Gayle A. Brazeau, Ph.D.

Pain Upon Injection: Fundamentals of

Subcutaneous Anatomy and Physiology for

Pharmaceutical Scientists

Tuesday May 17, 2016 7:00 am

Three Key Considerations:

• Anatomy/Physiology Subcutaneous Tissues and

Adjacent Tissues

• Mechanisms Pain and Pain Pathways

• Optimizing Vehicles and Excipients for Parenteral

Formulations for Subcutaneous Injections.

Optimizing Injection Route

J.F Jin, The optimal choice of medication administration route regarding intravenous, intramuscular,

and subcutaneous injection, Patient Preference and Adherence 5:923-942, 2015

Subcutaneous Tissue

https://en.wikipedia.org/wiki/Subcutaneous_tissue#/me

dia/File:Skin.png

• Space beneath the

epidermis/dermis and

skeletal muscle

• Also called hypodermis

• Lowermost layer of the

integumentary system

• Composed of fibroblasts,

adipose cells and

microphages

Injection Site Reactions

Key Questions

• What is the physiology associated

with pain upon injection?

• What are the mechanisms

associated with tissue damage?

• What is the role of inflammation and

swelling/edema?

Pain and Injections

http://classroomclipart.com/clipart-view/Clipart/Medical/nurse-giving-patient-injection_jpg.htm

Oh No,

Not Again!

Nociceptors – General Overview

• Sensory Receptors – free (bare) nerve endings

that detect signals from damaged tissue and

indirectly respond to chemicals released from

damaged tissue and or inflammation

• C-Fiber Classes and A Fiber Classes

• Categories

– Mechanical

– Thermal

– Chemical stimulation

http://neuroscience.uth.tmc.edu/s2/chapter06.htm

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2964977/

Nociceptor - Types

• Mechanonociceptors – respond to pinching,

cutting or stretching

• Thermal nociceptors – respond to the above

stimuli and thermal stimuli

• Chemical nociceptors - respond only to

chemical substances

• Polymodal nociceptors - respond to high

intensity stimuli such as mechanical, thermal

and to chemical substances

http://neuroscience.uth.tmc.edu/s2/chapter06.htm

Tissue Damage and Injections

Factors Affecting Tissue Damage

• Lipophilicity vehicle/drug

• Concentration

• Osmolarity

• pH (SC Avoid below 3 and above 9)

• Particle Size

• Solubility and Precipitation

• Biochemical Mechanisms

* Wu et al., JPP 62: 873, 2010

Biochemical Mechanisms:

Tissue Site Reactions

• Disruption sarcolemma membrane

• Alteration in cellular homeostasis

–Calcium release and transport

–Cellular energetics

• Free Radical Damage

Tissue Damage and Nociceptors

Tissue

DamageNociceptor

Activation

Factors

Inflammation

Swelling/Edema

Tissue Damage - Factors Activating Nociceptors• Globulin and protein kinases

• Arachidonic acid

• Histamine

• Nerve growth factor (NGF)

• Substance P (SP) and calcitonin gene-related peptide

(CGRP)

• Potassium - K+

• Serotonin (5-HT)

• Acetylcholine (ACh)

• Low pH (acidic) solution

• ATP

• Lactic acidhttp://neuroscience.uth.tmc.edu/s2/chapter06.htm

Formulation Considerations: Tissue

Damage and Pain

W. Wang, Tolerability of hypertonic injectables, International Journal of

Pharmaceutics 490 (2015) 308–315

Important Considerations• Avoid drugs or formulations which are irritating

at the injection site

– Essential to initially screen for irritation

• Cell culture, isolated tissues (muscle, skin)

• Avoid drugs which may case vasoconstriction

• Avoid viscous suspensions

All can lead to induration, sloughing, abscess

formation or even necrosis

General Considerations SQ

• Sites

– Upper Arm

– Anterior Thigh Surface

– Lower Portion of Abdomen

– Upper Back

• Injection Volume 2-5 ml

• Needles 3/8 – 1 inch, 24-27 Gauge

Drug Release Factors

• Function of drug solubility

• Type of formulation – solution

versus suspension

• Formulation viscosity

• Particle size

• Changes in blood flow (minor)

Formulation Factors - Tolerability

• Specific Agent/Concentration

• Formulation Excipients and pH

• Formulation Tonicity

• Formulation Osmolarity

• Injection Speed

• Injection Volume

• Injection Site

• Particle Size

• Injection Frequency

Molecule Dependent: Oligonucleotides

Figure 2: All 21 sc administered ONs resulted in

ISRs. Incidence ranged from 22 to 100%. For 4 ONs

no incidence numbers were reported, namely

IsisApo(a)Rx, Isis113715, ATL-03 and IsisGCGR-Rx.

