern Med. 1980;93(5):701-4. doi: 10.7326/0003-4819-93- 
5-701. 
30. 
Barry M, Keeling PW, Weir D, Feely J. Severity of cirrhosis and the relationship of alpha 1-acid 
glycoprotein concentration to plasma protein binding of lidocaine. Clin Pharmacol Ther. 
1990;47(3):366-70. doi: 10.1038/clpt.1990.41. 
31. 
McNamara PJ, Slaughter RL, Visco JP, Elwood CM, Siegel JH, Lalka D. Effect of smoking on binding 
of lidocaine to human serum proteins. J Pharm Sci. 1980;69(6):749-51. doi: 10.1002/jps.2600690646. 
32. 
Lerman J, Strong HA, LeDez KM, Swartz J, Rieder MJ, Burrows FA. Effects of age on the serum 
concentration of alpha 1-acid glycoprotein and the binding of lidocaine in pediatric patients. Clin 
Pharmacol Ther. 1989;46(2):219-25. doi: 10.1038/clpt.1989.129. 
33. 
Routledge PA, Shand DG, Barchowsky A, Wagner G, Stargel WW. Relationship between alpha 1-acid 
glycoprotein and lidocaine disposition in myocardial infarction. Clin Pharmacol Ther. 1981;30(2):154- 
7. doi: 10.1038/clpt.1981.141. 
34. 
Burm AG, de Boer AG, van Kleef JW, Vermeulen NP, de Leede LG, Spierdijk J, Breimer DD. 
Pharmacokinetics of lidocaine and bupivacaine and stable isotope labelled analogues: a study in healthy 
volunteers. Biopharm Drug Dispos. 1988;9(1):85-95. doi: 10.1002/bod.2510090109. 
35. 
Thomson PD, Melmon KL, Richardson JA, Cohn K, Steinbrunn W, Cudihee R, Rowland M. Lidocaine 
pharmacokinetics in advanced heart failure, liver disease, and renal failure in humans. Ann Intern Med. 
1973;78(4):499-508. doi: 10.7326/0003-4819-78-4-499. 
36. 
Huet PM, Lelorier J, Pomier G, Marleau D. Bioavailability of lidocaine in normal volunteers and 
cirrhotic patients. Clin Pharmacol Ther. 1979;25(2):229-30. 
37. 
Bauer LA, Horn JR, Maxon MS, Easterling TR, Shen DD, Strandness DE Jr. Effect of metoprolol and 
verapamil administered separately and concurrently after single doses on liver blood flow and drug 
disposition. J Clin Pharmacol. 2000;40(5):533-43. doi: 10.1177/00912700022009152. 
38. 
Prescott LF, Adjepon-Yamoah KK, Talbot RG. Impaired Lignocaine metabolism in patients with 
myocardial 
infarction 
and 
cardiac 
failure. 
Br 
Med 
J. 
1976;1(6015):939-41. 
doi: 
10.1136/bmj.1.6015.939. 
39. 
Wang JS, Backman JT, Taavitsainen P, Neuvonen PJ, Kivistö KT. Involvement of CYP1A2 and 
CYP3A4 in lidocaine N-deethylation and 3-hydroxylation in humans. Drug Metab Dispos. 
2000;28(8):959-65. 

34 
 
40. 
Huang W, Lin YS, McConn DJ 2nd, Calamia JC, Totah RA, Isoherranen N, Glodowski M, Thummel 
KE. Evidence of significant contribution from CYP3A5 to hepatic drug metabolism. Drug Metab 
Dispos. 2004;32(12):1434-45. doi: 10.1124/dmd.104.001313. 
41. 
Ha HR, Candinas R, Stieger B, Meyer UA, Follath F. Interaction between amiodarone and lidocaine. J 
Cardiovasc Pharmacol. 1996;28(4):533-9. doi: 10.1097/00005344-199610000-00009. 
42. 
Isohanni MH, Neuvonen PJ, Olkkola KT. Effect of fluvoxamine and erythromycin on the 
pharmacokinetics of oral lidocaine. Basic Clin Pharmacol Toxicol. 2006;99(2):168-72. doi: 
10.1111/j.1742-7843.2006.pto_482.x. 
43. 
Olkkola KT, Isohanni MH, Hamunen K, Neuvonen PJ. The effect of erythromycin and fluvoxamine on 
the pharmacokinetics of intravenous lidocaine. Anesth Analg. 2005;100(5):1352-1356. doi: 
10.1213/01.ANE.0000148123.79437.F9 
44. 
Orlando R, Piccoli P, De Martin S, Padrini R, Floreani M, Palatini P. Cytochrome P450 1A2 is a major 
determinant of lidocaine metabolism in vivo: effects of liver function. Clin Pharmacol Ther. 
2004;75(1):80-8. doi: 10.1016/j.clpt.2003.09.007. 
45. 
Narang PK, Crouthamel WG, Carliner NH, Fisher ML. Lidocaine and its active metabolites. Clin 
Pharmacol Ther. 1978;24(6):654-62. doi: 10.1002/cpt1978246654. 
46. 
Thomson AH, Elliott HL, Kelman AW, Meredith PA, Whiting B. The pharmacokinetics and 
pharmacodynamics of lignocaine and MEGX in healthy subjects. J Pharmacokinet Biopharm. 
1987;15(2):101-15. doi: 10.1007/BF01062338. 
47. Strong JM, Mayfield DE, Atkinson AJ Jr, Burris BC, Raymon F, Webster LT Jr. Pharmacological activity, 
metabolism, and pharmacokinetics of glycinexylidide. Clin Pharmacol Ther. 1975;17(2):184-94. doi: 
10.1002/cpt1975172184. 
48  Wójcicki J, Kozłowski K, Droździk M, Wójcicki M. Lidocaine elimination in patients with liver 
cirrhosis. Acta Pol Pharm. 2002;59(4):321-4. 
49. 
Rademaker AW, Kellen J, Tam YK, Wyse DG. Character of adverse effects of prophylactic lidocaine 
in the coronary care unit. Clin Pharmacol Ther. 1986;40(1):71-80. doi: 10.1038/clpt.1986.141. 
50. 
Weinberg L, Peake B, Tan C, Nikfarjam M. Pharmacokinetics and Pharmacodynamics of Lignocaine: 
A Review. World Journal of Anesthesiology. 2015;4(2):17-29. doi: 0.5313/wja.v4.i2.17. 
51. 
Kirkland DJ, Sheil ML, Streicker MA, Johnson GE. A weight of evidence assessment of the 
genotoxicity of 2,6-xylidine based on existing and new data, with relevance to safety of lidocaine 
exposure. Regul Toxicol Pharmacol. 2021;119:104838. doi: 10.1016/j.yrtph.2020.104838. 
52. 
Davies PS, Galer BS. Review of lidocaine patch 5% studies in the treatment of postherpetic neuralgia. 
Drugs. 2004;64(9):937-47. doi: 10.2165/00003495-200464090-00002. 
53. 
Gimbel J, Linn R, Hale M, Nicholson B. Lidocaine patch treatment in patients with low back pain: 
results of an open-label, nonrandomized pilot study. American Journal of Therapeutics. 
2005;12(4):311-9. doi: 10.1097/01.mjt.0000164828.57392.ba. 