For ONs that were studies at different dose levels

and/or multiple trials, an average ISR occurrence

was calculated.

Figure 3: Dose-dependent occurrence of ISRs

after administration of four different ONs. Higher

dose levels result in increased incidence of ISRs

up to 100 % at the highest dose level. The dose

levels tested for ISIS32566 and Mipomersen [10]

were placebo (0), 50, 100, 200 and 400 mg. For

IMO-8400 dose levels were: placebo (o), 0.075,

0.15, 0.3 and 0.6 mg/kg, and for ON_CHDR2 dose

levels were placebo (0), 0.5, 1.5 and 5 mg/kg.

L. van Meer et al. Injection Site Reactions after subcutaneous oligonucleotide

therapy, doi: 10.1111/bcp.12961

pH Effects

Local Tolerance of Subcutaneous Injections- Fransson & Espander Jansson, J. Pharm. Pharmacol., 1996

• Non-physiological

pH, buffer strength

should be kept as low

as possible to avoid

pain on injection.

• Pain due to transport

of K+ causing

depolarization of

nerve endings.

pH P NaCl VAS

(mM) (mM)

6.0-A 50 112 30.9

6.0-B 10 145 9.7

7.0-C 50 112 6.5

7.0-D 10 145 16.3

6.0-E 5 150 15.1

6.0-F* 5 150 38.4

6.0-G* 10 145 12.2

8.0-R 0 150 7.2

* - with 5 mg/ml HIGF-1

Excipients: Buffers• Goal Physiological pH (better to err on the more

basic versus acidic)

• Most common:

– Citrate (5-15 mM), Higher concentration 50 mM

can case pain (chelation of calcium)

– Acetates – Lower pH (concern with

lyophilization)

– Phosphates – need to be cautious complexation

• Alternatives: lactate and tartrate

Y. Mehmood, U. Farooq, Excipients Use in Parenteral and Lyophilized Formulation Development, Open

Science Journal of Pharmacy and Pharmacology 3(3): 19-27, 2015

An In Vitro Muscle Model

An isolated rodent extensor digitorum longus or soleus muscle

Carbogenated 37oC BSS

Creatine kinase activity is

measured with

spectrophotometric kinetic

assay at 340 nm

15 ml Test

Solution

Injected into

the muscle

or muscle

incubated in

the presence

of the test

compound

pH and Buffers*

• Carboxylic Buffers

– Acetate

– Succinate

– Citrate

• Type of Buffer

• pH Effect

– pH 2, 4, 6

• Buffer Capacity

– 0.1, 0.01, 0.001

*Napaporn et al., PDT, 2000

1. Buffer solutions with low concentrations should be

made isotonic.

2. For acetate buffer, formulate a solution at low buffer

capacity and near physiological pH. Although the

myotoxicity of acetate buffers is not significantly

different from normal saline, a trend towards

increased myotoxicity was evident at higher buffer

capacity and lower pH.

3. For succinate and citrate buffers, formulate near

physiological muscle pH as the myotoxicity is

minimized in this range.

General Formulation Considerations

Tonicity/OsmolarityConsequences: Enhanced tissue site irritation and

pain, tissue permeability and possible damage

• For intradermal, subcutaneous, and intramuscular

injections

– Ideally – around 300 30 mOsm/kg

– Preferably < 600 mOsm/kg

• Mitigation

– Avoid extreme pH and high buffer concentration

– Reduce injection volume

– Use anesthetic agentW. Wang, Tolerability of hypertonic injectables, International Journal of Pharmaceutics 490

(2015) 308–315

Injection Speed/Volume• 82 Individuals Type 1 or 2 Diabetes, 39% Females

• SC injections in the abdomen or thigh of 0.9% sodium chloride

• Injection speeds: 150, 300 and 450 µl/s

• Volumes: 400, 800, 1200 and 1600 µl

• Pain; Visual Analogue scale (VAS)

• Results:

– Lower end of the VAS scale

– Speed of injection was not significant

– Larger injection volumes (1200 and 1600 µl) were associated

with greater pain

– More pain associated with injections into thigh versus

abdomenHeise et al., Impact of injection speed and volume on perceived pain during subcutaneous injections

into the abdomen and thigh: a single-centre, randomized controlled trial, Diabetes, Obesity and

Metabolism 16: 971–976, 2014

Viscosity, Injection Volume and Flow

C. Berteau et al., Evaluation of the impact of viscosity, injection volume, and injection flow rate on subcutaneous

injection tolerance, Med Devices (Auckl) 8: 473–484, 2015.

•Normal Saline with nonanimal

hyaluronic acid injections into the

abdomen

•24 subjects, 122 injections

•6 injections per individual

•22 technical incidents in 12

subjects

• Equal number of males/females

Fluid Depot Location

Injected fluid location after injection (N=144).

Notes: (A) Example of typical 2D-ultrasound B-mode echography of injection site in the

abdomen, before (left) and after (right) injection in the same subject. The white arrow measures

the injection depth. (B) Fluid location evaluated by echography images, percentage of fluid

located exclusively in SC tissue. Significant difference (*P=0.0213) between 2 and 3 mL

injections: fluid located exclusively in SC tissue more frequently with 3 mL injections.

C. Berteau et al., Evaluation of the impact of viscosity, injection volume, and injection flow

rate on subcutaneous injection tolerance, Med Devices (Auckl) 8: 473–484, 2015.

Particle Size – Tissue DamageDrug

Suspension Concentration

(%w/v)

Suspension Size

Classification pka Solubility Log P or cLogP

Bupivacaine5%

SmallMediumLarge

Local Anesthetic 8.05 Free Base Solubility:0.7 mg/mlat pH 7.4HCl Salt:40 mg/ml

3.41

Phenytoin10%

0.2 m1.2 m4 m6 m

Anticonvulsant 8.3 0.032 mg/mL 2.52

Diazepam10%

0.4 m1.7 m2.1 m

Anticonvulsant, sedative, and

muscle relaxant

3.4 Free Base: 0.06 mg/mL13

3.86

0

500

1000

1500

2000

Norm

al S

alin

e

Bupiv

acai

ne (s

mal

l)

Bupiv

acai

ne (m

ediu

m)

Bupiv

acai

ne ( l

arge)

Dia

zepam

(0.4

µm

)

Dia

zepam

(1.7

µm

)

Dia

zepam

(2.1

µm

)

Phenyt

oin (0

.2 µ

m)

Phenyt

oin (1

.2 µ

m)

Phenyt

oin (4

.0 µ

m)

Phenyt

oin (6

.0 µ

m)

Cu

mu

lati

ve C

K

(Mean

+ S

EM

)

* *

*

**

**

*

Data was log transformed for statistical analysis. All treatments are significantly different from Normal Saline.

(A) * p<0.05 versus Bupivacaine Small and Bupivacaine Medium.

Selective Suspensions

Unmodified Polystyrene Beads

0

200

400

600

800

1000

1200

1400

1600

1800

Normal Saline 83nm 520nm 1.247 µm 2.642 µm

Particle Size

Cu

mu

lati

ve C

K (

Mean

+ S

EM

) U

/L

* *

#

*#

#

**

Data was log transformed. *p<0.05 versus Normal Saline. #p<0.05versus Unmodified 83 nm particle size with n = 6-7.

Carboxyl Modified Polystyrene Beads

0

300

600

900

1200

1500

1800

2100

Normal Saline 104nm 424nm 1.036 µm 3.06 µm

Particle Size

Cu

mu

lati

ve C

K (

Mean

+ S

EM

) U

/L *

*

#

#

#

#

*

*

*

Data was log transformed. *p<0.05 versus Normal Saline. #p<0.05versus Unmodified 83 nm particle size with n = 6-7.

Minimizing Injection Pain SQ Injections

• Medication should be at room

temperature

• Warm the injection solution

• Let alcohol dry before injection

• Optimize injection depth

• Injection sites should be rotated

• Message the site after injection

Conclusions – SQ Injection Formulations

• Complex site with potential for pain and/or tissue

damage

• Ideally goal is to minimize the concentration of API,

excipients, injection volume, pH, tonicity and

viscosity needed for formulation stability and release

• Look at the pharmacological properties of the API or

excipients which could cause pain or tissue damage

• Screen early in the development process for potential

pain or tissue damage.

Questions

Thank You!

Gayle Brazeau

gbrazeau@une.edu

University of New England

College of Pharmacy