35 
 
54. 
Sun Y, Li T, Wang N, Yun Y, Gan TJ. Perioperative systemic lidocaine for postoperative analgesia and 
recovery after abdominal surgery: a meta-analysis of randomized controlled trials. Dis Colon Rectum. 
2012;55(11):1183-94. doi: 10.1097/DCR.0b013e318259bcd8. Erratum in: Dis Colon Rectum. 
2013;52(2):271. 
55. 
Zink KA, Mayberry JC, Peck EG, Schreiber MA. Lidocaine patches reduce pain in trauma patients with 
rib fractures. Am Surg. 2011;77(4):438-42. doi: 10.1177/000313481107700419. 
56. 
Kim CH, Yoon JU, Lee HJ, Shin SW, Yoon JY, Byeon GJ. Availability of a 5% lidocaine patch used 
prophylactically for venipuncture- or injection-related pain in children. J Anesth. 2012;26(4):552-5. 
doi: 10.1007/s00540-012-1360-3. 
57. 
Bai Y, Miller T, Tan M, Law LS, Gan TJ. Lidocaine patch for acute pain management: a meta-analysis 
of 
prospective 
controlled 
trials. 
Curr 
Med 
Res 
Opin. 
2015 
Mar;31(3):575-81. 
doi: 
10.1185/03007995.2014.973484. 
58. 
Bartley EJ, Fillingim RB. Sex differences in pain: a brief review of clinical and experimental findings. 
Br J Anaesth. 2013 Jul;111(1):52-8. doi: 10.1093/bja/aet127. 
59. 
Ogungbenro K, Aarons L. How many subjects are necessary for population pharmacokinetic 
experiments? Confidence interval approach. Eur J Clin Pharmacol. 2008;64(7):705-13. doi: 
10.1007/s00228-008-0493-7. 
60. 
Julious SA. Sample size of 12 per group rule of thumb for a pilot study. Pharmaceutical Statistics: The 
Journal of Applied Statistics in the Pharmaceutical Industry. 2005;4(4):287-91. 
61. 
Yamamoto LG, Boychuk RB. A blinded, randomized, paired, placebo-controlled trial of 20-minute 
EMLA cream to reduce the pain of peripheral i.v. cannulation in the ED. Am J Emerg Med. 1998 
Nov;16(7):634-6. doi: 10.1016/s0735-6757(98)90163-2. 
62. 
Winek CL, Wahba WW, Winek CL Jr, Balzer TW. Drug and chemical blood-level data 2001. Forensic 
Sci Int. 2001;122(2-3):107-23. doi: 10.1016/s0379-0738(01)00483-2. 
63. 
Rowland,\ M, Tozer TN. Clinical pharmacokinetics and pharmacodynamics: Concepts and 
Applications. 4th edition. Philadelphia: Lippincott Williams and Wilkins; 2011. 
64. 
Greg Snow. blockrand: Randomization for Block Random Clinical Trials. R Package version 1.5. 2022. 
https://CRAN.R-project.org/package=blockrand 
65. 
James KE, Bloch DA, Lee KK, Kraemer HC, Fuller RK. An index for assessing blindness in a multi- 
centre clinical trial: disulfiram for alcohol cessation--a VA cooperative study. Stat Med. 1996 Jul 
15;15(13):1421-34. 
66. 
Bang H, Ni L, Davis CE. Assessment of blinding in clinical trials. Control Clin Trials. 2004 
Apr;25(2):143-56. doi: 10.1016/j.cct.2003.10.016. PMID: 15020033. 
67. 
Aronson JK. Medication errors: what they are, how they happen, and how to avoid them. QJM. 
2009;102(8):513-21. doi: 10.1093/qjmed/hcp052. 
68. 
Code of Federal Regulation, Title 21, Section 312.32 (21CFR312.32). (2020). Accessible from: 
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch. cfm?fr=312.32. Date of access: 05 
/ 06 / 2022. 

36 
 
69. 
Naranjo CA, Busto U, Sellers EM, Sandor P, Ruiz I, Roberts EA, Janecek E, Domecq C, Greenblatt 
DJ. A method for estimating the probability of adverse drug reactions. Clin Pharmacol Ther. 
1981;30(2):239-45. doi: 10.1038/clpt.1981.154. 
70. 
Must A, Spadano J, Coakley EH, Field AE, Colditz G, Dietz WH. The disease burden associated with 
overweight and obesity. JAMA. 1999;282(16):1523-9. doi: 10.1001/jama.282.16.1523. 
71. 
Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health 
Organ Tech Rep Ser. 2000;894:i-xii, 1-253. 
72. 
Cook RD. Detection of Influential Observation in Linear Regression. Technometrics. 1997;19(1):15– 
18. https://doi.org/10.2307/1268249 
73. 
Belsley DA, Kuh E, Welsch RE. Regression diagnostics: Identifying influential data and sources of 
collinearity. New York: Wiley; 1980. 
74. 
Muhammad Irfan Abdul Jalal , Kai Shen Ooi , Kai Cheong Foo et al. Transdermal Maltose-Based 
Microneedle Patch as Adjunct to Enhance Topical Anesthetic before Intravenous Cannulation of 
Pediatric Thalassemic Patients Receiving Blood Transfusion: A Randomized Controlled Trial Protocol 
J Clin Med 2022 Sep 8;11(18):5291. doi: 10.3390/jcm11185291. 
75. 
EMLA Cream 5% (5g pack). Summary of product characteristics. 
https://www.medicines.org.uk/emc/product/871/smpc/print 

37 
 
 
APPENDIX A: PATIENT INFORMATION SHEET AND CONSENT FORM 
(ENGLISH VERSION) 
 
PATIENT INFORMATION SHEET 
 
Research Title: Transdermal microneedle lignocaine delivery versus EMLA patch for topical 
analgesia before venepuncture procedure to adults in a clinic setting 
 
Introduction: 
You are invited to participate in a clinical research study. Before participating in this study, it 
is crucial that you read thoroughly and understand the information provided in this sheet, 
whereby the study will be verbally explained to you, and you will be allowed to ask questions. 
After you are adequately satisfied that you understand this study and you wish to take part 
or continue to participate in this study, you must sign this informed consent form. You will be 
given a copy of these patient information sheet and consent forms to take home with you. 
 
Purpose of Study: 
Vein-puncturing procedures elicits much pain and is regarded as the most frequently 
traumatic experience encountered among patients in the regular practice of the health care 
settings. Such pain may be reduced by applying topical anaesthetic drugs. For example, 
lignocaine cream is a non-invasive and convenient approach of administering anaesthesia to 
the body skin surface. The usual recommended duration of time taken for lignocaine cream 
to work is around 30 minutes. However, in a busy clinical setting, the time is often shortened 
to 15 minutes for a slight anaesthetic effect. As such, a transdermal drug delivery system 
(TDDS), the microneedle, has been introduced as an alternative to enhance the delivery of 
topical anaesthesia by puncturing the skin at a very superficial level to create multiple tiny 
tracts for faster action of anaesthetic drug absorption. The microneedle patch is a prototype 
device that is still being tested and not registered. It contains multiple micron-sized needles 
made of sugar (maltose) that will dissolve into the skin and subsequently achieve the 
objective of delivering the anaesthetic drugs. As such, our current research study aims to 
determine the safety and tolerability of lignocaine-embedded microneedle patch for patients 
requiring frequent venepuncture or intravenous (IV) cannulation. Besides, this research also 
aims to obtain preliminary information on the efficacy of lignocaine-embedded microneedle 
in reducing pain associated with venepuncture or intravenous cannulation. 
 
What will the study involve? 
For this research, you will be participating in either one of the following components of the 
trial: i) blood levels of lignocaine over 3hours or ii) lignocaine-microneedle versus EMLA patch. 
It is emphasised that you do not get to choose which group you will be in if you agree to 
participate. We use a selection system that the project investigators are abiding to. 

38 
 
 
i) Blood level of drug study over 3 hours 
In this study, you are will undergo a venepuncture for blood collection during an outpatient 
visit at the Ophthalmology Clinic, HCTM. A medical doctor will perform an intravenous 
cannulation on the back of your right hands (dorsal side) that is intended for blood collection. 
Subsequently, you will be receiving a topical anaesthetic administration via lignocaine- 
embedded microneedle which will be applied on the skin surface of the back of your left hand. 
This will result in the numbness to that particular region of the hand. A small amount of blood 
(about 3 mLs each time) samples will then be collected from the cannula at six time points 
(time, t= 0, 30, 60, 90, 120, and 180 minutes) which will be sent to the laboratory for 
determining the level of lignocaine in your blood. In total, you are expected to be at the clinic 
for 3 hours, which is the average time taken for a normal visit to the eye clinic. 
 
ii) Lignocaine-microneedle versus EMLA patch 
 
If you are selected as a participant of this part of the research, you will first undergo a routine 
clinical examination on the trial day. You may be chosen to rate your baseline pain score using 
a ruler-styled scoring system (VAS) and a pain monitoring device. The lignocaine-embedded 
microneedle or EMLA patch, depending on which group you will be allotted to, will be 
introduced on the identified surface of the back of your hand. After 30 minutes, venepuncture 
or intravenous cannulation will be performed by a trained medical officer. You will be then 
asked to evaluate the degree of your pain using the VAS score. You will also have a pain 
monitoring device attached to your palm for measurement purposes. You will then be 
monitored for one hour following the procedure to identify whether you experience any 
adverse effects associated with the lignocaine-microneedle application. 
 
Risks: 
This investigation possesses minimal risk to participants and is unlikely to cause side effects. 
Although topical skin anaesthetics are applied, you may still feel some pain from the needle/s, 
although this will be much reduced. Topical lignocaine is a widely used drug and little adverse 
reactions have been reported. Nevertheless, the possible side effects reported from the 
lignocaine application include: 
 
• pallor 
• redness 
• alterations in temperature sensation over the application area 
 
With regards to EMLA patch, special precautions are advised in G6PD deficient individuals 
who may be at increased risk for developing symptoms as a result of methaemoglobin rise in 
blood causing temporarily a bluish tinge to the skin and decreased oxygen in the blood. If you 
are G6PD deficient, please inform us about this condition prior to the study. 
Additional risks associated with microneedle application might include possible mild-to- 
moderate irritation, especially in those with sensitive skin, such as 
 
• redness 
• swelling 
• itching 
• blistering 

39 
 
 
Adverse side-effects reporting is part of the outcomes of this study, but If you encounter any 
problems or side effects experienced during the study, we will be giving you medications to 
reduce the side effects accordingly. 
 
Benefits: 
The information collected from this clinical study may contribute to the advancement of 
medical knowledge on the safety and efficacy of dissolving microneedle in delivering local 
anaesthetic agents, which will benefit patients in the future. 
 
Do you have to take part? 
Your participation in this study is absolute voluntarily. Your medical care will not be affected 
if you decide not to participate in this study. You will still have the usual standard of care 
according to the day-care protocol. 
 
If you agree to participate, you will be asked to sign the “Informed Consent Form”. You will 
be given a copy of the informed consent form and this patient information sheet. Should you 
decide to participate, you cannot decide which group you will be assigned to, but you are still 
free to withdraw from the study at any time without giving a reason or penalty. If you decide 
to cease from participating in this study, you must inform your study investigator and no new 
data will be further collected from you. The researcher may also remove your participation 
from the study for various reasons. In this event, you will not lose your rights as a patient and 
will still receive the usual standard of care. 
 
Data & Confidentiality: 
Participant’s confidentiality will be maintained throughout the investigation. Your personal 
data well be anonymized as your identity be always kept confidential. Data collected and 
entered into the Case Report Form will remain as the governed property of UKM. In the event 
of any publication generated from this study, your identity will be remained confidential to 
the public. 
 
By signing the Informed Consent Form attached, you (or your legally acceptable 
representative, if relevant) are authorizing such access to your study records. 
 
Payment and compensation: 
You do not have to pay, nor will you be paid to participate in this study. You do have to pay 
for the usual hospital charges. 

40 
 
 
Whom can I ask about the study? 
If you have any questions about this study or your rights, please contact: 
 
Principal Investigator: 
Prof. Dr Cheah Fook Choe 
Department of Paediatrics 
UKM Medical Centre 
Phone Number : 03-9145 5391 
 
Co-investigator 
Prof Dr Mae-Lyn Catherine Bastion 
Department of Ophthalmology 
UKM Medical Centre 
Phone Number: 03-9145 5983 
 
Dr Lam Chenshen 
Department of Ophthalmology 
UKM Medical Centre 
No. Telefon: 03-8921 6520 
 
Signatures 
To be selected into this study, you must sign and date the signature page [ATTACHMENT A] 

41 
 
ATTACHMENT A 
 
 
Patient/Subject Information and Consent Form 
(Signature Page) 
 
 
Research Title: Transdermal microneedle lignocaine delivery versus EMLA patch for topical analgesia before 
venepuncture procedure to adults in a clinic setting 
 
 
Researcher's Name: Prof. Dr Cheah Fook Choe, Prof. Dr Mae-Lyn Catherine Bastion, Dr Lam Chen Shen 
 
To become a part of this study, you must sign this page. By signing this page, I amconfirming the following: 
• 
I have read and understand all the information in this Patient Information Sheet and Consent Form, including 
any information regarding the risk stated in this study and I, have given sufficient time to consider about this 
study. 
• 
All of my questions have been answered to my satisfaction. 
• 
Hereby, I voluntarily agree to be part of this research study, to follow the study procedures, and to provide 
necessary information to the doctor, nurses, or other staff members, as requested. 
• 
I may freely choose to stop being a part of this study at any time. 
• 
I have received a copy of this Patient Information Sheet and Consent Form to keep for myself. 
 
 
 
 
Patient Name (Print or type) 
Patient Initials and Number 
 
 
 
Patient I.C No. (New) 
 
 
 
Signature of Patient 
Date (dd/MM/yy) 
(Add time if applicable) 
 
 
 
Name of Individual 
Conducting Consent Discussion (Print or Type) 
 
 
 
 
 
Signature of Individual 
Date (dd/MM/yy) 
Conducting Consent Discussion 
 
 
 
 
 
Name & Signature of Witness 
Date (dd/MM/yy) 
 
Note: 
i) 
All subject/patients who are involved in this study will not be covered by insurance 

41 
 
APPENDIX B: CASE REPORT FORM (CRF) 
 
 
CASE REPORT FORM 
 
Research title: Transdermal microneedle lignocaine delivery versus EMLA patch for topical analgesia before  
  venepuncture procedure to adults in a clinic setting 
 
Research Investigators: Professors Dr Cheah Fook Choe, Mae-Lynn Catherine Bastion, Dr Lam CS. 
 
 
 
Randomization code: 
 
 
A. SUBJECT’S DETAIL 
 
Subject’s name: 
 
 
 
 
Age 
: 
 
years    ________ months 
 
Registration No 
(RN)  : 
 
 
Telephone no : 
Handphone: 
 
 
 
. 
 
 
 
 
 
 
 
 
 
Home: 
 
 
 
 
 
 
 
 
 
 
Date of birth: 
 
/  
/  
 
 
 
 
 
 
 
 
 
 
 
 
Date of consent: 
 
/  
/  
 
 
 
 
 
 
 
 
Gender : 
 
Male 
 
Female 
 
 
 
 
 
 
 
Ethnicity: 
 
 
Malay 
 
Chinese 
 
 
Indian 
 
 
 
 
 
 
 
 
 
Others (please specify): 
 
 
 
 
 
Hospital Sticker 
Subject ID 
 
 
 
 
 
 
Date: 
 
 
/  
 
/  
 
 
 
 
Day 
 
Month 
 
Year 
 

41 
 
B. FAMILY HISTORY OF ILLNESS 
 
Has any of the family members having any significant disease in their history of illness. 
 
 
First degree  
Yes 
No 
 
 
 
 
 
 
 
 
Father 
 
 
 
 
 
 
 
 
 
Mother 
 
 
 
 
 
 
 
 
 
Siblings 
 
 
 
 
 
 
 
 
 
 
 
Others: 
 
 
 
 
 
 
 
If Yes, please specify the types of diseases and age at diagnosis: 
 
 
 
 
 

41 
 
C. REVIEW OF MEDICAL HISTORY / MEDICATIONS CHECKLIST 
 
 
Relevant medical history 
 
Diabetes  
 
: 
 
 
 
Hypertension 
 
: 
 
 
 
Kidney disease 
 
: 
 
 
 
Autoimmune disease 
: 
 
 
 
 Hepatic diseases  
: 
 
 
Others 
 
 
: 
 
 
 
Medications (please specify the name, indications, dosage and administration frequency) 
 
Name 
Reasons 
Doses 
Frequency of 
administration 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medication checklist 
Erythromycin 
 
Ciprofloxacin 
 
Amiodarone 
 
Beta-adrenergic blockers 
(timolol eyedrops, metoprolol etc) 
 
 
 
Has subject ever experienced any allergy or adverse event from any medications? 
 
 
Yes *  
 
 
No 
 
 
 
 
 
 
 
 
*If yes, please specify the type of medication: 
 
 
 
 

41 
 
 
 
 
Please specify the type of ocular diagnosis and the planned operation  
 
Diagnosis: 
 
Date: 
 
  
 
 
Surgery: 
 
Phacoemulsification: 
 
Left eye 
         Right eye 
 
 
 
 
 
 
 
ECCE: 
 
 
 
Trabeculectomy: 
 
 
 
Glaucoma drainage device: 
 
 
 
Pars plana vitrectomy: 
 
 
 
Corneal transplant: 
 
 
 
Others: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

41 
 
D. CLINICAL DATA 
 
 
Anthropometric measurement 
 
1. Weight : 
kg 
 
 
 
2. Height  
: 
cm 
 
 
3. Body mass index (BMI) 
: 
kg/m2 
 
 
 
Vital signs 
                To be applied if needed 
Unit 
Clinic visit 
    PK Study 
 
 
Date: 
     __ / __ / __ 
Date: 
__ / __ / __ 
Blood Sample (Y/N) 
Volume (mL) 
Systolic pressure 
mmHg 
 Time: 0 min 
Diastolic pressure 
mmHg 
 Time: 30 mins 
Heart rate 
bpm 
 Time: 60 mins 
Dextrostix 
mmol/L 
 Time: 90 mins 
Pain scale (VAS) 
 Time: 120 mins 
Pain score 
(Pain Monitor) 
 
 
 Time: 180 mins 
Samples sent to the 
Malaysian 
Department of 
Chemistry (Y/N) 
 Date: 
   
 Time: 
Initials and the name of assessor 
 
 

41 
 
ADVERSE EFFECTS TRACKING LOG 
 
No. 
Date 
reported 
Adverse event 
description 
Start 
date 
End 
date 
Ongoing 
(Yes or No) 
Outcome1 
Severity 
/ grade2 
Serious 
(Yes or No) 
AE 
treatment3 
Expected 
(Yes or No) 
Intervention 
Attribution / 
Relatedness4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scales: 
Outcome1 
Severity / grade2 
AE treatment3 
Intervention Attribution / 
Relatedness4 
0- Fatal 
1- Not recovered / Not resolved 
2- Recovered w/sequelae 
3- Recovered w/o sequelae 
4- Recovering / Resolving 
0- Mild 
1- Moderate 
2- Severe 
3- Life-threatening 
4- Death / Fatal 
0- None 
1- Medication(s) 
2- Medication TX 
0- Definite 
1- Probable 
2- Possible 
3- Unrelated 
4- Not applicable 
(did not receive intervention) 
 
Verified by (Prior to data entry): 
 
Signature: 
 
Name: 
Date: