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Physical and Chemical Properties Various authors have reported the water retention OH OH properties of hyaluronic acid. Hyaluronic acid has a greater capacity to hold water than any other natural or synthetic polymer. One gram of hyaluronic acid can hold up 8to 6 L of water. 2C0 9 - C=0 / 3CH Polymer Network n The structural factors underlying hyaluronic acid’s unique properties are its high molecular weight and Figure 1. Structure of hyaluronic acid. 4The repeating units, identical to the structure in brackets. large molecular volume.’ The large molecular vol ume forces the overlap0 of individual hyaluronic acid molecular domains, resulting in extensive chain entanglement and chain—chain103interaction. is hyaluronic acid, the rest being water which is Concentration (expressed in g/cc) and intrinsic mechanically immobilized within the coil. The rate viscosity (limiting viscosity number, expressed in of diffusion through the network is inversely related cc/g), which are measures of molecular volume, are to the size of the polysaccharide molecules. The related to the ability of hyaluronic acid to form vis structure of the disaccharide is energetically verysta coelastic polymeric networks. The product of con ble. The domain structure of hyaluronic acid allows centration and intrinsic viscosity has been called small molecules such as water, electrolytes, and the coil overlap parameter, which expresses the nutrients to freely diffuse through the solvent within degree of network formation) Concentration and the domain while large molecules such as proteins molecular volume (size)4 are totally interdependent will be partially excluded from the domain because in determining the physical properties of a hyaluro of their hydrodynamic sizes in solution. nic acid 13solution. They stated that the smaller the In the presence of K±, 4+,NH Rb±, and Cs+, an size of the individual hyaluronic acid molecules, the antiparallel double-helical structure forms with higher the concentration necessary for a viscoelastic hydrophobic and hydrophilic pockets between adja network to form. cent double helices. Under physiological conditions The carboxyl groups fully dissociate at physiolo (pH 7), this 5water soluble molecule exists in its salt gical the structure is sensitive to ionic 5pH’ strength form as sodium hyaluronate. The structure of hya and ;pH. The molecule expands at lower ionic luronic acid is shown in Figure i. strength due to repulsions between the charges. This 6 16,17 According to the International Cosmetic Ingredi increases viscosity. ent Dictionary and Handbook, hyaluronic acid is also The chains, when entangling, also interact with known as hyaluronan. Trade names for hyaluronic each other and form stretches of double helices so acid and trade7 name mixtures containing hyaluronic that the network becomes mechanically more acid are listed in Table 1. firm) Each glucuronate unit carries an anionic Sodium hyaluronate (CAS no. 9067-32-7), also ”89charge at physiological pH associated with its carbox known as hyaluronic acid sodium and hyaluronate ylate group. There are often hundreds of negative sodium, is the sodium salt of hyaluronic 7acid. charges fixed to each chain. These charges are Other technical names are: hyaluronic acid, sodium balanced by mobile cations such as Na±, K±, Ca, salt; sodium hyaluronate (1); sodium hyaluronate and Mg++. The charges are important in determin (2); and sodium hyaluronate (3); and sodium hya ing solubility in water because hyaluronic acid, luronate solution. Its trade names and trade name converted into an uncharged polymer by fully esteri mixtures are listed in Table 1. Potassium hyaluro fying with methyl groups, is insoluble. The molecule nate (CAS no. 31799-91-4) is the potassium salt has the properties of a highly hydrophilic (polyhy of hyaluronic acid, also known as hyaluronic acid, droxylic) material simultaneously with hydrophobic potassium 7salt. domains characteristic of lipids. Hyaluronic Acid, Potassium Hyaluronate, and Sodium Hyaluronate / Becker et al 7

Table 1. Trade Names for Hyaluronic Acid and Sodium Hyaluronate and Trade Name Mixtures Containing These Ingredients Trade Names 7of Hyaluronic Acid AEC Hyaluronic Acid Biomatrix Hyaluronic Acid SOL Trade Name Mixtures of Hyaluronic Acid Amisil-HA BioCare BHA-i0 BioCare HA-24 Bio BioCare Polymer BHA-i0 BioCare Polymer HA-24 BioCare SA Biosil Basics HMV - Hair Moisture Complex Biosil Basics HMW - Hair Moisture Complex Cromoist HYA Cromoist WHYA Glycoderm P Lipocare HAJEC Molecularsource LPC PHYTO/CER.HA Trade Names of Sodium Hyaluronate Actimoist AEC Sodium Hyaluronate Avian Sodium Hyaluronate Powder Avian Sodium Hyaluronate Solution Bio-HE Dekiuron HTL MYP Hyaluronic Acid Hyaluronate Na F93 Hyaluronate Na Fi00 Hyaluronate Na 1.0% Gel Hyaluronate Na P85 Hyaluronate Na P90 Hyaluronate Na P93 Hyaluronate Na P100 Hyaluronic Acid FCH- 150 Hyaluronic Acid FCH-200 Hyaluronic Acid (Na) Hyaluronic Acid, Sodium Salt Hyaluronsan HA-L5 10 Hyaluronsan HA-M5070 Hyaluronsan HA-Q Hyaluronsan HA-QSS Hyaluronsan Solution HA-Q 1P Hyasol Hyasol-BT LMW Hyaluronic Acid Na Salt Nikkol Sodium Hyaluronate OriStart SH RITA HA C-i-C RITA HA C-i-P Saccaluronate CW Sodium Hyalronate HA-Q Restylane Hylaform Trade Name Mixtures of Sodium Hyaluronate Actiglide Advanced Moisture Complex Aragoline Atecoron Belisilk HA Biocrystal Brookosome H Chronosphere FHC/HA Blend Chronosphere Hyaluronic Collagen-Hyaluronic Collagen-Hyaluronic Acid-Jelly Desaron EASHAVE Essential Vital Elements - S Gelhyperm (Avocado Oil) Geihyperm (Jojoba Oil) Gelhyperm (Macadamia Nut Oil) Geihyperm (Seabukthorn Oil) Gelhyperm (Wheat Germ Oil) HA-Sol 2% Hyaluronic Acid 1% Hydraiphatine 3P Hydroxan Hydroxan BG Hydroxan CH Iricalmin Polyson HQ Quiditat NwH Ritacomplex DF 15 Ritacomplex DF 26 Rovisome H A Saccaluronate CC Saccaluronate LC Spherica HA Thioglycans Toshiki BINS-3

Physical Form weight on drying and normally gives not more than Hyaluronic acid is available to the cosmetic formula 20% of residue on incineration. A 0.2% water solution tor as a highly purified, freeze-dried powder or as an of sodium hyaluronate can have a pH range of 5.5 to aqueous solution, and as its potassium or sodium 7.5. During the purification of sodium hyaluronate, 2salt. In this form, hyaluronic acid slowly but fully which involves the removal of lipids, proteins, and °dissolves in water to give viscous, clear to slightly nucleic acids, its molecular weight quickly drops. opalescent and colorless solutions, which must be Sodium hyaluronate dissolves slowly but completely preserved in cosmetic usage. The viscosity can vary in water to give a clear to faintly opalescent colorless with the method of its preparation, decreasing and highly viscous solution. The salt is soluble in sharply in the presence of electrolytes. Sodium hya sodium chloride solution but almost insoluble in luronate is supplied as a white fiber-like or creamy organic solvents.Up to 90% ofthe salt can be insoluble white powder with a very faint odor. According to in ethanol. This author also stated that aqueous solu this author, it usually contains not less than 98% of tions of sodium hyaluronate must be preserved, using the salt, although there are products with lower lev 0.4% to 0.75% phenoxyethanol, for example, as the els. It does not usually lose more than 10% of its material is a rich source of nutrients for microbes.

Cus dysgalactiae,

micus

ism

carbon

that found

industrially type

and

combs, lastic

Hyaluronic used but

by

ties

rooster

sources. 24

concentration cifically is ging. The vitreous

ampules, hyaluronic

acid. 8 by purity

completely

1800 European of

and

tracheas present

sodium umbilical

Hyaluronic from

are

Hyaluronic

8

then

bacterial

these

ultraviolet

International

pyogenes.

From

have

a

In

Two

Billek

derived

Method

produces

produce

II

C

are

proteins

higher

properties

kD. 25

are

The

human

and

A

cosmetics,

in

are

stain combs,

adjusted

and

hemolytic

for

a

in

of bodies

with

hyaluronate

patent

molecules

easily

and

the

and

more

Bacteria

from

and

countries

Streptococcus

cord

Hylaform

remaining

the

difficult

fermentation,

the

acid

These

acid

produced

concentration

from acid

dermal

acid

nitrogen

Staphylococcus “protein-free”

D39R. 2632

31

capsules

hyaluronic bovine

radiation

of

sterilized.

umbilical

(6

hyaluronic

form

Billek

has

hyaluronic

molecular

Journal

33-37 elastic

perivascular

of

issued

degraded

have

bacterial

to and

that

and

mg/mL),

of

Manufacture/Sources

also

the

rooster

pig

a

2

a

Streptococci

are

high

augmentation

low

is

to

physiological

an

processes

described

vitreous. 22

a

is

is

sodium

oniy

of

tendency.

eyes

can

sources. 9 ’ 22 ’ 31

has

clear,

for

of

from

more

in

of

prepare

easily

produced

known

and

insoluble

molecular

equi

Toxicology

cords. 2 ’

molecular high

Bacterial

has

acid

combs

Germany

acid

the

bacteria

by

acid fermentation

and

(20

sources

weights

be been

by

viscous

oxidative

solution

connective

aureus,

Streptococcus

a

highly

hyaluronate

process

free

where

lowering

broken

separated

mg!mL),

lower gels

molecular

include

its

a

are

and

as

Restylane

because

derived

Hyaluronic

process

but

and

viscoelastic

Hyaluronic

by

pH

/

complex

manufactured

of radicals

hyaluronic are

Wharton’s

described

weight

such

and

Vol.

weight

between

molecular

tendency.

lactic

viscous

the

and

can

hyaluronic

extraction

Other

of

by

Pneumococcus

of

agents.” 3 ’ 23

of

from

the

Streptococcus

and

28,

several

purifying

microorgan

tissue

7.0,

to from

by

and

as

shear

be

long

Streptococ

weight

is

but

historically

hyaluronic

acid pH

produce

zooepide its

No.

produced

produced

has

Group

with

centrifu

prepared

different

acid

bacteria

solution

put

below.

proper

rooster

a

viscoe

jelly.’ 3

acid

5

weight

acid

bovine

chains

to

forces

of

4S,

lower

other

from been

from

acid

into

spe

4.2.

and

and

and

the

the

the

July/August

is

A

is

a

household with

Ridgefield, harmless tion

ethyl linking

molecules lene—bridge-protein—methylene g

U to called acid

number hyaluronic

of duces

polysaccharide

the

N-acetyl 2 product

hyaluronate

Hyaluronic

in

acid). hyaluronate

was

(LMW) was

Balazs

methanol.

(50% zation.

the added

terminated,

maximum lected

S

Sakaguchi

specific

of

(both

0.63

zooecli4emicus

form

cosmetics. 8 ’ 38

Hylan

Hyaluronic

polysaccharide

Crosslinking

From

To

mixture

is

disaccharide. 40 then

the 2009

are

precipitated

crosslinks.

to

a By

hylan

from

stabilized

to

cc/g

process

was

produce

After

sodium

less

of

hydroxyl in

as

an

permanently

carbon 400%

groups. 39

the

heating

filtered

groups

B

Cockscombs

form

After

glues,

flask

acid,

NJ)

the

0.15

compared high

cell

a

acid

was

and

gel,

infinite

dense

fluid.

extracted

issued

and

surface

growth

activated

polysaccharide

for

acid is

relative

containing

utilizes

resulting

hyaluronate.

an

benzyl

thoroughly

dried

with

sold

chains

hyaluronic

titer,

chains molecular

of

N

groups

which

the

was

until

the

broken

by

This

The

in

tissue

structure

elastoviscous

the

This

NaCI).

It

a

used

network

swab

media

as

such

rest extract

carbon

with

the

is

isolated

to

associated

from

patent

transparent.

the

molecule, alcohol

other

in

vinyl

to

product

Hylaform

an

hydrolyzes

are

of carbon

a

in

augmentation

crosslinking

down,

for

hours.

as

addition

of

hyaluronic white

a

the

culture

the

epoxy,

a

Because

weight

removing

cockscombs

and

culturing

acid,

crosslinked

way

than

guinea

to

bridges

partial

hyaluronic

low

sulfone,

soft

through

in

polysaccharide

chains

and

extracted was

bond,

100°C,

agitated,

and

leaving

powder.

as

contained

solution,

through

the

also 1981

molecular

that

bacteria

tissue

medium. 3 ’

(Biomatrix,

added

(HMW)

irreversibly to

transferred

Hyaluronic

of

pig

specific

only

every

carboxylic

alumina

acid’s

the

of

not

used

which

the

process

of

process

sulfonyl-bis

sodium

conjunctiva.

is

some

for

part

to

acid

to

hyaluronic

augmenta

hyaluronic

the

methanol,

for

the

affect

stabilized

a

a

hydrated

which

2

was

in stabilize

0.57

be

volume

sodium

sodium

protein

limited

methy

to

weight

sterili

of

native

reacts

of

cross-

At

chain liquid

many

Inc.,

were

used

pro

into

acid

to

500

and

and

was

col

the

the

the

the

cc!

is a Hyaluronic Acid, Potassium Hyaluronate, and Sodium Hyaluronate / Beckeret al 9

polysaccharides, and the resulting unsaturated Hyaluronic Acid Hyaluronic Acid polysaccharide polysaccharide disaccharides can be analyzed within 1 rig. Hyaluronic acid can be detected and measured to within 1 ng in biological samples by specific radioas

¶? say making use of proteins with affinity for hyaluro —CH—O—CHj—CH —CH—CH 2O—CH — nic acid that are present in 44cartilage. Proteins — 0 2 with affinity for hyaluronic acid also can be used for 2 detection of the polymer using techniques similar to immunohistochemical methods .‘ An absolute, weight average determination of Figure 2. Chemical structure of the crosslink in insoluble molecular weight is obtained by using light scatter Hylan B 4gel. ’ 46ing. Milas et al suggested using this method to determine the range of molecular weights in a hyaluronic acid. The biomaterial retains the bio 4 sample in combination with steric exclusion chroma compatibility’ and biological properties of hyaluronic 22 tography and refractometric detectors acid, but its residence time in dermal tissue is increased by introduction of sulfonyl-bis-ethyl cross- links between the polysaccharide chains as shown in Figure 2. Impurities Restylane (Q-Med Scandinavia mc, Uppsala, When hyaluronic acid is derived from animal sources, Sweden) is produced in cultures of Equine strepto proteins may be present, which can affect the nonim cocci by fermentation in the presence of 24sugar. The munogenic and immunogenic properties of the hya fermentation material is then alcohol precipitated, luronic acid preparation. To achieve high purity, a filtered, and dried. The hyaluronic acid chains are certain degree of depolyrnerization occurs, resulting then chemically stabilized through permanent epoxi 6 in a lower grade product. For example,bovinehyaluro dic crosslinks that the manufacturer reports to alter nic acid (prepared by Etapharam, Vienna) contained only 1% of the hyaluronan molecular network. The approximately 10% of its total macromolecular con material heat is sterilized in its final container. tent in 47protein. et al compared Manna hyaluronic acid deriva Proteins that bind to hyaluronic acid include tives from rooster combs (Hylaform, to subjected hyaluronidases, antibodies, proteoglycans, and link crosslinking) and streptococcus (Restylane, stabi proteins, cell-surface receptors for hyaluronic acid, lized as declared by the were manufacturer), which and hyaluronic acid—bindingproteins isolated from developed for soft tissue augmentation. The tissues and fluids. researchers found that Hylaform42 functions as a 2 Balazs was granted a patent for “ultrapure” strong hydrogel, has a minor quantity of crosslinked sodium hyaluronic acid. The essential step that was hyaluronic acid (about one fourth that of Restylane), 8 different from other processes was to treat the and contains a low amount of protein (about one hyaluronic acid solution, which had already had the fourth of Restylane). functions as a that Restylane proteins largely removed, with chloroform for up to weak hydrogel and contains protein resulting from 5 days. The purpose is to eliminate “a relatively bacterial fermentation or added to enable crosslink undefined fraction (‘inflammatory [H]yaluronic ing reaction. Hyaluronic acid and sodium hyaluro [A]cid’)that is supposed to cause inflammation after nate may be crosslinked. 43 injection into the eye.” The purity of hyaluronic acid may vary from one Analytical Methods commercial batch to 48another. The batch of human umbilical cord hyaluronic acid used in this study had Originally it was necessary to purify hyaluronic acid <2% protein and <3% chondroitin sulfate. Using from tissue in order to measure the amount of hexo chondroitinase and protease digestion followed by samine or hexuronic acid, which results in an accu thermic denaturation and dialysis against dissociat racy within 100 2Ig. Hyaluronate lyase from ing buffer to get rid of smaller fragments obtains an Streptomyces hyalurolyticus was also employed to electrophoretically homogeneous hyaluronic acid degrade hyaluronic acid in the presence of other preparation.

Administration

category

tions Dictionary

of As

tary

Hyaluronic increasing

as

Use from

ture

rooster impure

H5N1 hyaluronic

contains

rooster

ducibility,

acid purify per

Hyaluronic sources. 31 weights

tested impurities

of gical

facturers

that did presence

the

led

stated hyaluronate; mation but

formulation. 49

cous

10

use

DNA

a

given

Cosmetic

Based

Micheels

Three

Hyaluronic

to

Clinically

visoelastic

meet

International

may

milligram

this

industry

from

reached

skin-conditioning

cockscombs

solutions

up sources.

and

that

(source

a

as

virus

combs

comb

after

based

ranging

in

recall

are

only episode

to

of

be

in

a

use

a

may

standards

and

on

agent In

in

high

of

the

the

acid

acid

polymer

acid

bacterial

endotoxins

reported

is

related

cataract

the

long-chain

contrast

Alcon

and

low

concentration

in

concluded

on

reports

the

evident

not

can the

Handbook,

of

of

inflammation

sensitive

range

(FDA)

Use

Reports

acid

of

material,

have

Journal

yields,

from

is

extracted

for

in

highlights

the

International

sodium

cited

that

will

levels

bacterial

given)

The

observation

be

hyaluronic

used

7

cosmetic

is

tissue

to

Laboratories,

free

for

of extraction

source

in

extraction

in

include

immunologically

hyaluronic

500

also

product

easier

inflammatory

to

to

studies

of

or

available

impurities

agent and

of

0.03

Table

of

to

HMW

in

purity.

hyaluronate

animal contained

detecting

although

Toxicology

of

that

other

the

hyaluronic

to

severe

kill from

impurities. 24

heat,

augmentation

cosmetics

the

sources

as

high

proteins

to

exhibits

was

mg

>20

formulations. 7

use

and/or

2.

the

US

a

in

acid

hyaluronic

that

produce,

that

polymers

difficulty

Cosmetic

with

protein

function

The

sources

sources,

of to

the

data

probably

intraocular

000

in

1987.

degree

acid

virus. 5 ’

or

/

Fort

Food

the

0.15

significant

showed

and

is

hyaluronic

223

Vol.

with

Viscoat

avian

reactions

superior

70°C

are

acid

authors

from deficiencies

as

base

in very

on

reactive. 40

lots

impurities

preparations

The

Worth,

28,

products.’ 2

extract,

mg

eliminating

uncommon

concentra from

other that

a

hyaluronic

of

of

and

Ingredient

frequency

from

molecular

due

functions

influenza

acid

pure

tempera

pairs. 5 °

viscosity

bacterial

No.

product

involved

that

(sodium

of

purity. 6

authors

Volun

inflam

manu

repro

to

stated

biolo

to

levels

Drug

DNA

than both

4S,

acid

was

and

and

TX)

the

vis

the

in

in

July/August

The

of

face

cles may

diseases respiratory

tion

sure respiratory inhaled

determination

tant chemical cation

of hyaluronate

O’Brien Hyaluronic included

from in

opean acid,

hyaluronate, products. 7 ” 2

cosmetic

by

gory

agent—miscellaneous

products. 7 ’ 52

quency application

hyaluronic

agent—miscellaneous

sodium

permeable form

obstructed

surface.

solutions, parations. 6

to

to

the

deposition

accelerate

industry. 53

increase

cosmetics

There

Cosmetics

Potassium

Sodium

Hyaluronic

within

and

aerosol area,

be

factor

to

is

use

sodium

2009

exposure,

a

Union. 56

reported

an

may

related

of

hyaluronate

are

deposition

aerosols,

reviewed

hydrated

in

among product

or

species,

are

use

aerosol

when

system. 57

affecting

a

acid/cm 2

acid,

system.

to

rate

cosmetic

the

LMW

are

while

properties associated

the

be

damage

The

certain

hyaluronate,

hyaluronate

No

no

in

and

in

as

The

Aerosols

of air

to

hyaluronate

used

acid

and

moisture

mass

in

by

of

a

the

sodium

restrictions

the

Japan. 55

applied

the

the

use

maximum

category

requires

function

and

the Table

the which frequency

hyaluronic

viscoelastic

it

potassium

1

of

The

aerosol

the

the

is

is

respiratory in

substances

of

Particle

region

repair.’ 4

mg/cm 2

products

number

health

and

and

deposited.

“fixes”

concentrations

the

used

cutaneous

concentration,

with 2%

potential

associated products

skin

site

hyaluronate,

location

toxic

2.

to

level

aerosol

is

Hyaluronic

or

depend

is

an

is

of

in is

the

of

of

concentration

in

the

is

spray. 52 ’ 53 is

of

reportedly

consequences

reported

potassium

size reportedly

of

cosmetic

moisture

of

analysis

a

of

HMW

acid

a

hyaluronate

deposition

action contributed.

marketed “anti-aging”

the

of

the

a

film. 13

body

use

surface

deposition

listed

system

for

a

particles,

Many

of

adverse

is skin

within

damaged

skin

with

respiration

product,

use

respiratory

on

has

the which

deposition.

as

hyaluronic

lotion. 53

and

were

of

in

of

as

acid,

product

of

the

occupational

a

conditioning

the

conditioning

most

the

The

used

Jensen

to been

hyaluronate

are

of

used

function

the in

Table

the

an

hyaluronic

prohibited

within

potassium

effects

their

the

of

duration

the

the

the skin

of

reported

of

skin

location

0.02

The

are

particle

specific

sodium

aerosol

human

inhala

film

impor

use

in

shown

appli

is

parti

in

tract.

At

expo

cate

2.

Eur

skin,

The

sur

acid

and skin

pre

601

fre

and

the

not

not

mg

for

an

11

of

of is Hyaluronic Acid, Potassium Hyaluronate, and Sodium Hyaluronate / Becker et al 11

Table 2. Cosmetic Product Uses and Concentrations for Hyaluronic Acid, Sodium Hyaluronate, and Potassium Hyaluronate Product Category Ingredient 52Uses Use 53(%)Concentrations Hyaluronic Acid Bath products

Other - 0.00 1 Eye makeup Eye shadow 15 0.02 Eye lotions 5 -

Eye makeup remover 2 0.00 1 Other 11 0.07 Fragrance products Powders I - Noncoloring hair care products

Conditioners 2 -

Permanent waves 1 -

Tonics, dressings, etc 1 - Hair coloring products

Rinses 1 - Makeup Blushers 7 0.02 Face powders 5 0.00005 Foundations 24 0.002 Lipsticks 0.01 Makeup bases 22 Other 0.001 Nail care products

Cuticle softeners 1 0.001 Other 1 0.01 Personal hygiene products Other I - Shaving products Aftershave lotions Shaving creams 3 0.3 Skin care products

Skin cleansing creams, lotions, liquids, and pads 6 0.00 1 Face and neck creams, lotions, powders, and sprays 8 0.1 Body and hand creams, lotions, powders, and sprays 12 0.001-1

Foot powders and sprays 1 Moisturizers 37 0.001-0.1 Night creams, lotions, powders, and sprays 17 0.02

Paste masks/mud packs 13 0.00 1 Other 23 0.001 Suntan products

Suntan gels, creams, liquids, and sprays 1

Indoor tanning preparations 1 0.00 1 Total uses/ranges for hyaluronic acid 223 0.00005-1 Sodium Hyaluronate Baby products Shampoos - 0.5 Lotions, oils, powders, and creams - 0.5 Other - 0.5 Bath products Oils, tablets, and salts - 0.5 Soaps and detergents 1 0.00 1-0.5 Bubble baths - 0.00 1-0.5 Capsules - o.s

(continued)

Aftershave

Feminine

Shaving

Other

Underarm Other Nail

Personal Nail

Nail

Creams

Cuticle Other

Basecoats Makeup

Nail Rouges Leg

Makeup Foundations Tints

Lipsticks

Makeup Bleaches Tonics,

Face Other Lighteners

Blushers Rinses Wave

Color Dyes

Hair

Other

Rinses

Shampoos Permanent Conditioners

Sprays/aerosol

Other

Straighteners Noncoloring

Perfumes Other Mascara

Sachets Fragrance

Powders

Eyeliners

Eye Eyebrow

Eye Eye Product

Other

12

Table

and

polish lotions

polishes

extenders

care shadow

makeup

coloring

powders

and

International

sprays sets

softeners

dressings,

and

products

body

bases

fixatives

hygiene

Category

products

pencils

deodorants

colors

and

deodorants

lotions

products 2.

with

and

waves

lotions

hair

and

paints

products

(continued)

undercoats

fixatives

enamel

color

products

enamels

care

etc

Journal

products

removers

of

Toxicology

/

Vol.

28,

No.

4S,

July/August

Ingredient

2009

96

27

20

17

15

10

15

16

11

6

3

2

6 - 1 -

-

5 - - 9 -

-

6 - - 4

- 1

3 - 1

- 1 ------

-

Uses 52

Use

Concentrations 53 (%)

0.00

0.5

0.5

0.001-0,5 0.5 0.5 0.5 0.0001-0.5

o.s o.5

0.5 0.01-0.5 0.002-0.5

0.0 0.0002-0.5 0.001-0.5 0.0005-0.5

0.0001-0.5 0.5 0.001-0.5 0.5

0.5

0.5 0.5 0.5

0.5 0.02-0.5

0.5

0.5 0.5 0.001-0.5

0.5 0.00

0.00 0.5

0.5 0.0002

0,5

0.5 0.0001-0.5

0.5 0.0001-0.5

0.001-0.5

0.00 0.5

0.0001-0.5

0.001-0.5

(continued)

5-0.5

1

1-0.5

1-0.5 1-0.7 Hyaluronic Acid, Potassium Hyaluronate, and Sodium Hyaluronate / Becker et al 13

Table 2. (continued) Product Category Ingredient 52Uses Use 53(%)Concentrations Beard softeners - 0.5 Men’s talcum - 0.5 Preshave lotions - 0.5 Shaving creams - 0.001-0.5 Shaving soaps - 0.5 Other 2 0.5 Skin care products Skin cleansing creams, lotions, liquids, and pads 20 0.000001-0.5 Depilatories - 0.5 Face and neck creams, lotions, powders, and sprays 48 0.005-1 Body and hand creams, lotions, powders, and sprays 25 0.0001-2

Foot powders and sprays - 1 Moisturizers 151 0.000001-1 Night creams, lotions, powders, and sprays 11 0.0001-1 Paste masks/mud packs 12 0.005-0.5 Skin fresheners 10 0.05-0.5 Other 38 0.001-1 Suntan products Suntan gels, creams, liquids, and sprays 4 0.000001-1 Indoor tanning preparations 1 0.001-0.5 Other 3 0.001-0.5 Total uses/ranges for sodium hyaluronate 601 0.000001-2 Potassium Hyaluronate Skin care products Face and neck creams, lotions, powders, and sprays 6 - Moisturizers 4 -

Paste masks/mud packs 1 - Total uses/ranges for potassium hyaluronate 11 None reported size and density. The parameter most closely associ Hyaluronic acid has been tested as a treatment ated with this regional deposition is the aerodynamic for lipodystrophy in AIDS 62patients. Injections of diameter, da, defined as the diameter of a sphere of hyaluronans is considered effective in relieving the unit density possessing the same terminal setting pain and effects of osteoarthritis 6364(OA). Hyaluro velocity as the particle in question. These authors nic acid application also has potential in facilitating reported that the mean aerodynamic diameter of the regrowth of severed nerves. 65’ respirable particles is 4.25 ± 1.5 urn. This value may be compared with diameters of anhydrous hair spray particles of 60 to 80 l.Lm(typically, <1% are below 10 Eye. Hyaluronic acid at 0.1% provides therapeutic um) and pump hair sprays with particle diameters of benefit in dry eyes, which is because hyaluronic acid >80 58u.tm. has a long residence time in the conjunctival sac and prolonged contact with the 66cornea. It also has been suggested for use in glaucoma”6 and other eye Noncosmetic Use surgery 687techniques. ’ General Medical Hyaluronic acid has been used to simulate neutrophil Surgery. ’ Hyaluronic acid has also been used in function in patients with extreme susceptibility to bac cochlear implant surgery to lubricate the electrode terial 5960infections. Hyaluronic acid is also used in on insertion as well as tympanic membrane repair ophthalmic surgery, treating joint inflammation in and 7274otosurgery. Hyaluronic acid also has been racehorses, drug delivery,orthopedics, cardiovascular used in urological surgery, particularly for vesicour aids, and’ wound ’6healing. eteral reflux 75Krauss. 14 International Journal of ToxicologyI Vol. 28, No. 4S, July/August 2009

Table 3. Summary of Drug Delivery Applications of Hyaluronic 277Acid Route Role of Hyaluronic Acid Therapeutic Agents

Ophthalmic Increased ocular residence of drug, Pilocarpine, tropicamide, timolol, gentamycin, which can lead to increased bioavailability tobramycin, arecaidine polyester, (S) aceclidine Nasal Bioadhesion resulting in increased bioavailability Xylometazoline, vasopressin, gentamycin Pulmonary Absorption enhancer and dissolution rate modification Insulin Parenteral Drug carrier and facilitator of liposomal Taxol, superoxide dismutase, human recombinant entrapment insulin-like growth factor, doxorubicin, paclitaxel (Rosato et a1278 Implant Dissolution rate modification Insulin ) Gene Dissolution rate modification and protection Plasmid DNA/monoclonal antibodies

Sodium hyaluronate may be the solution best facilitate manipulation inside the eye with reduced suited to provide a sufficiently thick mucosal layer trauma to the corneal endothelium and other ocular by endoscopic injection for endoscopic mucosal tissues. Healon GV also can be used to efficiently resection resulting in fewer complications because maneuver, separate, and control ocular tissues. the raised effect is maintained 76longer. However, Synvisc (Genzyme Corporation, Cambridge, hyaluronic acid may stimulate cell growth of any resi MA) and Nuflexxa (Savient Pharmaceuticals, Inc., dual tumor 77cells. Hyaluronic acid has potential use East Brunswick, NJ) are indicated for the treatment in anesthesia because its presence prolongs the of pain in osteoarthritis of the knee in patients who effects of tetrodotoxin blocks on rabbit 78nerves. have failed to respond adequately to conservative nonpharmacological therapy and simple analgesics, for example, acetaminophen. Respiratory Function. Inhalation of hyaluronic acid Restylane8384is used in cosmetic surgeryfor the correc (at respirable particle sizes) by persons with pulmon tion of wrinklesand facial sulci, including perioraland ary emphysema may improve the elastic properties of periorbitalwrinkles,wrinklesin the genal region,lower the lungs thus improving respiratory function. lip elevation, and’ droopingof the nasogenian sulcus. It 79 is also used in lip contour and augmentation as well as correcting depressed scars. It is contraindicated in Drug Delivery. Hyaluronic acid is used as a drug deliv patients with severeallergiesmanifested by a historyof eryagent. Hyaluronic acid maybe used as a skin sub anaphylaxis,historyor presence of multiple severealler stitute8 delivering antibiotics to ° burns or skin dressing gies,or allergiesto gram-positivebacterial 85proteins. and to accelerate 8182healing. The uses of hyaluronic Hyaluronic acid’s use in correction of glabella acid in drug deliveryare shown in Table 3. wrinkles (ie, between the eyebrows, above the nose) ’ is controversial because this region is crisscrossed with relativelylarge blood vessels near the skinsurface FDA Approvals. Deflux (Q-Med Scandinavia mc, and can lead to the possibilityof severe complications, Uppsala, Sweden) is used to treat children who have including blindness. Current and potential noncos vesicoureteral reflux, an abnormal condition in which 86 metic uses for hyaluronic acid are listed in Table 4. urine flows backwards from the bladder to the kid 83neys. This condition causes repeated, severe urinary tract infections, which can harm the child’s kidneys. General Biology This treatment should not be used in patients who have one kidney that does not work normally, an Natural abnormal pouch in the bladder wall, an extra ureter, Occurrence a urinary tract infection, or abnormal urination. Hyaluronic acid naturally occurs in the human body Deflux should never be injected into blood vessels. in the avascular body compartments like the synovial Healon GV (Abbott Medical Optics, mc, Abbott fluid and vitreous 67humor. It is also abundant in Park, IL) is indicated for use in anterior segment tendon sheaths and bursae and found in the small ophthalmic surgical 83procedures. Healon GV cre amounts of fluid in the “serous” cavities (pleura, ates and maintains a deep anterior chamber to pericardium, and peritoneum) and in the less well-

woman

kg

Dividing total

acid

calculated

mated

order

the

of resides

average

dermis

(—0.l between

persists

Vocal Allergy

septa

defined

Drug

Drug

Drug

Hyaluronic Antiadhesive

Joint

Silver Rheumatoid

Possible Arthroscopic

Encapsulate

Ear Trauma

Nose Trabeculectomy

Spacing

Vitreous

Retinal

Application

Penetrating

Comfort

Cataract

hyaluronic

(132

Based

The

surgery

epidermis

resides

delivery

delivery

delivery

mobilization

and

of

cord

hyaluronate

in

of

response

to

reattachment

mg/g

applications surgery

in

human

(-.-0.5

adult

substitute

has

approximately

surgery

eye

between

Ib)

largest

skeletal

planes

magnitude

throat

the

be

implants

muscle

acid

skin

on

(eg,

keratoplasty

arthritis

cells

that

applications drops

surgery

woman

vehicle

vehicle vehicle

in

wet

weight

a

-0.5

this

human,

applied

use

provides

prevention

acid

surgery in

to

(estimated

total

tissue,

the

mg/g for

amount

during

provides

approximately

of

glaucoma protect

-

and

tissue). 4

based

the

information,

bodies

Healon

(eg, implantation - -

-

Table

mg/mL).

skin.

in

nasal

skin

topical

tissue

is

of

higher

topically

surface

wet

(corneal

cardiac

individual

support

fallopian

bone

after

the

where

skin

on

skin

endothelium

9000

slow

patch

of

spray

50%

Approximately

4.

management

proven

and

The

tissue)

matrix

abdominal setting

to

movement

hyaluronic

by

(CTFA

release

than

or

to

transplantation)

Current

it

area

g

be muscle. 8 ’

of

tube

skin,

in

the

allow

actual

the

is

of

0.6 effects

the

fibers,

dressing

2-4

in

present

around

and

surgery)

of

Hyaluronic

and

skin.

area

of

CIR

2006),

hearing

total

surgery

the

mg/g

Ag±

Hyaluronic

and

mg/mL)

in

concentrations

maintain

16900 acid

such

the

of

spindles,

Expert

horses

dermis

The (microbiocidal)

15%

in

Potential

the

in

hyaluronic

skin

and

of

yielding

epidermis

(7-8

the

both

as

Acid,

perforated

average

of

cells

anterior

cm 2 . 88

in

are

yields

Panel

body)

those

Wound

(esti

g

a

hand

Osteoarthritis

acid

and

Ophthalmic

the per

Orthopedic

Potassium

Noncosmetic

60

an

in

Surgical

a

Other

chamber

tendon

into

ear

healing

cluded

the

sis. 90

tors

Synthesis ments

of eye,

noted

daltons, skin

following:

of

skin

skin/cm 2 .

a

drums)

wounds

weight

hyaluronic

surgery

Hyaluronate,

blood

Biosynthesis dome

cells HMW

of by

=

and

When

Applications

that

are

blood

0.318

weight

that

is per

during

were

vessels,

in

By

of

highly

0.6

hyaluronic

HMW present

area

synthesis

hyaluronic

hyaluronic

synovial

using

vessel

mg acid

mg

of

arrested

and

surgery

of

approximately

hyaluronic

angiogenic

hyaluronic

while

of

hyaluronic

in

Sodium

in

the

skin:

growth.

skin

Hyaluronic

Tan

Laurent 28 ° Tan

Scuri

cartilage, Surendrakumar Tan

Brown

Goa

Tan

Goa Tan

Tan

Tan Tan

Laurent 28 °

Tan

Tan

Tan

Goa Tan

acid, Goa

Goa

Goa

fluid

was

estimate

in

LMW

acid

9000

by

acid

et

et

et

et

and et et

et

and et

et

et

et

et

and

and

and

and Hyaluronate

152

mitosis.

in

a1 6

et

blocked

al 6

area

al 6

al 6

a1 6

al 6

a1 6 al 6

al 6

a1 6

al 6 a1 6

joints. 89

acid/cm 2 ,

and

acid

acid/g

Benfield 279

Benfield 279

increased

Benefield 279

Benefield 279

Benfield 279

al 145 ; Benfield 279

the

g/

was

hyaluronic

Acid

in

0.6

of

is

Reference

16900cm 2

are

range

inhibits

estimated

the

Brown

hyaluronic

The

et

mg/g,

required

x

by

potent

al 153

/

These

vitreous

0.53

Becker

of

authors

an

during

and

the

the

millions

acid

inhibitor,

g/cm 2 /l

Jones 277

to stimula

=

amount

authors

for

et

growth

acid

be

of 0.53

mito

al

frag

con

the

the

the

of

in

15

g g 16 International Journal of Toxicology/ Vol. 28, No. 4S, July/August 2009 detachment of the cells from their substrate before cornea, neural tube, mammary ducts, placenta, and they could divide. Hyaluronic acid is biosynthesized epidermis.’ in the outer cell membranes.’ In the skin, hyaluronic acid is synthesized pri marily by dermal 9fibroblasts and by epidermal kerati Immunological Effects nocytes. In fibroblast cultures, the rate of hyaluronic9 acid biosynthesis is regulated in part by There are divergent reports on the effect of cell’ density. At low cell densities, biosynthesis is hyaluronic acid on macrophages. Relatively high high, and cell motility and cell proliferation are also concentrations of HMW hyaluronic acid inhibit the high. At high cell densities, cell proliferation is low, movement and phagocytosis2 of macrophages, pre and hyaluronic acid biosynthesis is shut 92down. sumably due to the viscosity of the medium, while lower concentration enhance their phagocytosis Biological Functions and pinocytosis.’°” Suppression37 of the formation of humoral preci Biological functions involving hvaluronic acid and pitating’’ antibodies to certain major classes of pro its salts include water retention in the matrix, tis teins3present in the peritoneal fluid from a patient sue hydration, water homeostasis, lubrication, with Wilm’s tumor was 138reported. Their findings solute transport, cell migration, neutrophil adhe were interpreted to suggest that hyaluronic acid can sion, cell interaction, cell division, bone resorp interfere with both the elicitation of a complete tion, fetal wound healing, wound healing, antibody response and the formation of “normal” development, and red blood cell aggregation and patterns of antigen-antibody precipitates in labora 1,12,74,93-115 adhesion.’ tory tests. The authors stated that their results Chondrocytes are dependent on a hyaluronic support the possibility that hyaluronic acid may play acid coat for the deposition of the cartilage 116matrix. an immunoregulatory role by masking potential In egg fertilization, a layer of hyaluronic acid must be immunogens. penetrated by the sperm, accomplished by virtue of Hyaluronic acid functions in U937 macrophage hyaluronidase on the sperm head.” Sliwa suggested proliferation and 39death.’ Hyaluronic acid inhibited that hyaluronic acid plays a7part in the attraction of U937 macorphage proliferation by impeding cell sperm to the ovum. 18 proliferation and inducing apoptosis. Hya]uronic acid in the extracellular matrix binds Hyaluronic acid plays an important role in muco to cells through specific cell-surface receptors, sal host defense in the lungs.’ Hyaluronic acid including CD44 and the receptor for hyaluronan retains and regulates enzymes°4 important for homeos mediated motility (RHAMM), leading to intracellu tasis at the apical mucosal surface of the lungs, releas lar signaling and modification of cell behavior.” ing them and activating some of them into the airway 9 lumen at times of insult to the epithelium when hya Role in Development luronic acid is degraded. Because LMW hyaluronic acid increases ciliary action in the lungs, simulta Hyaluronic acid plays a role in mammalian develop neous with the release of enzymes, smaller hyaluronic ment. For example, the concentration of hyaluronic acid fragments stimulate ciliary beating (through its acid is high during the morphogenetic phase of interaction with RHAMM) and hence the clearance development and removed by hyaluronidase during of foreign material from mucosal surfaces. differentiation.’ 20 Possible functions for hyaluronic acid during development include promoting the detachment Infection Control ”2of cells from a substrate, disruption of intercellular junctions’ permitting cell migration, reduction of Pericellular hyaluronic acid of human synovial cells the effect of fibronectin on spreading and motility can interfere with infection by the Newcastle disease of neural crest cells, increased cell motility, vascu 41virus.’ The authors hypothesized that cell-bound larization control, cell protection, and embryo hyaluronic acid occurs as a dense gel and would be Hyaluronic acid has been expected to impede the access of viruses to the shown to be important to the development of the plasma membrane.

ments

were The

exposure

increasing products

effects

cantly

None for form powder

rect

for

dyl

solution methacryloyl

to

of 0.04% form vascular

tetramethyl-rhodamine The

assessed.’ 42

Dulbecco’s thoracic

Complex

1

Complex

Complex

Simple

System

5-rhod.

ceramide

ceramide

methacryloyl-dex

a

form

48

Photoreactivity

methacrylate

Cytotoxicity

contact

effects

theoretical

cytotoxicity

HyA6O.

a

of

tested.’ 43

less

system

hours.

of

(from

(w/w)

was

system

1%

system

system

the

in

that

HyA

smooth

on

Dex

hyaluronic

IV

III

viable aorta

complexity

the

viscous

of

dissolved

Modified

with

other

(A)

(D)

(C)

Sodium

irradiation-induced

in

The

was

ratio

with

with

Cells

(B)

was

S

hyaluronic

presence

l5-HyA.

substitution

an

than

Liposomal

zooepidemicus)

of

(GMA)

rat

solutions/gels muscle

of

solutions/gels

free-radical

incorporated

aqueous

(dex-rhod/dextran)

exposed

mixture

in

hyaluronan-based

cells

acid

in

hyaluronate

cells

Sprague-Dawley

Eagle

were

DMEM

Table

and

DMEM

of

HyA

was

Ceramide

Cholesterol -Linolenic

Ceramide

Dipalmitoyl

Cholesterol

Cholesterol

Dipalmitoyl

Dipalmitoyl c-Lino1enic

c-LinoIenic

with x-Linolenic

and

cells

acid

iron

from

Phosphatidylcholine

Phosphatidyicholine

Phosphatidyicholine

dextran

environment

of

to

to

skin

used

Medium

5.

added

hyaluronic

HyA6O.

was

photopolymerized

nutrient

form

HyA6O

ions.

and

with

were

harvested

into

had

the

Lipids

to

Hyaluronic

lipid

Compositions

under

IV

III

lipid was

to

form

acid

acid

acid

acid

were

combined

its

control

to a

The toxic

photoinitiators

HyA.

placed

The

20%

quantify

to

peroxidation

were

(DMEM)

hydrogels

the

dissolved

degradation

supplement

models

a rats

then

ultraviolet

molecular

mixed

acid

form

effects.

from

resulting

1% Dextran

Acid,

solution

solution

groups.

in

signifi

glyci

of

(w/v)

frag

indi

were

with

Dex

in

the

Potassium

the

Skin

of

to

in

to

to

a

Concentration

Models

within

semisolid

hyaluronic are

ence.

suggested nic

the mine

the on resonance

dation model

samples

were

spectrometry of

which

(P to imal

(MDA) acid.

reactive

ton and

fate

tion weights

mined

skin

and

the Hyaluronate,

<

stable

able EPR

200

200

100 200

hyaluronic 100 100

100

determine

acid

100 100

100

100

100

was

catalyst

type 6,

preparations

shown

erythemal

Used

.05)

A

irradiated

The

the

TBA

corresponds

systems.

the

by

of

3.3,

in

thiobarbituric

to oxygen

added

of

signal

treated

nor

(tM)

formulations

free

reaction. laser

effects

hyaluronic

each

that

authors than

by

skin.

reduce

(EPR) reaction

the

acid

in

3.2,

of

Trommer

its

ion

and

Table

radicals.

intensities

to

the

acid

light

S

exclusive

The

exposed

species

the

the

dose

and

with

enzymatically

with

in

batches

the

signals

of

were

Sodium

spectroscopy

stable

concluded

Haber-Weiss

to

products

or untreated Samples

authors

cosmetic

scattering,

amount

5. 2.2

hyaluronic

samples

Liposomes

Liposomes

Liposomes

Shaking

with

hyaluronic

a

acid.

2

et

could

its

Ten

acid

produced

method

method

method

(ROS)

There

to

UV-B

sample.

a1 143

demonstrated

x

free Hyaluronate

topical

between

of

fragments

3

and

Manufacturing

millimolars

Electron

hyaluronic

reported

(TBA) for

be times

was

as

radicals.

of

that

was

of

generation

and

prepared

prepared

prepared

dose

samples

degraded

g/mol.

120

protective

were

without

was

an

The

reaction

acid

each

lower

with

administration

acid

malondialdehyde

the

neither

no

electron

the

mm

pharmaceutical

assay

/

of

used

and

concentration

1

Becker

paramagnetic

that

on

by

difference by

samples

by

the

The

preparations

The

human

composition

photodegra

for

x

0.25

ferrous

for

Process

acid,

hyaluronic

the

the

the

fragments

the to

via

ROS

was

106

for composi

to

the

all

hyaluro

4

authors

donator

all

thin

thin

et

thin

initiate

J/cm 2 ,

a

model

deter

deter

lipids refer of

g/mol

al

mass

min

used

with

Fen

skin

and

layer

sul layer

layer

the

of

in 17

phically.

confined blood

terstaining from killed

to penetration

area with

44.7-63.1 experiment, of

mice,

mum beled

above.

3 highest

at to upwards, of

tions, 4

authors was

[ 3 H]hyaluronan, no number

Spraque-Dawley depth Absorption,

skin. overall lated.

( 3 H-glucosamin; significantly and

control.

applied

Sprague-Dawley

Hyaluronic and

(1350-4500

1

1

18

or

cm 2

hours,

a

a muscle

16 4

x

adverse

Autoradiography

Absorption!Dermal

International

killed

depth maximum

of

was

12

excised

the

The

nonradioactive

hours depth

6 Excretion

hyaluronic

vessels

and

aged

The

circle

40-jim

5

of

mean

applications

values,

to cm

The

to

of

Radiolabel

also

outermost

treated

the

dissolved

between

hyaluronic

the

mg)

and

addition

136

3

a

of

effect

6

3

blood

showed

examined

after

the

area

days

similar

Da)

cm 2

of

just

shaved

blood

higher

rats

to

and

shaved

acid

500

skin applied

thick,

processed

that depth

Distribution,

in

jim

mice

Journal

6

hyaluronic

acid

rat

with

the using on

10

on

was

above

were

after

vessels

of

months,

processed

to

rats. 144

excess

fixed

vessel

in

of

layer

was

collagen was

from

area

beneath

that

the the

jiCi/mL)

on

in

under

of

was

the

were 700

last

were area

acid

was

of

gel.

in

hyaluronic

NaOH,

applied

radioactive

radiolabeled

treated of

the

approximately

treated

mouse

the

the

and dorsum

morphology

found approximately

gently

the

elsewhere

down

per

application, Toxicology

for

silver number shaved

used of

jim,

found

penetrated

was

treated

In

Penetration

were

final

human

general

cut

The

back

platysma

blood

examined

400

acid,

frozen

for

unit

bundles.

in

the

and

the

deeper in

mainly

twice

excised

to

rubbed

to

skin.

skin

from

grains

of to

used. 145

treatment.

a

Metabolism,

of

a

dpm,

acid

histology,

citric

backs

per

the

detect

every

gel

counted. citrate

epidermis.

length

vessel

penetrate

the first

/

in

citric

anesthesia.

on

one

hyaluronic Vol.

sectioning.

umbilical

of

than

daily

muscle. SKh/1

millimeter

lymphatic

to

the

that

the to

and

50

in

trunk.

autoradiogra

the

the

800

were

on the

to

were

There

part

12

young

28,

a

buffer

rat

of

the

experiment

the

mg

One buffer

buffer

was

mice

the

maximum

for

fatty

untreated

a

the

the

hours back

skin.

The

jim.

form

Radiola No.

skin

11

and

hairless

marked

dermis,

rat

evident

Twelve

(range,

dermal

clearly

of

Coun

5

calcu

These

group

depth

maxi

other

After

adult

were were

male

4S,

Sec

days

skin acid

vein

The

side

skin

The

and

to to

as

was

had was

the

the

for

of

July/August

a

a

a

The

were The

kidneys,

pregnant

entire

after

nant

were

(number sodium eral its 4

evidence relatively restricted weight

of labeled after

applied

2 identified

cluded imately utes

recovered

(3.6 significant authors keratinized

30 onward. was

ered hair lymphatic

mouse

the at penetrate

trated them, findings

layers

grains

hours

hours,

the

[ 3 H]

minutes

metabolic

Distribution

In

brain,

found

transfer

dermal

applications

but

follicles.

the

application,

from

x

ra.s

collected found,

2009

body

2-hour

of

within

or

a

aggregated

profile

studies

in

acetyl

to

after

suggested

second

that

was

adrenal

hyaluronate

1

were

injection.

the

of but

that

Because

rats degradation

little

injected

as from

to

the

sinuses. 145

Harder’s

these

in

the

the

hour.

rats

were

layer

after

macromolecular

matrix,

Da) epidermis.

of hyaluronic

similar

the

the

but

smaller

application.

hyaluronic

time

cell

The

were

passage

group

an

skin

of

degradation

hyaluronic

the

skin

skin

within

radioactivity

not

experiment

from

gland,

same

authors hyaluronic

taken

areas,

of

indication

was

did gel

increased

In

boundaries

mainly

at

authors

that

point,

The

fraction

the

intravenously

after

provided).

injected

gland,

surface

the

(4

to

of

hair

and

the

application,

polymers.

demonstrate

within

the

was

the Grains

only

distribution

control

hyaluronic

x

through levels

at

quantity

dams

The

placenta,

skin

acid.

more

12

acid

with

follicles

stated bloodstream,

in

as

acid

10’

Mice skin

1,

dams.

lungs,

stated

examined.’ 46

of

using

had

to

slightly

amounts

was

authors

on

that

into

acid

4,

and the

the

these

were

had

equilibrated

were

Because

in

Autoradiograms

hyaluronic

Da),

the

content

was

was rapid

after

16

24,

day

receiving

that

the

From

begun

small

the

the of

there

heart,

metabolites

in in lining

Three

with

and

been

that

10

hours.

which and

radiolabel

acid.

killed

high

found

within

48,

areas.

lower

also

the

lower

17 the

the

1

basal

stated

hyaluronic

mice,

fetuses

transit

there

of

to

skin

amniotic

the

in

the

from

was

and

4

absorbed

of

‘ 4 C-SL-lOlO

authors

after

liver,

As

there found

as

bloodstream

radioactivity

within rudimentary

cells

8

to

by

the

both

intact to

one

acid

proportion

pregnancy

epidermis, the

SD

keratinous

hours,

at

molecular

radiolabel

than

that

in

was

failure

72

was

freezing.

early

5

8

concen

of

through

8

authors

30

approx

spleen,

serum,

or

dermis

of

of

whole

was

recov

of in

hours

of

hours

series

hours

preg

these

clear

fluid

after min

con

1

acid

that

sev

and

the not

the

the

the

the the

to

as

to a

binding

into

and was

milk with

plasma

time.

blood

the

ing

caudal

follows

ether-anesthetized radioactivity

experiment. and pregnant

14 C-SL-1010 24 sues was

4 were

radioactivity

48 Whole

Amniotic

24

fetuses Adrenal

Fetal

Placenta

Spleen

Kidney

Lung

Liver Heart

Harder Brain Plasma

Blood

Sample

hours

Table

hours

hours

hours

In

birth

The

same

nontoxic

distributed

the

liver

from

of

time

total

collected

supernatant

The

body

from

gland

another

vein.

the per

was

after

the

fluid

casein

plasma

6.

authors

after

rats

then

1,

time.

after

2

until

authors

casein,

entire

dam

the

4,

same

lactating

highest

in

Tissue

of

administration

Lactating

to

Milk

on

sodium

the

and

reduced

from

the

throughout

the

the

ocular

fraction, the

Radioactivity

experiment,

24 were

day

was

body

repeated

injection

general

on

was

Concentration

24

stated

fetuses

injection,

casein

tissues

dams

fraction

hours.

at other

18

hyaluronate

the

homogenized,

obtained.

rats

hours

fundus

over

after

of

collected

rats

1

15th

hyaluronic

and

hour

pregnancy.’ 46

and

that

pattern

fetuses

increased

of

is and

The

the

fraction,

the

and

time.

72

after

(n

the

Sodium

shown

in

the

offspring.

after

vein

to

divided

body

1

was

sodium

and

hours.

The

211.791

radioactivity

decreased

126.571

h(n=2)

above of

=

the

21.166

22.195

26.396

20,529 31.667

14.753

12.217

Hyaluronic

17th

authors

(16-200

0.913

0.608 0.2

dams

6.839

2,797

(10

in

administration

Radioactivity

seen

was

maintained

3)

and

of

decreased

19

in

milk

the

distribution

binding

over

Hyaluronate)

calcium

acid

days

mg/kg)

the

the

Radioactivity

were

Table

injection

hyaluronate

were

collected

At

3

in

litters.

time

increased

to

dams

in

collected

1 .iL),

fractions

after

fraction,

Acid,

1,

the

S

the

in

mildly

killed,

4,

to

6

livers

After

with

with

(Ca) until

until

Radioactivity

first

The

and

and

and

giv

I

the

the

Potassium

tis

of

to

of

at

to

to

Fetus

Intravenous

Dam

Rats

chamber

was

nic

addition, humor

original weight-average

the luronic

a The

luronic

saline ing

New Approximately

(3.8

was

3.4

10 supernatant

Ca of

21% tion, tivity

cetylpyridinium

on

number

Hyaluronate,

the

(tg

mg/mL.

acid

binding

117.343

it

Metabolism

kg.’ 47

Day

24.712

29.369

mixture 35.159 20.462

reached.

90.098

18.271

turnover 13.970

16.915

4h(n3or4)

mL)

4.347

2.141

contained

0.870

of

7.966 2.314

into

0.2%

of

equivalent

Zealand

removed

radiolabel.

Administration

containing

mixture,

acid

the

each.’ 46

acid

18

volume

was

with

the

1

The

±

±

± ±

±

±

± ± ±

± ±

±

±

±

average

of

casein

Two

radiolabel.

to

fraction.

0.443

0.438

0.552

2.962 4.214

(Healon),

2.910

rabbit 2.208 2.749

0.212

1.339

3.530 29.934

was

1.687

19.890

of

The was

Pregnancy’ 46

mixed anterior

and

of

20%

a

test

molecular

56%

and

hyaluronic

White

0.5%

chloride,

and

Total

rabbits

SL-1010/g

trace

of

A Sodium

determined

hyaluronic

fraction

dissolved

of

material

replaced

with

was

to

1%

subset

of

2

of

commercial

chamber

more

80000.

casein

63%

The

(almost

then

the

‘ 4 C-SL-

amount

rabbits,

had

Hyaluronate

dimethyl

and

1.93

injected

wet

weight

of

acid

of

radioactivity

hyaluronic

of

had

concentrated

in

was

by

0.05

contained

that

tissue)

measured

acid

the

g

that,

by

[ 3 H]Hyaluronic

1010

of

was

the

phosphate-buffered

0.2 background

of

of

purified;

weighing

5 the

was

radiolabel

contained

radioassay

concentration

commercial

hyaluronic

in

labeled

mL

(A

tested the

mL

sulfoxide.

[ 3 H]hyaluronic

/

24h(n=

eyes

Becker

29.072

17.103

13.256

Formulation

the 3.432 0.185

6.427

920 3.267

6.500

3.135 0.633

3.932

2.071

3.082

acid

1.624

replaced.

77%

the

casein

of

was

by

of

until

000

[ 3 H]hya-

hyaluro

aqueous

anterior

radioac

fraction

± ± et

± ±

±

and ± ±

± ±

± ±

±

±

2.7 ±

around

to

female

inject

in

level).

to

3or4)

al

0.499 0.008

0.173

2.048 0.184 0.121

0.108

0.141

0.098

6.048 0.209

1.028

1.108

1.190

frac

hya

84%

acid acid

The

and

the

the

the

be

In

of

to 19

linear,

both

acid

taken

taken acid bolus Three

taken

is

(range, approximately followed was with

to of

additional

[ 3 H] carotid

samples blood increased

30 for

ewes, lism

clear

authors

had

acid

[ 3 Hjhyaluronic approximately

125 exponentially. in to injections

maximum collected

after back

milliliter larly

50 hyaluronic

[ 3 Hjhyaluronic acid

20

14

1

0.12

other

unlabeled

13

blood

hours,

give

an

minutes

The

hours

Hyaluronic

were

International

taken;

.tg/min

infusion

of

with

venous

in

a that

elimination

of

days.

2,

the

sampling

at

dose. to

weighing

the

1,

approximate

1-compartment,

hyaluronic

artery,

±

half-life

the

3.3-6.5

the

stated

5,

were time

4

rabbit

baseline,

a

after

5,

then

by

daily blood

had

blood

the and

and,

and

hyaluronic hours

0.05

levels

20,

3 H

then

injections.

acid;

thigh

within

steady-state

(2

neck

Twenty

10,

after

and

hyaluronic

a

was

it

drawn

given,

0.2

not

1

respectively.

8 approximately

and radiolabel

the

mL/min). injection

The

for

that

in

40-minute

was

acid with

Journal

ig/mL.

acid

between

minutes).

ig/mL

takes

hours.

samples

later,

a

20,

acid

of

of

was

arterial

with

1

with

the

curves

stopped,

mL

intramuscularly

been

acid

injection 20

4

5,

40

45-minute

rabbit

these

half-life

sheep

2

injected

half-life

had

approximately

and

micrograms days

and

the

at

acid

right

10,

minutes ig

a

for

investigated. 148

0.1

injections

0.1 of

was

to

The

Arterial in

days

Blood

kinetics

2

2,

acid

mixed

into

This

a

arterial was

intravenous

Toxicology

plasma

20

catheters

were

0.055

obtained

mg

and

40

then

2

excess

the

times

was

at

mL

The jugular

half-life

mL

4,

at

intravenous

and

A

ig/mL;

subcutaneously

to

given.

was

and

a

was

intravenous

of

1,

detectable

the subcutaneously

minutes

baseline

and

6,

liver.

normal

dosage

with

15

rate

delay

later.

injected

with

every

of

samples

collected

2,

authors blood

blood

determine

mL

blood the

model.

should

8,

hyaluronic

5.3

was

eye.

38

of

given

minutes

the

4,

concentration

vein

/

injected

of

of

for

fitted

the

the

then

10, Vol.

30

the

The

Arterial

8,

due

[ 3 H]hyaluronic injections,

kg,

other

[ 3 H]hyaluronic

±

was

tracer

approximately

mixture.

approximately

approximately

samples

samples

It

levels

blood

postinfusion. samples

and

infusion

and Ten

normal

mixture,

immediately

intramuscu

1. 15,

After 2 28,

be

hours.

infusion

commercial

stated

were

reached were

to

hyaluronic

bolus

before

to

well

1

estimated

decreased

after

day

24

half-life.

the

adjusted

minutes No.

metabo material

20,

injected

dose

Merino

3

sample acid

and

in

bolus

blood

hours hours

fitted

were

were were

Two-

right

for

level

dose

to

4S,

then

that

The

and

and

was

and

the

the

the

its

of

of

of

to

at

a

July/August

9

when

were

other

samples space lowed found

and 2.2 24 eye carpine maximum

8, the

the decreased injection.

monkeys 3 H was

3 animals was

into weight-average

24 4 bility.

dure which

used. tion

of mentioned

were

levels with

nic Laurent

[ 3 H]hyaluronic N-acetyl-glucosamine

nic

of injection,

metabolic

10

3.32

18.2-43.5

hours)

additional

both

12,

[ 3 Hjhyaluronic

hours

hours

in

to

when 1 iL

[ 3 HjHyaluronic

Laurent

mean

acid

acid

injected

pilocarpine-treated

the

withdrawn,

the

of

also was

±

2009

day

of

metabolized,

Injections no

a

the

2.7

in

by

about

Unlabeled

was

and

at

sexes,

0.30

treatment

anterior

6 10

were

half-life

not

et

molecular the

labeled

but

a

was

and

adverse

kg. both

for

treated blood

after

It repeated

collected

adult the

half-life

minutes).

4%,

more

rate

reproducible

exponentially.

experiment.

24

a!

mg/mL

concentration,

reached

the and

bloodstream

given

tg/min/kg

into

showed

Injections

the

monkeys

mean

weighing

often

on

taken

2.42

hours

volumes

injection.

acid

dropped

New

molecular

was

could

were

rapid

same

chamber.

and

with

next

Fraser

cynomolgus

hyaluronic

of

the

with

effects

acid

was

for

pilocarpine

the

using

plasma

every acid

was

weight

a

with

±

The

a

from

Zealand

0.062

26.9

after

a

was

50

slower eye through

week.

maximum

3 H

time be

turnover

the

were

lag

between

3 H

body

Twelve at

0.48

ranged

21

pilocarpine.

were

was

after

repeated

mixed

into

authors

the

animals. 1 iL

detected

The

day 75

produced

of

in

of or

the

an

The

Aqueous

phase

from

injection.

8

weight

hours concentration

for

±

was

±

The

injected

the

weight.

the

the

of

addition

White 5

monkeys

treated

j.tL

rate

ig/mL.

acid

raised

used

7

ear

after

plateau

the monkeys.’ 49

authors

the

monkeys.

the

from

cynomolgus

7.0

0.009

and

months

as

during

authors

[ 3 H]hyaluronic

2.3

sheep.

the

6 on

at

acetyl

authors

reported

of

vein

later. and

the

with

[ 3 Hj

peripheral

eye

2

in

half-life

(average

The

was

4 2

x

and

rabbits

minutes

and

humor

a

3

at

hyaluronic

The

hyaluronic 21.1

to

into

to

After

with

days

Blood The

of

the

few

level

experiment

igJmL/min

106.150 monkeys,

9.5

without

reported

the

at

at

3

1

a

purified.

group

3

showing

pilocarpine,

4.9

4.2

researchers

stated

in

elimination

The

hours

stated

to the days

concentra

day

0, of

previously

hours

and 1, maximum

that

hours

following

50

weighing

from

reaching Hyaluro

(100 monkeys occurred

of

46.3

kg,

hyaluro

samples

2,

pleural

plasma

x

8,

cornea

(range,

proce

in

Blood of

in

tL

21

every

there

4,

pilo

were

after

acid

that acid

that

acid

that

The

106,

and

and

fol

sta

j.tL)

the

the

the

rig,

the

for

±

6, of

of or

weight

with

tional White

weight

to

saline The

thioglycolate, suspensions cavity

acid pared

layers

proposed

forms

delivery

dermal

mal. 277

numbers was

Brown hyaluronidase. lower region

acid digestion umn

dose. A

bile at ing

treatment,

male The

administered

in the

to

appears

polyethylene

induce

24

the

the

Hyaluronic

Inflammation

Effect

the Excretion

amount

effect

collected

up

half-life

100

presence

because

rats

peak The

hours.

with

in

(epidermis).

was

(PBS).

loss

5

8-

of

rabbits,

chromatography

centered

of

penetration

They

and

excretion

to

as

system

control

the

mL

to

20

2.9

that

method.

under

peritonitis.

of

bile

2.9

of

that

24

investigated)’ humans

[ 3 H]H 2 0

on

‘ 4 C-sodium

1 of

Jones,

deeper

or

The

of

to

intraperitoneal

kg,

for

hyaluronic

noted

5-hour

The

at

and

every

hyaluronic it

hours

and was

Penetration

[ 3 H]hyaluronic

tube

kg,

eluted

PBS

male

solutions

45

may

10

rabbits,

ether

pleural

were

Acid

disappeared

on

cumulative

of

2%

authors

A

was

were

analyzed

mg/kg

turnover

where

that was

layers

in

studies

2

bile

at

in

range.

the

was

depend

major

and

The in

injected

starch

hours

anesthesia. 146

1,2, Produced

the

a

confirmed

hyaluronate

inserted

acid

hyaluronic

the

in

approximately

male

fraction

lavaged

0.4%

acid’s

and

review

of

into

of

female,

it

irritant

used

blood

mice,

Nineteen

3,4,

peak

of

others

infection

using

forms the for

0.1%

from

of

in on

in

excretion

with

acid

with

and

the

hyaluronic

of

phosphate-buffered

the

influence Other

the

a

into

the

Hyaluronic

skin

or 10

and

article,

the

eluted

without

number

peritoneal

the

the

gel

was

with

from

During to

caudal

latex

injected

in

female,

5

irritating

the

acid

hours

intraperitoneal

species

(SL-

the

After

is

days.

drug

(dermis)

be

hyaluronidase

on

New

pleural

more

filtration

administered

rate

Chemicals

proportional

made

1

the

addition

an

varied

bile

at

beads,

hyaluronic

hyaluronic

1010)

hour

compared

vein.

as

and

acid

Acid,

reservoir

An

previous

confirm

5

Zealand

a

and

into

fraction

average

average

shallow

of

duct

1.

a

agents

lavage

space.

LMW

up

addi

com

drug

after

then

with

ani The

Bile

and

was

col

Potassium

was

3%

the

of

of

of

All Allen

researchers. respiratory

manifested Zealand nic

and be

adverse provided).

cluded was

different injected

sodium animal). removed were

sodium the injected

3 Animal

sodium

96

space

sodium sodium

Denlinger tion. 31 mg/kg

No

ual in

tress. the

rabbit injection.

1000-fold

Hyaluronate,

the

x

6 used

hours

peritoneal

Pleural

Peritoneal Acute

variations.

Oral

deaths

2.7

remaining

acid

then

rabbits

killed

et

lavage

10

of

All

hyaluronic

died

No

that

hyaluronate hyaluronate

kg. 15 °

al

effects.

hyaluronate

hyaluronate

fluid hyaluronate

The

with

from

as

White

from

into

Toxicology

random-bred

after

The

injected

gently

increase

The

further

the

and and Toxicity

in

cell/animal

no

stress.

within

highly

Space a

tolerated

fluid

count

was

wash

and

After

10

surgical

a

ICR

sham

the

count

animals

evidence

the

blood

injection.

injected

large

Balazs

rabbits

massaged, mL

Sodium

on

extracted.

acid

21.1

details

purified

in

24

decreased

mice

reached

pleural control

No

15

leukocyte

at lavage

injection

days

returned

part

of

collected

hyaluronic

(n

(n

the

hours

96

days,

tool

were

j.tg

injected

had

white

toxicity

and

buffer. weighing

= of

=

irritants

orally

Hyaluronate

pleural

were

The

hours.

2

of

to

was

sodium

and

level.

its

pneumothorax,

space

in

10)

no

12),

the and

the

The

46.3

produced

after

was

at

count

male

peritoneal

to peak

(55

at

made

the

provided.

administered

clinical

The

3,

animals

48

into

abdominal

was

acid

The

0.5

after

injections control

leukocyte

or

12,

‘g

The

with

caused

the

of

not

between

eye hours ±

hyaluronate

at peaked

I

rats. 21

abdominal

of

highly

up

24,

highly

concentrations

mL the

Becker

noted

24

6

4

injection,

the

authors

by

large

[ 3 H]hyaluro-

without

significantly

signs

of

were

x

adult

48,

level

hours

(count

cavity

peritoneal

of

fermenta

PBS.

a

skin

well wall, count

106

Animals

purified

purified

at such

et

individ

72,

by

2.2

buffer,

>1200

10-

of

killed. in

al

36

New after

con

cell/

wall

One

and

was

was

and

can

any

dis

not

the

the

the

but

kg

as

± in

21 to 22 International Journal of Toxicology/ Vol. 28, No. 4S, July/August 2009

Inhalation seen around one of the 12 control implants. Micro scopic examination revealed Sheep allergic to human neutrophil elastase (HNE) that there was minimal to slight chronic inflammatory cell were used to test hyaluronic acid as a way to block infiltration at both types of implant sites. The fibrous bronchoconstriction.’ Using a disposable medical membrane was graded minimal to slight around the nebulizer, the sheep inhaled a placebo then the HNE control and 52 minimal to marked around the Restylane implants. (dissolved in PBS) 30 minutes later to establish a baseline. The sheep later inhaled 3 or 15 mg LMW hyaluronic acid (150 kD) or HMW hyaluronic acid Chronic Toxicity (300 kD) dissolved in PBS. After either 0.5, 4, or 8 hours, the sheep were then challenged with HNE. Implantation Inhaled HNE in untreated sheep caused a short-lived A study conducted by Q-Med AB of Sweden on bronchoconstriction reaching its peak quickly Deflux was reported. The product is composed (0-5 minutes) 155 after challenge and resolved within of microspheres of crosslinked dextan suspended in 30 minutes. Aerosolized LMW hyaluronic acid a carrier gel of nonanimal, stabilized hyaluronic acid. blocked the FINE-induced airway response for the A 2-year implant study of Defuix was conducted 0.5- and 4-hour time period. The 8-hour time period using 22 rabbits. The objectives of this study were was only partially effective at the 3 mg dose but was to determine the biocompatibility and migration completely effective at 15 mg. There were no ill potential of Deflux when implanted into the rabbit effects reported from the hyaluronic acid. In another bladder submucosa. In each rabbit, 1 g of the test set 6, of trials, 7.5, or 15 mg HMW hyaluronic acid article was injected submucosally into each of the was administered 8 hours before the HNE challenge. following bladder sites: right and left bladder neck The 15 mg of HMW hyaluronic acid was completely and right and left bladder wall. Follow-up evalua effective while the 6- and 7.5-mg doses were less tions were conducted at 1 week and 1, 3, 6, 12, and effective in blocking bronchoconstriction. There 24 months. The urinary bladder, pancreas, kidney, were no toxicities reported. liver, lung, draining lymph nodes, and brain were removed and examined histologically for inflamma tion, infection, irritation, foreign body responses, tis Short-Term Toxicity sue necrosis, and scarring. Other organs were Inhalation examined for gross abnormalities. The microspheres induced a fibrous tissue reaction around each micro- Male Beagle dogs were used to test the effectiveness sphere without any adverse inflammatory reaction. of using a mixture of hyaluronic acid and recombi Another 2-year implant study of Deflux was nant human insulin in dried powder form as an reported.’ Twelvedogs were implanted with 2.5 g of delivery 55 inhaled drug system for insulin.’ Hyaluro 53 the test article in the followingsites:right and left urin nic acid formulations containing insulin (10% w/w) arybladder neck and right and left urinary bladderwall. were found to extend the mean residence time in The follow-upevaluations were conducted at 2 weeks blood and terminal half-life when compared to spray and 3, 6, 12,and 24 months. In each animal, the urinary dried pure insulin. There were no toxicities reported. bladder, pancreas, kidney, liver, lung, draining lymph nodes, and brain were removed and examinedhistologi Implantation callyforinflammation,infection, irritation, foreignbody Four samples of Restylane (20 mg/mL nonanimal responses, tissue necrosis, and scarring. Other organs stabilized hyaluronic acid) and 4 samples of high were examined for gross abnormalities. The micro- density polyethylene reference standard were spheres remained in the tissue in all injection sites for implanted into the paravertebral muscles in each of at least 2yearswithout causing anyadverseforeignbody 3 anesthetized 54rabbits.’ After the rabbits were reaction. There were no inflammatoryreactions. observed for 4 weeks, they were killed, and implant sites were examined macroscopically and microsco Ocular Toxicity pically. A white focus (possibly a deposit of hyaluro nic acid) was seen at 3 of the 12 Restylane implant A highly purified special fraction of sodium hyaluro sites. Encapsulation, with a thickness of 1 mm, was nate was used in an experiment testing the amount of HyaluronicAcid, Potassium Hyaluronate, and Sodium HyaluronateI Becker et al 23 inflamation caused when using hyaluronic acid to 24. Repeated implantations of sodium hyaluronate replace the liquid vitreous in the eyes of owl monkeys did not increase the severity of the haze and flare (Aotus trivirgatus) and rhesus monkeys (Macaca or any immunogenic response. 21inulatta). Sodium hyaluronate from 2 sources of In a continuation of the above studies, highly rooster combs and from human umbilical cord were purified HMW factions of sodium hyaluronate were also tested. The molecular weight varied between 1.2 injected into 6 eyes of 5 owl monkeys. Each and 3.0 x 106, andtheconcentrationwas 10.0 ± 2.0 received 2 to 4 injections over 5.52 years in 2 eyes, mg/mL. Sodium hyaluronate was dissolved in a phy 6.5 years in 2 eyes, and 9 years in’ 2 eyes. All eyes siological balanced salt solution that was also used as were completely normal, having no pathology in the the control substance. Sixty-seven owl monkeys and anterior segment, lens, vitreous, retina, or choroid. 15 rhesus monkeys were used in this experiment. Akasaka et al reported a “negative” result of an In owl monkeys, 1 mL of the liquid vitreous, repre eye stimulus test based on the standard Draize test senting approximately half of the total, was slowly of hyaluronic acid produced by the fermentation. withdrawn from 1 eye and replaced with the test No further details were provided. solution. The same was done with the rhesus mon 3 keys except that only 0.5 mL vitreous liquid •was ’ Skin Irritation replaced. Evaluation for inflammation by visual examination and leukocyte count was carried out Hyaluronic acid did not cause irritation in a single- 48 hours after surgery and at day 7. The leukocyte stimulus skin test using Japanese rabbits and Hartley count in the aqueous humor varied between 0 and guinea 3pigs. No further details were provided. 115 cells/mm in owl monkey eyes and between 0 ’ and 853 cells/mm in rhesus monkey eyes. The mean Skin Sensitization values were3 28 ± 7 and 38 ± 13 cells/mm respec tively. The difference between these3 values was not Potassium hyaluronate prepared from human umbi significant (P .4). Only 2 eyes had slight turbidity lical cords was tested for antigenicity using rab and slight haziness in the vitreous, in the owl mon Potassium hyaluronate was 6bits.” purified by keys. Only 2 rhesus monkeys had slight turbidity and repeated extraction with 90% phenol solution and haze. In both species, these reactions completely dis by repeated precipitation from aqueous solution at appeared within 72 to 96 hours after injection. pH 9 to 10 by 1.25 vol of ethanol saturated with These authors also injected the highly purified potassium acetate. Potassium hyaluronate was then sodium hyaluronate into the eyes of an undisclosed centrifuged after treatment with p-nitrobezyl bro number of owl monkeys that had previous reactions mide and acidified to hyaluronic acid. The amino to concanavalin A, endotoxin, and a less purified benzyl ether of the hyaluronic acid was coupled to sodium hyaluronate after the eyes had been clear of horse-serum albumin and whole rabbit serum (3.6 the reactions for 1 21month. The highly purified g protein to 1 g compound). Three rabbits were sodium hyaluronate implantations did not cause any immunized with the horse serum—hyaluronic acid significant inflammatory reaction in the previously preparation by injection at 30 mg, 60 mg, and 120 inflamed eyes. The leukocyte counts after a first and mg subcutaneously at weekly intervals for the first second implantation of sodium hyaluronate were not course. The rabbits were bled 1 week postinjection. significantly different from normal owl monkey The second course consisted of 4 intravenous injec aqueous after highly purified sodium hyaluronate tions of 20 mg at weekly intervals. These animals was injected 1 or 2 times. were bled (second time) 10 days postinjection. Preci These authors repeatedly, up to 6 times, injected pitin tests were carried out in bulk. Complement sodium hyaluronate into owl monkey 2eyes. The fixation was carried out using 2 minimal hemolytic experiment started with 76 eyes and finished’ with doses of complement. Agglutination was tested on 11. The average time between each injection was a washed suspension of cells from a young culture 5.5 months. The leukocyte count 48 hours after the of a capsulated group C Streptococcus whose cap first injection in 102 eyes varied between 0 and sules were known to be dissolved by hyaluronidase. 200 cells/mm The mean and standard error of the A positive agglutination reaction was reported with mean3 were 20 ± 3 cells/mm In 71% of these the horse serum albumin but not to test substances implantations,. the cell.3 count was between 0 and that contained rabbit serum. The presence or 24 International Journal of Toxicology/ Vol.28, No.4S,July/August2009 absence of hyaluronic acid did not change the skin-sensitizing antibodies was performed with the reaction. No adverse effects from the injections were sera of rabbits that had received streptococcal or reported. umbilical cord hyaluronic acid. Test sites were The possibility of an antigenic response to injected with 0.1 mL of each rabbit’s serum intrader hyaluronic acid derived from different sources was mally into a normal rabbit. Forty-eight hours later, 157tested. Hyaluronic acid was prepared from 2 dif each prepared site was injected with 0.01 mL of the ferent extractions from human umbilical cords and same hyaluronic acid, which had been employed to NY 5 strain of Streptococcus. The Streptococcal vaccinate the animal supplying the serum. The derived hyaluronic acid was free of protein. The results were read at 30, 45, and 60 minutes. The skin bacterial antigens were derived from 3 strains of test revealed slight erythema lasting for 2 days about group A beta hemolytic streptococci belonging to ser the injections site in one rabbit, which had received ological types 1, 4, and 14 and a strain of hemolytic Streptococcus strain 1, and in one, which had Streptococcus aureus. Young adult rabbits weighting received hyaluronic acid from S aureus. A large area between 5.5 and 6.5 kg were inoculated with the of erythema, fading gradually in 3 days, was observed antigens either by injection intravenously in the mar about the injection site in one rabbit, which had ginal ear vein, intramuscularly, or subcutaneously (n received umbilical cord hyaluronic acid, and a small 3 for each route). For the intravenous injections, area of erythema lasting I day was in another rabbit, each rabbit received 1 injection of 0.5 mL of antigen which had received the same antigen. A small raised daily for 3 days during the first week (1 mg umbilical area of induration, lasting for 2 days, and a similar cord hyaluronic acids or 1.3 mg streptococcal hya reaction, plus a small amount of erythema, was luronic acid adjusted to 1 mg after the first week). observed in 2 rabbits, which received streptococcal They were allowed to rest 4 days, then received 1 hyaluronic acid. Erythema fading in 3 days was mL daily for 3 days then allowed to rest for 4 days. observed in one rabbit, which received streptococcus This continued for 7 more weeks for a total of 27 A-i plus hyaluronic acid, and in one, which received injections except that the doses of the seventh week S aureus plus hyaluronic acid. Erythema was were administered subcutaneously in a dose of 0.5 observed at 1 hour in 2 rabbits, which received crude mL. Each rabbit was bled from the ear before treat extract plus streptococcus A-4. At 24 hours, the area ment and after the injections of the third, sixth, and of erythema was reduced, and there were small areas eighth day after the ninth week of injections. The of induration. The induration remained for several serum was separated, merthiolate added, and stored days. The results of the tests for nonprecipitating, at 4°C to 8°C. For the intramuscular and subcuta skin-sensitizing antibodies were entirely negative neous injections, the hyaluronic acid content was 1 during the 1-hour observation. The authors did not mg/mL; the dose was 0.5 mL 3 times per week for report any findings for the 24-, 48-, and 72-hour an average of 23 injections. Each rabbit was bled observations. before treatment, at 2-week intervals and 10 days The antigenicity of streptococcal-derived hya after the last injection. The sera from the rabbits in luronic acid was tested on 58rabbits) each treatment were tested for the presence of anti Streptococcal-derived hyaluronic acid was from 5 bodies against the group-specific C carbohydrate and sources (isolated by other researchers). Each rabbit for antitype-specific M protein. None of the rabbits was immunized with 1 mL (2 mg/mL isotonic saline) had any noticeable physical reactions following of streptococcal-derived hyaluronic acid emulsified injections of antigens. None of the rabbits developed with I mL Freund’s complete adjuvant. Injections precipitating antibodies against either umbilical cord were given in multiple sites (intramuscular, subcuta or streptococcal hyaluronic acid. neous, and intradermal). One month later, the rab These authors performed skin sensitization tests bits were injected again with 1 mL (1 mg/mL) on rabbits with the same hyaluronic acid prepara hyaluronic acid without adjuvant. Preimmunization tions as 57above.’ Each rabbit was skin-tested with and postimmunization sera were compared. The sera 0.1 mg in 0.1 mL of the antigen that it received: were analyzed by double diffusion studies using anti 2 types of umbilical cord hyaluronic acid or the serum to streptococcal-derived hyaluronic acid. The streptococcal hyaluronic acid. Injection sites were authors stated that the findings indicated that the observed immediately and at 24, 48, and 72 hours for rabbits formed precipitating antibodies to the strep erythema and induration. A test for nonprecipitating, tococcal hyaluronic acid, which, they speculated,

effect

was

the with

erately

admixture

nounced,

same

obtained

and men. body

hyaluronic luronic

than adsorbed with

used

ethanol jugate,

weeks

solutions hooded

pollen

complete derived

(n responses

In during

of ing,

No

anaphylaxis rooster

human

cetylpyridinium

umbilical bits

umbilical intervals was

50, cords

tained

derived crossreac may

=

the

the

In

experiments

formation

The

and

The

no

hyaluronic

received

saline.

8),

and

hyaluronic

indicate

those

experiment

evaluated

responses,

for

An

first

was

a

4

of

0.28%

or

enhanced

later,

umbilical

Lister

enhancement

proteins

(n

acid

precipitated

(2)

from

comb

500

second

from

immunogenicity

hyaluronic

(P

effect

after

ts

using

adjuvant

to

antibody

after

estimation

enhanced

of

cord

(1) systemic

=

to

experiment,

cord

used.

acid

adsorbed

obtained

Adsorption <

with

(PCA)

A1(OH) 3

jig.

hyaluronic

8).

produced

egg

4

rooster

.01)

and

of

that

to

egg

coprecipitated

rooster

hyaluronic

immunization

immunization. hyaluronic

intramuscular

acid.

acid

1

with

Passive

experiment,

Rabbit

or

hyaluronic

PCA

using

of

proteoglycan

0.57%

with

which

Hyaluronic

cord

albumen,

of

was

chloride

secondary

BNW/FU

by

the

reactive

was

by

albumen

responses

acid

hyaluronic

hyaluronic

the

(n

toAl(OH) 3

by

with

coprecipitation

birch

secondary

Enhanced, combs

reactive

of

birch and

of

hyaluronic

combs

ethanol

streptococcal

subcutaneously 1

=

rabbits.

to

tested. 16 °

comparable

sera

hemaglutination

of

acid

egg

were

following

jig

hyaluronic

preparations

of

antibodies.

9),

A1(OH) 3

acid,

acid

protein

CPC,

coprecipitated

antibodies

pollen

egg

dog

rats

pollen

purified

were

acid,

(CPC),

albumen

the

and

(10

to

antibody

acid

was

for

with

and

antibodies

were injections

from

stronger

denatured

albumen

dog

at

500

(n

of

albumin,

response

acid

acid

Hyaluronic

authors

mg/mL)

although

(4)

acid—albumin

tested

passive

500

protein.

preparations

precipitated both

=

dose human

formulas:

Hyaluronic

or

as

resulted

injected

Hyaluronic

hyaluronic

cartilage.

albumin.

secondary

obtained

jig/dose;

acid denatured

of hyaluronic hyaluronic

9),

peptides.

antigen, in

Egg

500

admixed

was

on

responses

jig/dose;

on

dog injected

sexes.

before,

(3)

guinea

(P levels

in

at

testing

against

tested

compared cutaneous

was

with

and

egg

was

umbilical

antibody less

albumen

Freund’s

observed

Acid,

antibody

4-weekly

albumin

admixed

<

in

jig/dose;

in

human

human

In

by

Three

mod

there

.00

albu

used. birch

of

anti

with

with pigs.

Rab

hya

pro

con with

acid

also acid dur con

into

was

acid

acid acid

and

the the Potassium

any

the

an

in

1)

5,

nate

None with

tained pared

represented period

immunized lower

of nized were

the exhibited The

and the chased

a before

acid—specific gens

comb inflammatory

ter neously

sodium groups

4, response.

test. 31

the

to response

hyaluronic with

(P additional dose

and

taneously prior

enhanced

injections. response

(0.044 (P tion

or

direct

Hyaluronate,

this

4.44

12,

saline

<

comb

<

Rabbits

In

anti-chicken

hyaluronic

3

resultant

preparations

4.440

had

of

that

sera to

.0

of

with

killed

of

with

protein

of

.05)

maximum

a

18,

to

of

in

No

group

1),

dog

mg/kg)

each of

purified

mg/kg.

hyaluronate, dog

coat

binding

third

the

at

the

was

a

preparation.

an

several

seen

in

to

There

the

from

the

rabbits injected

and

independent

the

the

details

detectable

those 4 mg/kg

both

Hyaluronic

hyaluronic

with

albumin

reactivity

rabbits

albumin

4

at

the

were

anti-chicken

acid

dog

enzyme-linked

injected

separate

immunization

antibody

responded

preparation

experiment,

chicken

secondary

positive

rabbits

and

heterogeneous

reactions

using

24.

The

at

week

priming

dose

the

were rats. 160

hyaluronic

assay

in

the

obtained

weeks.

albumin.

protein

optical

doses

at

were

(n

Sodium

elicited

They

induced

used

with

immunized

enhancing

the

hylan-immunized

separate

or dog

=

crude level

was

Hylan

to

response

immunized

29.

1

no

protein

Serum sites

control,

to

response.

acid

provided. or

4)

of 1

acid.

only

the

of

were

primary response

Hyaluronic

The to

albumin

after

to

injection

reactions

day

detect

given

density

Hyaluronate

protein

titer

was

3

A

the

hyaluronic

were

within

with

0.044

This

a

rooster

per

investigate

days

first

administered

G-F

Hylan

acid

immunoassay

an

a

and

heterogeneous

before

authors

samples skin

immunized

significant

crude The

preparation time

subset

prepared

only

crude

was

effect

to

with

sera

rabbit

14

immunized

antibodies.

before

was dose

20,

enhanced

All

and

were

mg/kg,

with

markedly

before suspended

response.

the

chicken

readings

sites. 160

days

in

various

of

protein,

G-F

3

of

sustained

a

from

sodium

acid

4

rooster

or

rooster I

of

independent

was

injections, acid

a

range

of

stated

administration.

second

were

the

rabbits

Becker

at

Hylan

rabbits,

used

maximization

the

dog

proteins a

and

severe

later

dog

20.31

0.44

weeks

crude

the

(1.1

with

discernible

the

hyaluronic

I

protein. 3 ’

was

and

secondary

plates

first

A

compared

collected

Two

obtained

to

doses

probably

purified.

subcuta

of

hyaluro

None

immune

that

albumin in

protein,

protein.

chicken albumin

as

et

without

booster

G-F

antigen

rabbits

rabbits

immu

mg/kg,

over

mg/kg)

simul

3

Three

a

either

0.044

al

saline

acute

injec

com

roos

anti

0,

con

pur

days

of

and

the

for

20

of

of

of 25

1,

4 a 26 International Journal of Toxicology/ Vol. 28, No. 4S, July/August 2009

Table 7. Treatment Groups of Guinea Pigs to Compare the Sensitization of Noncrosslinked (SupArtz) and Crosslinked (Synvisc)Hyaluronic6Acid’ Group Test ’ Substance Dosage (mg/kg) Concentration (mg/mL) Volume (mL/kg)

1 Synvisc 0.5 0.5 1

2 Synvisc 2.5 2.5 1 3 Synvisc + CFAa 2.5 2.5 2a

4 SupArtz 2.5 2.5 1 5 SupArtz + CFAa 2.5 2.5 2a 6 No injection NA NA NA

7 Egg albumen + CFA 1 mg/animal 2 1 mL/animal NA, not applicable. a Equal amount of complete Freund’s adjuvant (CFA) and test substance.

Hartley guinea pigs were used to compare the were treated with SupArtz and both had + grade sensitization by noncrosslinked (SupArtz, Smith and reactions. At 24 hours, 6 of the animals treated with Nephew, London, England) and crosslinked 0.5 mg/mL SupArtz had + grade reaction and 1 had a (Synvisc, Genzyme Corporation, Cambridge, MA) +++ grade reaction. Four of the animals treated hyaluronic 6acid.’ In the first part of the experi with 2.5 mg/mL Synvisc had a + grade, 1 had a ment, the guinea’ pigs (n = 8) were anesthetized with ++ grade, and 3 had — grade reactions. All 8 of the isoflurane, weighed, and subcutaneously injected animals in the 2.5 mg/mL noncrosslinked (SupArtz) with hyaluronic acid or egg albumin according to the hyaluronic acid had — grade reactions. Seven of the doses in Table 7. Injections were administered on animals in the group receiving 2.5 mg/mL noncros days 0, 7, and 14. Controls were not given injections. slinked (SupArtz) hyaluronic acid + CFA had — grade Sera were prepared from blood collected from the reactions, and 1 had a + grade reaction. Of the ani medial saphenous vein 12 days after the final sensiti mals treated with egg albumen, 6 had a ++ grade zation injection. A skin allergy test was performed on reaction, and 2 had a +++ grade reaction. Two con the sensitized guinea pigs. Fourteen days after the trol animals were treated with crosslinked (Synvisc) last sensitizing injection, each abdomen was shaved hyaluronic acid, and both had ++ grade reactions. and 2 injections of phosphate buffered saline (PBS) Two other control animals treated with noncros were given. This was followed by 2 injections of 0.1 slinked (SupArtz) hyaluronic acid had — grade reac mg hyaluronic acid sensitizing agent that the animal tions. Forty-eight hours after the injections, 4 was sensitized to or 0.01 mg egg albumen if the ani animals treated with 0.5 mg/mL noncrosslinked mal was sensitized to egg albumen. The guinea pigs (SupArtz) hyaluronic acid had — grade reactions, 3 were observed at 3, 24, and 48 hours after the injec had a +, and 1 had a ++ grade reaction. Five animals tions. Changes in skin conditions were measured and treated with 2.5 mg/mL crosslinked (Synvisc) hya reactions recorded as follows: <1 mm, —; 1.0 to 5.0 luronic acid had + grade reactions, 1 had a —, and mm, +; 5.1 to 10.0 mm, +±; 10.1 to 15.Omm,+++; 2 had ++ grade reactions. Four of the animals and >15.0 mm, ++++. At 3 hours, 7 of the 8 ani treated with 2.5 mg/mL crosslinked (Synvisc) hya mals in the group treated with 0.5 mg/mL cross- luronic acid + CFA had ++ grade reactions, 2 had linked (Synvisc) hyaluronic acid had reactions of +, and 2 had — grade reactions. All of the SupArtz ++ grade and 1 animal had a reaction of +. Six of the treated group had — grade reactions. Seven animals 8 animals treated with 2.5 mg/mL crosslinked (Syn treated with egg albumen had +++ grade reactions, visc) hyaluronic acid had reactions of ++ and 2 had and 1 had a ++++ grade reaction. Two of the non a reaction of +. Three animals treated with 2.5 mg/ sensitized animals were treated with crosslinked mL noncrosslinked (SupArtz) hyaluronic acid had — (Synvisc) hyaluronic acid; 1 had a + grade reaction, grades, 3 with + grades, and 2 animals with ++ and the other had a ++ grade reaction. Two nonsen grade reactions. Six of the animals receiving 2.5 sitized animals treated with noncrosslinked mg/mL noncrosslinked (SupArtz) hyaluronic acid + (SupArtz) hyaluronic acid had — grade reactions. CFA had + grades, and 2 of these animals had ++ Eighteen days after the final sensitizing injection, the grade reactions. Two of the nonsensitized animals animals were anesthetized, weighed, and given an

Twenty

twice

the

used

Hearing

caused

compared passive

nic

production

tested.

lactic by

hyaluronic the venous

and

1 (SupArtz) that diameter nous

animal nea

mals

albumen kg nously

crosslinked were

received and

Evans hours

injections

animals.’ 61

of form

hours veins

anaphylactic

the

acid, intravenous

acid,

5-fold stance

.9%

noncrosslinked a

Ototoxicity

acid—specific

same

Twenty

photographed. CFA).

competitive

bulla

were

(7)

nonserisitized

pigs.

that

hyaluronic injections,

to an

3

or

response

anesthetized,

after

at

of

for

dilution;

Blue

The

egg

the in

to

injection

adult

and mLikg

cutaneous

antibody

sera

test

tests

of

+

1

2-day

3

intravenous

the

what

diluted

with

received additional

each:

hyaluronic

The

production

tympanica albumen.

acid

the

in Animals

CFA-sensitized

the

mL/kg

(Synvisc)

injected

mL/kg

The

addtional

(8)

(1%

from

the

injection

pigmented medial

were symptoms

the

intradermal

the

blue

animals

noncrosslinked

in

the

(5)

caused

sera

intervals.

the

(1)

acid.

of

guinea

immunoglobins

ELISAs.

solution

titer

ototoxicity

5-,

Synvisc-sensitized

guinea

guinea

(SupArtz)

The

egg

“native”

sera

and

guinea

crosslinked

0.4

animals

performed

0.1

anaphylactic

spots

guinea

weighed,

egg

Thirty

in

symptoms.

hyaluronic

25-,

that saphenous

injection.

This

acid

guinea

on

of

was

albumen,

guinea mL/kg

were

a

a

(6)

mL

into

from

pigs

albumen

higher

then

Sprague-Dawley

the

passive

pig

125-fold

on

and

pigs

with

Five

pig;

received

animals

injections,

sera

application

form. minutes

or

pigs

Neither saline;

were

completely

their

observed

hyaluronic

the

sera

of

serum SupArtz-sensitized received

pigs

Evans

pigs

125-fold

and

observed

crosslinked

at

on

(3)

(SupArtz)

(Synvisc)

in

Hyaluronic

saline)

numbers

acid

days vein

were

hyaluronic

Hyaluronic

cutaneous

sensitized

(P matching

from

medial

response.

skin

any

did

a

dilution. were

the

(2)

that

received

and

injected

after

animals

25-fold was

=

sera

for

Blue

with

noncrosslinked

the

killed,

after

for

elicit

undiluted

was

concentration

8

animals

was

the

.0005) and

also received

(4)

acid

used

animals

again

filled

determined intradermal

the

of

guinea

3

hyaluronic

hyaluronic

saphenous

0.4

and

from

rats

measured

sensitized

(Synvisc)

repeated

hours

Acid,

sera

acid

Eighteen

hyaluro an

the

a the

dilution;

received

(without

5

anaphy

acid.’ 62

or

and

intrave

intrave

Synvisc

for

to

strong

mL/kg

mg/kg

when

1

intra

at

5

were

then

with

sub

sera

in

that

pigs

last IgG gui

ani sera per

mg/

mg/

Potassium

egg

the

for

24

a

cochlear was

middle

not

of in

that outer

exudate of group,

right a by

test in nistered

small

intact. was pigs

access similar molecular Twenty

sutured, middle

200 was

shaved 450 the

had application in

stem

cation. rial was

after

middle

observed last

the nic

of

cation. ears

teen application,

large

colorless

Hyaluronate,

the

one

the

an

hyaluronic

the

The

be

greater

or

acid,

hyaluronic

returned

not

5

tympanic

to opened

under

of

and

filled

ear

were

response

of

ear

the

hair

quantity

animals

right

overdose

to

harmful guinea

control

animal

the

In

300

ear

round

quantity.

behind

18

ears

The Five

ear

experimental

(10

in

these

toxicity

ototoxic

sensory

800

in

and

the

cavity

filled

in

application,

various

evaluated

a

cells

with

weight

animals

any

Duncin-Hartley

of

in

the

or

was

middle

iL

of

the

the

examined

control of

days

authors

by

a

right

g,

to

after

humans.

the

yellow

and

each

animals

window

pig

where

acid.

at

thresholds

membrane

groups.

new

of

as

the

of

hyaluronic

acid

caused with

to

drilling

tympanic

viscous middle

of

were

normal

filled of

tympanic

cells,

of

to

after

In

1

treated

exudate,

the a

Sodium

amounts

middle

administer of

did

pentothal

hyaluronic

right

and

ear

14

sex)

had

single hearing

the

administered

the

guinea

group

Hearing microscopically

viscous

viscous concluded

3.4

the

anesthetized.

procedure

animal’s

with

there

strongly

the

still

days,

not

There

cavity,

by

were

ear.

niche

3

inner

a

young

3

material.

a

test

ear,

ear.

months

area

Hyaluronate

months

dropped,

hole

ear,

x

the

membrane.

last

dose.

slight

were

exhibited

three

bulla.

of acid,

cause

hyaluronic

strain,

The

of

test

was

the

pigs

10’;

killed,

exudate,

group,

exudate.

and

sodium.

The

tests

was

hyaluronic ear.

another

hyaluronic

acid

but after

application

1

healthy

of

suggesting

left through

hyaluronic

had

that round

The

closed.

was

animals

had

some

amount the

was

outer

the

The after

into

destruction

after

19

authors

All

no was

nothing

weighing

were

administered

ears

but

Each

and

3

3

none,

viscous

for

left

hyaluronic

I

mg/mL),

performed.

a

auditory

sign

tympanic

perforations

and

performed

the In

animals

months. Becker

There

albino

Two

20

wounds

viscous window

the

the

large

acid

all

examined.’ 63

hair

its

of

the

their

given

One

ear

acid the

were

animal

acid

of

that

middle of

concluded

animals,

of 14

thresholds

was

and

either

last

acid

last

wall.

presence

of

between

(average

cell

hyaluro

material

was

amount exudate

et

missing

had

was Guinea

control

middle

except

month

of

it

in

brain-

giving

to

admi

had

appli

killed

mate al

appli

1 the

niche

bulla

After

were

were

may

acid

The

loss into

had was

the

the left

the

the Fif

ear

the

for

no

27

of

a

a 5 28 International Journal of Toxicology/ Vol. 28, No. 4S, July/August 2009

The hearing of 20 (10 of each sex)young, healthy change or any behavioral change were noted during albino Duncan-Hartley strain guinea pigs was tested the 3-week period after the epidural injection. One while under 64anesthesia.’ They weighed between saline injected rabbit had decreased appetite, activ 340 and 730 g. After half of the animals were tested, ity, and body weight. This animal also had wound their middle ear was slowlyfilled from the bottom via inflammation at necropsy. The hyaluronic acid group a thin catheter with 200 to 300 tL of hyaluronic acid had no pathological abnormalities by light micro (average molecular weight of 3.4 x 106; 19 mg/mL). scopy, whereas 2 rabbits in the saline group had The hearing test was readministered immediately abnormal findings (localized or diffuse meningeal and again after 28 days. The other half, the control inflammation, focal inflammation, and degenerative group, was also tested again after 28 days. The mean myelopathy in the white matter). No structural auditory thresholds shifted for the animals that were changes of neurons or glial cells were detected by administered hyaluronic acid in their middle ear. electron microscopy. The blood—brainbarrier, nuclei Histologically, 3 of the control animals had a small of neurons, nucleoplasm, cytoplasm, astrocytes, oh amount of brownish material at the stapes and/or godendrocytes, and basal lamina of capillaries were on the mucosal membrane of the right middle ear normal. The authors state that hyaluronic acid cavity, which was interpreted to a sequella of the sur administered epidurally to rabbits was not found to gery. Six of the test group animals had a small cause any sensory-motor dysfunction, behavioral amount of brown viscous fluid in a recess in the right change, or neurotoxicity by either light or electron middle ear cavity. One of these also had a small microscopy. amount of a similar fluid at the footplate of the stapes. Macroscopic examination of the middle ear Reproductive and Developmental Toxicity cavity revealed that hyaluronic acid was almost com pletely eliminated from the cavity 28 days after its Rats administration. Scanning electron microscopy revealed no morphological alterations of the hair Several similar reproductive and developmental toxi cells that could be related to the hyaluronic acid. city studies on hyaluronic acid and its salts were per Sodium hyaluronate was used as a lubricant in formed using rats. One 66study’ is described in detail the implantation of Silastic silicone rubber (Dow in this section and summarized in Table 8 with 10 Corning Corp, Midland, Michigan) electrodes with other studies. 4 platinum bands into the ears of 72cats. One ear had A multigenerational study of the effects of one drop of sodium hyaluronate instilled through the sodium hyaluronate derived from cockscombs on round window, and a drop was spread on the probes pregnant Sprague-Dawley rats and their offspring before insertion. This was repeated in the other ear was 166performed. In all cases, a 1%solution of hya with sodium chloride solution as a control. There luronic acid in physiological saline solution was were no ill effects of the procedure or of sodium hya used. After weighing, male and female 12-week-old luronate noted over the next 4 months. The effect of rats were housed together in pairs. From day 17 of sodium hyaluronate on the hearing threshold was pregnancy to day 20 after parturition, sodium hyalur inconclusive in that 2 cats had higher hearing onate was administered by subcutaneous injection in thresholds in the treatment ear, 3 cats had higher the back area to the dams at 7 mg/kg (0.7 mL/kg), 20 thresholds in the control ear, and no difference was mg/kg (2 mL/kg), or 60 mg/kg (6 mL/kg). The control detected in the sixth cat. group was administered 6 mL/kg physiological saline solution (n 21 or 22). Body weights were deter Neurotoxicity mined daily during pregnancy, and dams were exam ined on delivery day. Food consumption was The acute neurotoxicity of hyaluronic acid was tested measured on days 3, 6, 9, 12, 15, and 19 after par on 20 New Zealand White male rabbits weighing turition. Females nursed the pups for 21 days during between 2.0 and 3.0 65kg.’ An epidural injection of which the pups were observed for abnormalities 0.2 mL/kg normal saline or hyaluronic acid (Hyruan, (physical and behavioral) and weighed 3 times per LG Life Sciences, Seoul, Korea) (10 mg/mL; molecu week. The dams were killed on day 21, bled, and dis lar weight, 1100 kD; pH, 6.3-8.3) was administered sected so to observe the organs. Implantation marks (n = 10 per group). No signs of any motor or sensor in the uteri were also counted to calculate the live Table 8. Summary of Reproductive and Developmental Studies of Subcutaneously Injected Hyaluronic Acid Using Rats Study Source! Treatment Notes on F0 Notes on F1 — Notes on F2 NOAEI

Furuhasi Rooster combs/subcutaneous injection from Unabsorbed residue of hyaluronic acid at injection No neonatal abnormalities founds 60 mg/kg d et al166 day 17 of pregnancy to day 20 after sites; no effect on weight or food and water con parturition sumption; no effect on fertility Ota Rooster combs!subcutaenous injection to Unabsorbed residue of hyaluronic acid at injection Neonatal development normal; unabsorbed residue 50 mL/kg d et a1281 females from day 17 of pregnancy to day 21 sites; no effect on weight or food and water con of hyaluronic acid at injection sites; no effect on after delivery sumption; no effect on fertility; no effect found at weight or food and water consumption; no effect necropsy after delivery on fertility; no effect found at necropsy after delivery; no neonatal abnormalities found Tanaka Bacteria/subcutaneous injection 2 weeks Unabsorbed residue of hyaluronic acid at injection No neonatal abnormalities found 50 mL/kg d’ et al282 before mating and 1 week after mating sites; no effect on weight or food and water con sumption; no effect on fertility; no effect found at necropsy after delivery Tanaka Bacterialsubcutaneous injection to females Unabsorbed residue of hyaluronic acid at injection Neonatal development normal; unabsorbed residue 50 mL!kg d et al283 on days 7 to 17 of pregnancy sites; no effect on weight or food and water con of hyaluronic acid at injection sites; no effect on sumption; no effect on fertility; no effect found at weight or food and water consumption; no effect necropsy after delivery on fertility; no effect found at necropsy after delivery; no neonatal abnormalities found Ono Rooster combs/subcutaneous injection to Unabsorbed residue of hyaluronic acid at injection Neonatal development normal; unabsorbed residue 50 mL/kg d et a1284 females from day 7 to day 17 of pregnancy sites; no effect on weight or food and water con of hyaluronic acid at injection sites; no effect on sumption; no effect on fertility; no effect found at weight or food and water consumption; no effect necropsy after delivery on fertility; no effect found at necropsy after deIivery Ono Rooster combs/subcutaneous injection to Unabsorbed residue of hyaluronic acid at injection No neonatal abnormalities found 50 mL/kg d et a1285 females 9 weeks prior to copulation and sites; no effect on weight or food and water con first week of primary copulation sumption; no effect on fertility; no effect found at necropsy after delivery Ono Rooster combs/subcutaneous injection to Unabsorbed residue of hyaluronic acid at injection Neonatal development normal; reproductive ability 50 mL/kg d et al286 females from day 17 of pregnancy to day 21 sites; no effect on weight or food and water con normal; neonatal development normal after delivery sumption; no effect on fertility; no effect found at necropsy after delivery Matsuura Bacteria!intraperitoneal injection to females Unabsorbed residue of hyaluronic acid at injection No neonatal abnormalities found; neonatal devel 64 mg/kg d et a128’ day 7 to day 17 of pregnancy sites; no effect on weight or food and water con opment normal sumption; no effect on fertility; no effect found at necropsy after delivery Matsuura Bacteria/intraperitoneal injection to females Unabsorbed residue of hyaluronic acid at injection No neonatal abnormalities found; reproduction 64 mg/kg d’ et a1288 day 7 to day 17 of pregnancy sites; no effect on weight or food and water con ability normal; neonatal development normal sumption; no effect on fertility; no effect found at necropsy after delivery

(continued) L’) Table 8. (continued)

Study Source! Treatment Notes on F0 Notes on — Notes on F2 NOAEL

Hattori Not stated/daily subcutaneous injection to Unabsorbed residue of hyaluronic acid at injection No neonatal abnormalities found; reproduction 40 mg/kg d et a1289 males and females for 8 weeks during 2 sites; no effect on weight or food and water con- ability normala weeks of mating sumption; no effect on fertility; no effect found at necropsy after delivery Kumada Not stated/subcutaneous injection to females Unabsorbed residue of hyaluronic acid at injection Neonatal development normal; unabsorbed residue 40 mg/kg d et al29° from day 7 to day 17 of pregnancy sites; no effect on weight or food and water con- of hyaluronic acid at injection sites; no effect on sumption; no effect on fertility; no effect found at weight or food and water consumption; no effect necropsy after delivery on fertility; no effect found at necropsy after delivery; no neonatal abnormalities found a F2 observation not reported. birth indexes were ation. On ters

were Other and dentition (days skeletal (days ation was males reflex, dissected. and

6 the diagonal The nistered. before. viability, remaining birth. day

experimental and lower delivery. group 60 observed residue reticular made for severity gestation 20 group. sporadic case

controls

for

day

mg/kg mg/kg

During

electrical

Two-thirds

of

There

of

noted

There

kept

1

the

general number

measured

females.

killed

of

was index.

2,

16

and

Weight, was

pregnancy, of righting 10

on

measurements

The

increased

4,

were

No

After structures

at

severe

relative 3,

of

board) and There At

and

and

until (5 zone

3

(days

group,

the during

for group length,

The the

subjected at noted

3 the

was

were

other

adrenal

third and

week

of

week and

male,

litters

females

week

determined

The

shock

reduced

21

of 17). symptoms

nursing

F1 A

dams.

site

the

20th

the

pervasive

cells

reflex, remaining

10 were

sex,

on

of

no 10

was 4),

motor

treated

surviving histologically. weights were days and

no newborn

abnormalities

pregnancy

3,

of 10,

food

number

weighed, examined.

where

weeks newborn S

the 3

days

through

7 number

morbidity gland were avoidance

Fur

female).

and was

differences day the

performed and

no from

The

to

the

3

the

mg/kg

of and

nodular

higher indexes

dams

of test

included

consumption growth

other 4,

motor

with

nursing, pups

present malformations nodular 4

of

the remaining

at

of of were

3

dams

weights

grouped

effects

pups

each

avoidance

pups 7,

of fetuses for

in (rotating

hour

pregnancy. measured,

the

14), age.

group,

survivor

of or

were

On 60 Individual

14,

newborns,

macroscopic

than the

70%

aptitude males

culled

foci

were among

function

Nodular

group

were recorded were at (days

nursing.

were

heart mg/kg was

implantations,

between day

and

and

Sexual in Hyaluronic

auricle

16

foci 60 ranged

the

week

of

killed

was

(most

the

ethanol

into

pups

the

for

for

determined.

rod,

mg/kg

21,

and

reaction).

allowed opening found. the

21. weighed

from

divided

live

8 were in

on and

and

the injections

control and

test

counted.

the

body herplasia 2 through adrenal

4.

The

the

test

development

the were sex

A vertical, weeks

from cases) on were

Morbidities

dams

10 separations

birth

day

treated

every of

lungs At

differences

group.

control morbidity

the was

dissected. killed

gelatinous

Acid,

and

F1

ratio,

the

60

(cornea!

weights each

7 3

into

week

of

to

weekly

noted. gener 4 mated.

slightly

gener

group,

Three

index, litters

in mg/kg in admi

to

S

mg/kg

gland

mean

their

20th were

eyes day. Potassium were

after

12),

The

give

and and and

The

lit

and

one

the

live

the

as

7, or

of

birth with compared weight the abdominal testes, pared in compared were clymis the thymus tive ovaries groups no (P day and diagonal all ence 20,0 fetuses treated (P tions, the tions,

of sion group control, tail groups. mean implantations, of birth (P weight ber ment compared

compared the

Hyaluronate,

absolute <

dead < <

There

the control The

the

When

timing

weight

length

malformations

number of

the .05),

21 controls.

index, of

7.0 less mg/kg

.05).

.05).

in with

of

in

or

number

index, percentage

pups gestation

through

had

with placenta

were

and at

F2 and fetuses, male

skeletal (F2),

showed

There

control

the

the

and board

vaginal the

and than mg/kg

with

was

birth

with

hair, controls.

Of The

(P examining

group

with of of viability with generation, or

group, a

control

that

dams

uterus

F2

less

copulation

of

male and

the

the the <

viability

lower experimental the

relative

the

separation

controls.

no

the

were

was

of

controls.

generation, day

60.0

on number

corpora

.0

fetus examinations

compared odontiasis,

group.

length

had

and no controls. opening

the

Sodium

than

female relative

at

authors

bled

5).

live

difference

F1 administered

of

control

day 7.0 compared

maintained

weights index,

70

animals.

day

The

no

abnormalities. nor male/female

mg/kg

separation When

control

macerated

body offspring,

the

organ fetuses,

the

the

carcass mg/kg for

21,

index,

There

The differences

was

the

70

and lutea, Hyaluronate

relative of

placenta

There

and

in

of differences

or reported control

20.0

male group

to

In

or

length

F1

(P

there

newborns,

group

eye

comparing

groups of

treated

authors

weights motor longer

auricle,

external

in controls.

fertility

absolute

group

with

external

group <

the of

were

lactation

generation

was percentage

number the

at

were

opening,

.05). there of the and

mg/kg

sodium

the weight

fetuses,

(P

were

or

a sex

postnatal weight

group

had

auricle

the

less no 7.0 function

in

steeper

for no when I <

Balance and

change

(P weight, was

female

at

At

Becker

stated

no appearance

malformations

index

ratio

was

weights the .05); malformations

The

differences

control between

a a

sex

the

pups

difference mg/kg

groups

< all day

than

of

of

70

hyaluronate

higher

index,

longer

(P differences

lower

the

descent

percentage

was

on

compared .05); number

the

of

and no

implanta

treatment ‘7.0

newborns

ratio,

days,

angle

the < 21 et than

between

F1

of that

or

develop

showed

tests

control

day

on

resorp

female

al

.05).

differ

higher group epidi

of

group

mg/kg

com adhe

pups

body

than

rela when num

their

there

male

body

the

the

the the

3

live

for

the

the

31

in

of of

of

in

of

in 32 International Journal of Toxicology/ Vol.28, No. 4S, July/August2009 were no differences on day 2 or 4 for these 2 groups embryos and fetuses, and viable fetuses were and no difference at all for the 60.0 mg/kg group recorded. Body weight and placental weight of the compared with the control group. There were a live fetuses were measured. Then they were sexed higher number of pups with the appearance of and examined for external anomalies and intrathor abdominal hair on day 10 in the 20.0 mg/kg group acic and interperitoneal abnomalities. The intra (P < .05) compared with the control group but not throracic and interperitoneal organs of the fetuses with any other group or on any other day. There were were fixed, stained, and microscopically examined more pups with odontiasis in the 20.0 mg/kg group for anomalies, including dilation of the ventricle on day 11 when compared with the control group system. The skeletons were stained and examined for (P < .05); there was no difference on any other day skeletal anomalies or variations. The number of for all 3 groups. On day 17, a higher number of pups sacral and caudal vertebra with ossification was had opened their eyes in the 60.0 mg/kg group when counted as an indicator of progression of ossifica compared with the control (P < .05). There were no tion. There were no observed changes in health or differences in the other groups. The authors con general condition of the treated dams during the cluded that there were no adverse prenatal or postna pregnancy period. No miscarriages were observed. tal effects due to hyaluronic acid in rats; the NOAEL There was no body weight difference between the was reported to be 60 mg/kg. As noted in Table 8, control group and the S mL/kg d’ group throughout there was a slightly higher NOAEL in rats of 64 mgI the pregnancy. From day 15 to day 17, the mean kg d and a lower NOAEL in rats of 40 mg/kg d’. weight of the 15 mL/kg d’ group was higher than the control group (P < .05). The mean body weight gain Rabbits for day 6 to day 19 was also more than the control group (P < .05). There was no difference in mean A reproductive and developmental toxicity study was body weights for this group for the remainder of the conducted using KBL: Japanese white rabbits to pregnancy period. On day 12, the mean body weights determine the effects of SL-1010 sodium hyaluro of the 50 mL/kg d group was higher than the con nate (average molecular weight of 1.78 million, con trol group (P < .05); this continued through day 19 centration not reported) in isotonic sodium chloride (P < .01). The weight gain for day 6 to day 19 was 167solution. After 3 weeks of acclimation when the higher (P < .01) and lower from day 19 to day 28 females were 4 months old and the males were over (P < .01). There were no differences between the 5 months old, the nulliparous females and males treated and control groups during the remainder of were mated. When a pair had been observed to mate the pregnancy period. The increased body weight twice, that day was considered day 0 of pregnancy. was attributed to the unabsorbed sodium hyaluro Four groups of pregnant rabbits (n = 13 or 14) were nate accumulating in each dam; there were no differ grouped in a stratified random sampling method by ences in food consumption for the pregnancy period. body weight based on the weights on day 0 of preg There were no differences in any of the measured nancy. Sodium hyaluronic acid solution, 0.5, 15, and reproduction parameters or external anomalies in 50 mg/kg d’, was subcutaneously administered to their fetuses. There were no differences in skeletal the dams once per day from day 6 to day 18 of preg abnormalities found. There were no differences in nancy. The locations of the injections were rotated the visceral observations of fetuses. between 6 sites: the left and right sides of the neck, A developmental toxicity study was performed of chest, and lumbar. The doses were control (isotonic sodium hyaluronate using New Zealand White rab sodium chloride solution), 5 mg/kgd’, 15 mg/kgd’, 68bits.’ The rabbits were grouped into control, 8 and 50 mg/kg d’. The dams were observed for gen mg/kg d’, 20 mg/kg d’, and 50 mg/kg d’ and mated eral condition and health before and after each injec (n = 16 of each sex). In all groups but the 50 mg/kg tion. Body weight was measured on day 0, days 6 to d’ group, 13 females became pregnant. In the high 19, day 23, and day 28 of pregnancy. Food consump est dose group, 15 females became pregnant. The tion was measured every other day from day 1. The injections were made on the 6th through the 18th dams were killed on day 28 of pregnancy. The uteri day of gestation. Body weights of the dams were and ovaries were removed and the main organs measured on day 0, 6, 7, 8, 9, 12, 15, 24, and 29 observed macroscopically. The corpora lutea were of gestation. Food intake was measured every day counted. The number of implantations, dead of pregnancy. The dams were killed on the 29th day each counted. implantations, sied. of

fetuses and ities. ined no there injection In rial pared unabsorbed was was the

group. of were cental 50 there pattern, experimental anomalies

neous

ied females 50 groups old (NRD (SPF).’69 experiment).

were Based dosages pregnancy. days pituitary, killed, 20, 2 1 adrenal 18 10

gestation

of

the

of the changes

mg/kg

mg/kg

of mg/kg

The

retention,

on 15th related

not

22, observed

The pregnancy males

for

Skeletal

animal,

6 observed

4 was

pregnancy.

were with 101;

occasional weights generation 20

The

on 19th

were

to organogenesis

exsanguinated,

were 24,

groups an

and

were

abnormalities.

or

ovaries gland, Body

through site

d’, effects

d’

d1, mg/kg

in

thymus After 18 protrusion

a

molecular

indication

in

to

with were controls, Food 26,

50 56-

no

day

measured.

subacute they

each

The

sodium

and group

specimens

groups

containing

set

the not

the

retention for and

delivered

which

daily.

live

compared weight

mg/kg

before

and

(n differences

to

kidneys,

and 4

pentobarbital

d of

The

at

consumption

were

pregnant highest

paired by on general

the external group, of

separated gland,

external,

weeks =

the fetuses,

57-week-old

pregnancy

40

28

had

Body

and

the

the

days

and weight,

the which

general

d’ of around

treatment HMW

24th 17)

using

toxicity

hyaluronate and

mg/kg

of thought

and

The

number There

subcutaneously

of

increased

toxicity.

were uterus

but

authors.

dose the a

with lungs, pregnancy. authors with

uterus, condition weights the group

of

20,

and

sodium

females placental

and

gelatinous/foamy

day

and

live

necropsied. Japanese observed

the

visceral,

there

in acclimation,

1.9 conditions

d’, 50

control

the processed

22,

tested.

sodium

test

were

controls. was

sodium

for

housed to 25-

were

Hyaluronic

visceral

of

females and fetuses heart,

the

had

dead

mg/kg

authors million) of

This

interpreted were

20 The be

and

24, periphery

hyaluronate,

was body the

pregnancy

measured on

were

to of

corpora

no

solution. on

results The

removed

mg/kg increased

due weight

group. was fetuses

26,

white

between

and

rats, the

and NOAEL

8

no liver,

ovaries

measured

26-week-old individually. days d’

hyaluronate on

deaths

were

weights

of Otherwise,

placed abnormal and

The (the

mg/kg were

dams suggested

to

Acid,

and

statistical

also

animals.

12-week- the

days

necrop

groups,

d’,

the

skeletal

sponta

6

of rabbits of

of

exam spleen,

lutea,

mate on There

sexed

to

2 from were

brain, com

to

stud

28 dams

Potassium

true The were

were

and

pla

the into but was

the

6

this d’ the and test

age

day

on

be

18,

on

to

of

weighed. The each abnormalities, dead fixed in centa skeletal uted or measurements

adverse demonstrated. survival, examined

Genotoxicity Hyaluronic details tion coli hyaluronate

benzoflavone activation method (TA98,

positive Sodium

acrylamide, jig/plate, was 2-methoxy-9-acridinyl) of

in mice strain S9 1000 hyaluronate 1 were as as

(24

1%

150

Hyaluronate,

,3-propanediamine-N-(2-chloroethyl)-N

food

any

S9

the vivo Mix.

A

the

(WP2uvr

The

in

no

surviving method

animal.

to

fetuses,

hours and

mg/kg

were

(8

reverse

Mix

at

treatment

jig/plate

phosphate were injected consumption TA100,

negative

examination. increase

The

effect. the

hyaluronate weeks

positive

micronucleus genotoxicity

controls

The any

sex

125 was

in

necropsied.

(S9

and

measured.

acid

N-ethyl-N’-nitro-N-nitrosoguanidine,

(15.0 apart)

negative provided.3’

the sodium

concentration. ratios,

was

and

A)

uterus The jig/plate,

(molecular

and

(1%)

mutagenicity

induced and

fetuses

TAI

of

used

derived 2-aminoanthracene taken

Sodium

There

at

was

produced

in

fetuses.

control,

twice

group. Sodium

age).’7’ control.

were

inactive

buffer.

corpea

mL/kg),

body

surviving

the

2

535,

with

or

performed.170

and

was

differences

control

of hyaluronate, There

on

with

plates/test at

One-third

-dihydrochrolide 250 were The

mutation

were

any

test

from

sodium 2-(2-furyl)-3-

and into

Sprague-Dawley

There

weights hyaluronate

Hyaluronate

necropsy

ovaries

tested

and weight

0,

The

Phosphate

lutea,

by

in Salmonella

The

jig/plate,

or

remainder

of was

and

using TA1 test

was

no

75

fetuses

examined

phenobarbital

mutagenicity the

mitomycin

the

300 test

the

were

hyaluronate

at

experimental

teratological no

frequency that were purified 537)

mg/kg of

and absorbed

of

of

concentration. bacterial

without

31.5

The solution CD-i

other and demonstrated mg/kg

in

abdominal

maternal

the

bacterial

500

/

2.4

(5 no

could

buffer weights

were

Becker

and

did

the

removed

-nitro-2-furyl)-

was

preincubation

in

jig/plate,

fetuses

none

body typhimurium for

(7.5

jig/plate,

million)

water.

parameters

(ICR) Escherichia

C the

presence

rat

(30 -(6-chioro-

not

fermenta

in

embryos,

metabolic be

of

counted.

tests.

used

was et was

was

and

external

toxicity

absence

any sodium mL/kg),

weight

of

effects

sodium

of

attrib

al

mL/kg) livers).

groups

affect

There

from

were

cavity

in

pla male

62.5

used used

The 5,6-

the

and

and

for

was

test No

33 an

an

of 34 International Journal of Toxicology/ Vol. 28, No. 4S, July/August 2009

of the sodium hyaluronate solution. The positive results, but there was no difference in chromosomal control group received 10 mL/kg mitomycin C in dis aberrations between treatment and negative control tilled water injected once. Bone marrow cells were groups. sampled from 5 mice from each treatment group Multiple mutagenicity tests were performed on 24 hours, 48 hours, and 72 hours after the last injec HMW sodium hyaluronate (trade name NRD1O1; tion. Mice were killed by cervical dislocation and molecular weight, 1.9 73million).’ An Ames test used marrow extracted from the thigh bone using fetal calf S aureus strains TA100, TA98, TA1535, and TA1537 serum. The samples were fixed and stained. The and E coli. Sodium hyaluronate was provided by the number of blood cells with micronuclei among manufacturer (0.25%, 0.5%, and 1% aqueous solu 1000 polychromatic erythrocytes per subject and the tions) and diluted with distilled water to yield number of polychromatic erythrocytes in 1000 blood 312.5, 625, 1250, 2500, and 5000 jig/plate. Distilled cells were counted. No signs of toxicity were noted water was the negative control. The positive controls while the mice were alive, and there were no deaths were 2-aminoantracene, 9-aminoacridine hydro during the treatment period. The positive control chloride, sodium azide, and N-ethyl-N’-nitro-N produced an increase in micronuclei. There was no nitrosoguanidine disolved in dimethylsulfoxide. The difference found between the treatment groups test solutions were tested with and without meta and the control groups in regard to the number of bolic activation. Positive control groups produced polychromatic erythrocytes. expected results, but there were no differences in the A reverse mutation test using 1% sodium hyalur number of reverse mutation colonies between any onate in phosphoric acid (2 lots with molecular test culture and the negative control. weights averaging 2.12 million and 2 million) was In an in vitro chromosomal aberration test 72conducted.’ Histidine-requiring strains of S typhi using Chinese hamster lung fibroblasts, sodium murium (TA98, TA100, TA1535, and TA1537) and hyaluronate was tested at 62.5 jig/mL, 125 jig!mL, 5 tryptophan-requiring strains of E coil WP2uvrA 250 jig/mL, 500 jig/mL, and 1000 jig/mL for a treat were used. S9, fractionated from rodent livers, was ment time of 24 and 48 hours with and without used for metabolic activation. Hyaluronic acid doses metabolic 173activation. The controls were the same were 31.3 jig/plate, 62.5 jig/plate, 125 jig/plate, as above. Two hundred metaphase cells were 250 jig/plate, 500 jig/plate, and 1000 jig/plate. observed for each slide for gaps, breaks, exchanges, Isotonic sodium chloride solution was used as the and other chromosomal abnormalities. Positive con negative control. The positive control for TA98 and trol groups produced expected results, but there were TAI 00 was 2-(2-furyl)-3-( 5-nitro-2-furyl)acrylamide; no differences between the treatment groups and the N-ethyl-N’-nitro-N-nitrosoguanidine was used for negative controls. the positive control for TA1 535 and WP2uvrA, and Sodium hyaluronate was tested in a micronu 9-aminoacridine was used for TA1537. The tests cleus assay using 8-week-old ICR (Crj: CD-i) mice, were run for both 24 hours and 48 hours. There was treated with 90, 180, and 360 mg/kg once a day for no difference in the number of reverse mutations 1 day or for 4 consecutive 73days.’ Bone marrow spe between any of the test groups and the negative con cimens were collected 24, 48, and 72 hours after the trols. These authors also performed an in vitro chro final injection or the single injection after killing the mosomal aberration test using cultured Chinese mice by cervical dislocation. The number of poly hamster fibroblast cells with and without metabolic chromatic erythrocytes with micronuclei among activation. The dosages were 250 jig!mL, 500 jig! 1000 erythrocytes was counted. There were no mL, and 1000 jig/mL of 1% sodium hyaluronate. differences between any of the treatment groups and Isotonic sodium chloride solution was used as the the negative controls. negative control, and 5 jig/mL of N-ethyl-N’-nitro N-nitrosoguanidine and 20 jig/mL of benzo[a]pyrene Carcinogenicity were used as positive controls. Petri dishes received 5 mL of medium in which 4 x /mlZ cells were 310 Tumor Cells dispersed. On the third day, the test substances, positive controls, or negative controls were added. McBride and Bard used several types of cultured Incubation continued for either 24 hours or fibrosarcoma cells, lymphoblastoid cells, mammary 48 hours. Positive control groups produced expected carcinoma cells, VERO and BHK-2i cells, and Hyaluronic Acid, Potassium Hyaluronate, and Sodium Hyaluronate / Becker et at 35

Table 9. Metatstic Potential of HA-L and HA-H Cells Following Intravenous Injection of 1316-Fl Mouse Melanoma Cell Lines Into C57BL/6 6Mice” Lung Nodules/Lung

Cell Type No. of Cells lnjected(x l0) Mean ± SD Range

HA-L 2.5 3 ± 1.6 0-5 10 115 ± 35 80-150 HA-H 2.5 200 172 ± 66.5a 90->200 10 b> >200 a P < .01 compared with HA-L. b P < .05 compared with HA-L. fibroblasts from 14-day-old mouse embryos and (<0.1 6jig/lU cehls/48 hours) compared with human skin to study the hyaluronic acid halo pro mesothelial cells (10 to 72 6jig/10 cells/48 hours). duced by these 74cells.’ Cells of each type were When placed in conditioned media from the placed on cover slips or in 96-well microtiter plates mesothelioma cells, fibroblast and mesothelial cell at a density of 1 cells/cm C2Hf/Bu fibrosarcoma production of hyaluronic acid increased; a concen (FSA) cells displayed2 translucent halos ranging in tration of 50% of 10-fold concentrated conditioned diameter up to 17 .jim, averaging 8.8 jim. The halos medium in relation to culture medium (v/v) induced were not penetrated by spleen cells added to the cul a near maximal effect in mesothehial cells and 70% of ture. Motile cells had very prominent barriers around the maximal effect in fibroblasts. their trailing edge, but the halo tended to be much less The metastatic potential of tumor cells expres pronounced at their leading edge. The exclusion phe sing different levels of cell surface hyaluronic acid nomenon was seen with particles other than normal were 76examined.’ Flow cytometry was used to iso spleen cells. Halos also excluded immune spleen cells, late B16-F1 mouse melanoma cell lines expressing lymph node cells, thymocytes, normal peritoneal exu either high (HA-H) or low (HA-L) hyaluronic acid date cells, and erythrocytes. When fixedwith 1%formal on their surfaces. HA-H had approximately 32 times saline or 2.5% glutaraldehyde, the halos remained. more cell surface hyaluronic acid than HA-L cells. When dehydrated, the halos were no longer visible. After removal of hyaluronic acid from the cell sur The same results were achieved with 8 types of faces with testicular hyaluronidase, it was found that FSA cells from mice; 3 types of mammary carcinoma HA-H produced hyaluronic acid approximately 15 cells from mice; adenocarcinomas 1, TM,and 2 from times faster than HA-L; hyaluronic acid levels were mice and rats; 3T3 G and 3T3 S cells from mice, restored within 20 hours in both cell lines. The 2 cell BHK-21 hamster cells; mouse embryo fibroblasts lines had similar growth rates in vitro; when injected and adult human skin fibroblasts.’ The exception into syngeneic mice, the 2 cell lines gave rise to 74 that grew at similar rates. HA-H or HA-L was the 7 lymphoblastoid cells lines tested. Vero tumors cells also appeared not to have the protective halo. cells were injected into the tail veins of syngeneic 106 The authors note that halos <2 jim could not be C57BL/6 mice (2.5 x l0 or 1 x cells /mouse; detected. When bovine or ovine testicular hyaluroni n = 10; experiment was run 3 times). Mice were dase (10 TU/mL) was introduced, the barrier was killed 14 days later, and the lungs were fixed. Visible removed, and the spleen cells could approach the lung tumor nodules were assessed under a dissecting cell membrane. This was also the result with fungal microscope. Another set of mice were allowed to live (0.1-10 IU/mL) hyaluronidase. After removal of the to determine the mortality from HA-H and HA-L. enzyme, the cells regenerated their ability to repel The HA-H cells caused a greater number of lung spleen cells within 2 hours. metastases that were larger than the HA-L cells as The production of hyaluronic acid by 3 human shown in Table 9. Tn general, the nodules of the malignant mesothelioma cell lines (Mero-25, HA-L cells were smaller (0.1-0.5 mm) than the Mero-14, Mero-82) and 9 primary human mesothe HA-H cells (0.1-8 mm). The mortality rate was hal cell types were measured.’ The mesothelioma 50% at day 25 and day 32 for the HA-H and HA-L cell lines produced small7 amounts of hyaluronic acid cells, respectively. 36 International Journal of Toxicology/ Vol.28, No. 4S, July/August2009

Human mesothelioma cells (Mero-25) with and surrounding necrotic tissue, while the other 2 strains without transfected HAS2 gene (the gene controlling formed distinct tumors that were histologically simi the ability to produce hyaluronic acid) were corn- lar to the controls. 17/ pared. The cells were plated (5 x 104 cell/dish), The authors injected each of the above cell lines incubated for 24 hours, and then overlaid with 1 x (iO cells in 10 iL PBS) into the caudate nucleus of 7i0 formalin-fixed erythrocytes. The cells were syngeneic mice (n = 10) to induce intracranial observed through an inverted microscope and the tumors. /8 The mice were killed when they became size of the hyaluronic acid halos measured. Halos moribund1 or after 1 month; the brains were excised around the HAS2 transfected cells were larger than and frozen in embedding medium. The tumors and the halos around the unmodified cells (P < .05). The brains were sectioned for microscopic examination. HAS2 transfected cells produced 59.6, 103.8, and The experiment was repeated and extended to 187.2 4ng/10 cells/24 hours hyaluronic acid com 2 months. Unmodified SMA56O cells formed large pared with 14 4ng/10 cells/24 hours from unmodified intracranial tumors within 18 days. The mice cells. The peaks in the size range of hyaluronic acid injected with the modified cells did not result in any was 3.9 x 106 kD. Proliferation of both types of cells obvious disease within 8 weeks. Upon examination, it was compared by counting the cell number at 1, 3, 6, was observed that individual tumor cells were and 9 days after subculturing. The modified cells had observed at the injection site but no tumors. North about a 2-fold higher proliferative capacity than the ern blot analysis was performed on human gliorna unmodified cells. cell lines (D54, D270, D645, U373 MG, and U251 These authors analyzed the cell cycle profiles by MG). It was observed that U251 MG and D270 placing both types of cells in culture in starvation expressed HAS2 at high levels; U373 MG, U87 medium, centrifuged, and subjected to DNA content MG, and D645 expressed HAS2 at lower levels; and analysis and cell cycle kinetics using a flow cytometer D54 cell did not express HAS2 at all. The size of the for cell cycle 77analysis.’ After serum stimulation, hyaluronic acid halo, in general, corresponded with HAS2-transfected cells had a higher proportion of the level of HAS2 gene expression in D270 cells. cells in S phase (26.6%) compared with unmodified However, U25 1 MG cells had only a small hyaluro cells (18.9%). The cells were grown in soft agar for nic acid halo. 4 weeks, and colonies larger than 0.5 arbitrary units Balb/c nulnu male mice or AJJax mice were used were counted by microscope and their area measured to test the effects of hyaluronic acid on the growth of for a growth assay. The hyaluronic acid—producing injected LX-1 human lung carcinoma cells or TA3/St cells formed 2- to 3-fold larger colonies in soft agar 79cells.’ ALZET osmotic pumps were inserted under than unmodified cells. The shape of the colonies the skin in the dorsal region of the mice loaded with formed by the modified cells was more irregular than either PBS or hyaluronic acid (approximate molecu unmodified cells. Increased synthesis of hyaluronic lar weight, 2.5 x ;3i0 n = 5). Pumps delivered the acid was found to lead to an increased proliferation treatments at approximately 0.5 jig/U.05 jiL/h. On rate and to anchorage-independent growth in soft the day after insertion, 0.5 to 1.0 x 106 of LX-1 or agar. TA3/St tumor cells (in PBS) were injected in front The HAS2 gene was inserted into a murine astro of the pump. After 7 or 14 days of treatment, the cytoma cell line (SMAS6O) using the murine mice were killed and the tumor’s growth was mea mHAS2/pClneo 78plasmid.’ SMA56O cells and sured by weight. Hyaluronic acid inhibited LX-1 HAS2-modified SMA56O cells (3 strains) were sus tumor growth by approximately 50% to 80% and pended in PBS (106 cells/100 1iL) and injected sub TA3/St tumor growth by approximately 60% to cutaneously into the flanks of syngeneic VM/Dk 65%. In additional experiments, animals were mice (n = 10). When a tumor became palpable, it injected with LX-l cells and were untreated for was measured in 3 dimensions using calipers. When 7 days. They were then treated with hyaluronic acid the tumors reached 1 3cm the mice were killed and for 14 days. This experiment was run twice. The the tumors excised , and frozen in embedding growth inhibition was between 40% and 75%. When medium. Overexpression of HAS2 caused a reduc the treatment regime was reversed, treatment for 14 tion in tumor growth rate; the onset of tumor forma days and no treatment for 7 days, inhibition was tion was similar to unmodified cells. One strain of 68%. When treated for 7 days followed by no treat modified cells formed cysts with few tumor cells ment for 14 days, inhibition was 52%. In a soft agar Hyaluronic Acid, Potassium Hyaluronate, and Sodium Hyaluronate / Becker et al 37

Table 10. Growth Characteristics of the Transplantable 18Tumors Tumor °Grafts Transplantable Clone n ‘Weeks of Growth Wet Weight (g) Tumor Growtha

Mock 4 14.0 ± 0.8 4.2 ± 0.6 0.30 Has2-b 4 16.5 ± 2.0 4.2 ± 0.7 0.25 Has2-d 11 7.9 ± 0.5 3.4 ± 0.4 0.43 Hyall-f 3 17.3 ± 3.0 2.5 ± 1.0 0.14 Hyall-h 4 16.5 ± 2.5 2.6 ± 0.3 0.16 a Wet weight/weeks of growth.

assay, inclusion of 100 .ig/mL hyaluronic acid in the transfectants was slower (P = .029) compared with agar inhibited colony formation by LX-1 human lung mock transfectants. The authors stated that the expres carcinoma, HCT1 16 human colon carcinoma, and sion of Has2 enhanced tumor growth, whereas expres TA3/St murine mammary carcinoma cells by 80%, sion of Hyal1delayed tumor development. The amount 68%, and 72%, respectively. of hyaluronic acid in the tumors was determined by The effects of inserting hyaluronan synthase 2 microtiter-based assay. Tumors from the high hyaluro (Has2) and hyaluronidase 1 (Has 1) genes into non nic acid—producing clone contained an average of hyaluronic acid—producing rat colon carcinoma cells 150% more extractable hyaluronic acid compared with (PROb, a subclone of the cell line DHD-K12) were tumors derived from mock and Hyal1 transfectants tested.’ The genes for the synthase and the hyalur (P = .006). The mean vessel area, boundary length, °8onidase were inserted separately into the cancer cells and diameter did not differ among the tumor types using pCl-neo producing the Has2-b, Has2-d, examined, leading the authors to suggest a similar vas HyaIl-f, and Hall-h cell lines. Has2-b and Has2-d cular phenotype. The authors concluded that Has2 synthesized about 2.5 and 12 g hyaluronic acid per overexpression suppresses vascularization of the viable 1 x 106 cells/24 hours; Hyall-f and Hall-h exhibited tumor fraction. hyaluronidase activity of about 360 and 220 mU/mL, The effects of the 3 different genes controlling respectively. No hyaluronic acid or hyaluronidase hyaluronic acid synthesis on tumor cell metastasis activity was detected in the wild-type PROb cells or were 181tested. Aneuploid human breast adnocarci in mock transfected cells. Transfected cells (5 x noma cell line MDA-MB-231 was selected based 106 cells in 50 1iL PBS) were injected subcuta on its production of HAS2. Nontransformed rat cells neously into the right shoulder of BD-IX rats. The were transfected with HAS 1, 2, and 3. In the MDA subsequent tumors were measured with a caliper and MB-23 1 cell line, overexpression of each HAS iso- allowed to grow to approximately 1 cm in diameter. The form promoted the formation of a hyaluronic acid rats were killed and tumors were excised, cleaned of coat where HAS2 produced a larger matrix than nontumor tissue, and the wet weight was determined. HAS1 or HAS3. These authors investigated the The tumors were then snap-frozen in liquid nitrogen. importance of HAS2 expression in highly invasive Tumor growth was determined by dividing the wet breast cancer (MDA-MB-231) by characterization weight by the number of weeks of growth. Tumor pro of the antisense inhibition of HAS2 (ASHAS2). duction rates were different for each group as shown in ASHAS2 resulted in a 24-hour lag in proliferation Table 10. The tumors of mock transfected cells grew to that was concomitant to transient arrest of 79% of 4.2 g in 14 weeks, similar to the low hyaluronic acid— the cell population in 0G to .1G ASHAS2 did not producing Has2-b--derived tumors. The higher hya alter the expression of the other HAS isoforms, luronic acid—producing Has2-d tumors reached 3.4 g whereas hyaluronidase and the hyaluronic acid in 8 weeks. The growth rates of the Hyall-f and receptor, CD44, were downregulated. The antisense Hyal-h cells were lower compared with tumors of mock inhibition of HAS2 cells accumulated greater transfected cells; a wet weight of 2.5 g was reached amounts of HMW hyaluronic acid (>10 000 kDa) after 17 weeks. The growth rate of tumors from the in the culture medium, whereas mock and parental Has2-d transfectants was higher (P = .038), and the cells liberated less hyaluronic acid of 3 distinct growth rate of tumors from Hyali-f and Hyal1-h molecular weights (100, 400, and 3000 kDa). 38 International Journal of Toxicology/ Vol. 28, No. 4S, July/August 2009

Five-week-old CBA nude mice were used to gen transcriptase-PCR was used to detect hyaluronidases erate parental, mock, and antisense inhibition of HYAL1 and HYAL2 transcript expression. Enzyme- HAS2 181tumors. The tumor cells were harvested linked immunosorbent (ETESA)—like assay was used in the logarithmic growth phase by scraping, resus to detect hyaluronidase levels. Western blotting and 106), 1 pended (final concentration, 2 x and injected SDS-PAGE was used to detect hyaluronidase pro into the mammary fat pad of the same mice. Tumor teins. Hyaluronic acid levels were measured by growth was measured twice weekly. After 84 days, ELISA. Size profiling and purification of MIA the mice were killed and the primary tumor, liver, PaCa-2 hyaluronic acid was done by gel filtration kidneys, brain, and lungs removed at necropsy and chromatography. For the CD44 cleavage assay, MIA examined. Mice inoculated with parental or mock PaCa-2 cells were plated (5 x iOn) and cultured transfected MDA-MD-23 1 established primary overnight, then incubated with 10 jiM MG 132 for tumors with comparable growth over the experiment. 30 minutes to inhibit secondary cleavage of the Mice inoculated with ASHAS2 tranfectants did not CD44 intracellular domain. The cells were incu establish primary tumors. Metastasis in animals bated with various samples of hyaluronic acid for inoculated with parental and mock-tranfected cells 1 hour then lysed. Samples were separated by elec was most prevalent in the brain and lung but also trophoresis under reducing conditions and trans detected in kidney and liver transfectants. Mice ferred to a polyvinylidene difluoride filter. The injected with MDA-M13-231 ASHAS2 did not exhibit filter was incubated with HRP-conjugated anti- metastasis to any organs. rabbit IgG to detect the anti-CD44cyto pAb, or with Additional mice were injected in the left ventricle HRP-conjugated anti-mouse IgG to detect the with 1 x 5i0 cells.’ Animals inoculated with par anti--tubulin mAb. The secondary antibodies were ental and mock-transfected8 cells prevalent ’ had detected using ECL Western blotting detection spread of the cancer to the brain, liver, kidneys, lung, reagents. Immunofluorescence microscopy was per and bone. Mice injected with MDA-MD-231 formed. To perform a migration assay, 12-well ASHAS2 did not exhibit metastasis to any organs. Costar Transwell (Corning, Inc., Lowell, MA) cham Mice inoculated with parental or mock-transfected bers containing polycarbonate filters with a 12-jim MDA-MB-231 cells had a shorter survival period port size were used. Both sides of the filter were (72 and 77 days, respectively), compared with coated with 500 j.tg/mL 1000-kD hyaluronic acid. ASHAS2 animals (124 days; P = .000 1). The authors MIA PaCa-2 cells (2 x l0 cells/mL) were added stated that, collectively, these results strongly impli to the upper compartment and incubated for 3 hours cated the central role of HAS2 in the initiation and with or without BRIC23S or mouse IgG. Hyaluronic progression of breast cancer, potentially highlighting acid was added to the upper compartment at a final the codependency between HAS2, CD44 (hyaluro concentration of 50 jig/mL and incubated for an nic acid receptor), and hyaluronidase 2 expression. additional 15 hours. The cells on the upper side of The mechanism that human pancreatic carci the filters were wiped off. The filters were fixed in noma cells (MIA PaCa-2) use to create LMW methanol, stained, and mounted on glass slides. (—10-40 polymers) hyaluronic acid that induce Migrated cells were counted under a light micro angiogenesis, enhance CD44 (hyaluronic acid scope. The MIA PaCa-2 culture supernatant was receptor) cleavage, and promote the migration of found to contain hyaluronic acid—degrading tumor cells in a CD44-dependent manner was enzymes, which digested hyaluronic acid in a 82researched.’ MIA PaCa-2 cells show CD44 clea pH-dependent manner with the optimal pH of 4.0. vage in the absence of any exogenous stimulation at The presence of hyaluronic acid—degrading activities a readily detectable level; therefore the possibility in the culture supernatant was confirmed by a that these tumor cells may generate CD44 cleavage substrate-gel electrophoresis analysis. MIA PaCa-2 inducible hyaluronic acid oligosaccharides by expres cells expressed 2 of the known hyaluronidases, sing hyaluronic acid—degrading enzymes was tested. Hyal-1 and Hyal-2, at both the mRNA and protein To prepare the MIA-PaCa-2 culture supernatant, levels and secreted both of these proteins into the MIA PaCa-2 cells were cultivated in a flask (3 x culture supernatant. The researchers stated that 5i0 cells/flask) overnight. The culture medium was other studies noted that Hyal- 1 and Hyal-2 expres collected, centrifuged, and concentrated 50-fold. It sion was detected in a human prostate cancer cell was then filtered (0.22-jim pore filter). Reverse line LNCaP and in a human breast cancer cell line, HyaluronicAcid,PotassiumHyaluronate,and SodiumHyaluronate/ Becker et al 39

MDA-MB231, respectively. In this study, high levels of chemotherapeutic agents were added and the cells of hyaluronic acid were detected in the supernatant; incubated for an additional 72 hours. At that point, the cells generated hyaluronic acid ranging from 10 ig/mL hyaluronic acid (mixture of 3 to 8 repeating approximately 10 to 40 saccharide pairs, which are disaccharides in length) was either added or not to the similar in size to those that have been shown to medium, and the cells were incubated for another enhance Cdrr cleavage in CD44-expressing tumor 24 hours. Cells were harvested, and viable cells were cells. The addition of MIA PaCa-2 culture superna counted. The hyaluronic acid caused approximately tant induced the upregulation of CD44 cleavage in 55-fold sensitization increase in the MCF-7Adr cells MIA PaCa-2 cells, as evidenced by an increase in the but had little effect on the non-drug-resistant cells. membrane-bound 25-kD cleavage product in West The authors tested a range of concentrations for ern blotting analysis. This cleavage was strongly effectiveness and found that up to 250 ig/mL. there inhibited by the Fab fragment of the anti-C D44 neu was little or no effect of hyaluronic acid 84alone.’ In tralizing monoclonal antibody 13RIC235, indicating combination with doxorubicin, concentrations of the upregulated CD44 cleavage was because of the 10 jig/mL were effective on the drug-resistant can interaction between CD44 and its ligand. The cul cer cells. The authors repeated this experiment ture supernatant also enhanced the migration of using other drug-resistant cells and reported that MIA PaCa-2 cells in the Transwell migration assay; hyaluronic acid increased the sensitivity of cells this migration was almost completely inhibited by resistant to taxol by approximately 12-fold, 1,3- the anti-CD44 monoclonal antibody BR1C235 but bis (2-chloroethyl) -1-nitrosurea (BCNU) by approx not by mouse IgG, indicating that the enhanced imately 78-fold, and to vincristine by approximately tumor cell motility was also dependent on the 10-fold. Hyaluronic acid had little effect on the CD44—hyaluronicacid interaction. non-drug-resistant cancer cells. Hyaluronic acid treated MDA-MB23 I human mammary carcinoma Treatment cells, resistant to the folate analog methotrexate, Tumor were 133-fold more sensitive compared with non- The use of testicular hyaluronidase (PH-20) to treated cells. reduce the presence/production of hyaluronic acid The authors stimulated hyaluronic acid produc in cancer treatment was 183tested. Homozygous, tion in drug-sensitive MCF-7 cells by infection with female ICR SCID mice were injected with 5 x 106 a recombinant adenovirus driving expression of human breast carcinoma cells (MDA435) into the 84HAS2.’ In 3 runs, modified MCF-7 cells produced mammary foot pad. Hyaluronidase activity is 2.5- to 4-fold more hyaluronic acid than untreated expressed in relative turbidity reducing units cells or control cells infected with recombinant (rTRUs). The mice were injected intravenously with 3-galactosidase andenovirus. The increased hyaluro 75 rTRU hyaluronidase on days 0, 2, 4, and 6 or were nic acid production induced a 10- to 12-fold increase administered a single injection of 300 rTRU hyalur in resistance to doxorubicin, the opposite effect of onidase (day not specified). Control animals were continuous treatment with hyaluronic acid. This administered saline. Tumors were measured every experiment was repeated with emmprin, a member other day. After 4 days in both treatment regimes, the of the immunoglobulin (Ig) superfamily that is tumor volume decreased by 50; the tumors in the enriched on the surface of most malignant cancer controls continued to grow. After 1 month without cells and promotes tumor progression and regulates any further treatment, major differences continued hyaluronic acid production. MCF-7 cells infected to be observed between tumors in the treated and with recombinant emmprin adenovirus were nontreated animals. There were no apparent toxic approximately 10-fold more resistant to doxorubicin effects or changes in the behavior of the mice during treatment than controls. The effect of emmprin the experiment. was reversed by treatment with hyaluronic acid The effectiveness of the application of hyaluronic oligomers. The authors stated that this finding con acid to drug-resistant cancer cells was 84tested.’ firmed that emmprin increases drug resistance with MC F-7/Adr drug (doxorubicin)—resistant human hyaluronic acid. mammary carcinoma cells were grown in culture for The authors tested the effects of hyaluronic acid 24 hours in 24-well plates. Controls were the nonre oligomers on the phoshoinositide 3-kinase (P1 sistant parental MCF-7 cells. Various concentrations 3-kinase)/Akt cell survival pathway in MCF-7/Adr 40 International Journal of Toxicology/ Vol.28, No. 4S, July/August2009

84cells) Hyaluronic acid suppressed phosphoryla of hyaluronic acid resulted in a 5-fold reduced ability tion of Akt (protein kinase B) and stimulated of cisplatin to cause HNSCC cell death. Hyaluronic expression of protein tyrosine phosphatase acid plus anti-CD44 antibody did not inhibit tumor (PTEN) in the presence of doxorubicin, taxol, yin cell growth. For HSC-3 cells, the IC50 for cisplatin cristine, and BCNU. P1 3-kinase activity was also alone was 4 jiM and 20 jiM with hyaluronic acid. For inhibited, but there were no effects on total SCC-4 cells, the IC50 for cisplatin alone was 20 jiM levels of Akt. BAD is a member of the family of and 100 jiM with hyaluronic acid. apoptosis-regulating proteins. The authors expected Yin et 86al’ explored the inhibitory effects of hya hyaluronic acid treatment to lead to phosphorylation luronic acid with paclitaxel using tumor metastasis of BAD at serine residue 136 (BAD136), the site of and ascites formation. Female mice (strain no. 615) Akt-mediated phosphorylation. However, in MCF were inoculated IP with Ui 4 cells cervical tumor cells 7/Adr cells, there was little phosphorylation of (2.5 x 106 cells/mouse) then treated daily for 5 days BAD136 in the presence or absence of the drugs with saline, 30 mg/kg IP hyaluronic acid alone, or hyaluronic acid. 10 mg/kg IP paclitaxel alone, 30 mg/kg hyaluronic The use of hyaluronic acid in the endoscopic acid plus 10 mg/kg paclitaxel, or 15 mg/kg hyaluronic mucosal resection (EMR) procedure to remove acid plus 5 mg/kg paclitaxel (n = 10 each group). tumors was 77tested. Mice (n = 20) were injected Survival was recorded for 40 days; all mice were into the right back with hyaluronic acid (0.2 mL; killed after 40 days. This experiment was repeated 5%) or saline (0.2 mL). A wound was inflicted by except that chemotherapy was started on day 2 after using a surgical knife to remove a 5-mm circle of skin, inoculation of tumor cells and continued for 3 days. which was sutured closed. The mice treated with Nine days after IP implantation of tumor cells, the hyaluronic acid were then injected with transplanta mice were killed to observe for ascites formation. ble adenocarcinoma cell line colon 26 (1 x 106 Paclitaxel alone and both the lower and higher dose tumor cells) in 0.1 mL 0.5% hyaluronic acid. The combinations of paclitaxel and hyaluronic acid controls were injected with the same cells in 0.1 improved survival up to 40 days compared with mL PBS. Tumors were measured every 3 days. After saline control by 108.8%, 135.3%, and 135.3%, 2 weeks, the tumors were removed, weighed, respectively (P < .01). Both the combinations measured, and examined histopathologically. The improved life span when compared with paclitaxel hyaluronic acid group had larger tumors than the alone (P < .05). Hyaluronic acid alone did not controls on days 9, 12, and 14 (P .001, P = improve life span. Paclitaxel alone and both the .000 1, P .000 1, respectively). The tumor weights lower and higher dose combinations of paclitaxel and were also greater for the hyaluronic acid group on hyaluronic acid reduced ascites formation inoculated day 14 (P = .001). On day 14, the proliferating cell with U14 cervical tumor cells (P < .05). nuclear antigen-labeled index in cancer cells was These authors also implanted 7-week-old female higher in the hyaluronic acid group than in the C57BL/6 mice with Lewis lung carcinoma (LLC) control group (P = .0001). CD44 expression on the cells in the footpad (5 x 106 cells/mouse).’ After surface of the cancer cells was enhanced in the hya 12 days, the mice were anesthetized86 and the tumor- luronic acid group compared with the control group. injected foot removed surgically. The mice were vVestern blot analysis also revealed that CD44 pro grouped and treated daily for 5 days as above with tein expression was higher in the hyaluronic acid saline, paclitaxel, and/or hyaluronic acid (n = 10 per group compared with the control group. treatment group). On day 40, the mice were killed The use of hyaluronic acid in conjunction with and the lungs removed and fixed. Lung metastases cisplatin for the treatment of cisplatin-resistant head were counted and measured under microscopy. and neck squamous cell carcinoma (HNSCC) was Tumor metastases were reduced with hyaluronic 185investigated. Two cell lines were used: SCC-4 and acid alone (23.1%) and paclitaxel and hyaluronic HSC-3, both from primary oral tongue squamous acid at 1:1 (19.2%) and 1:3 (36.3%; P < .05). Blood cell carcinomas. Various doses of cisplatin along with samples were taken and analyzed for gene expres 50 jig/niL hyaluronic acid were used to treat plated sion. The combination treatment upregulated the cells (3000/plate), and the IC50 was calculated. Cis expression of vitamin 3D binding proteins, which is platin alone inhibited tumor cell growth; the addition a macrophage-stimulating activator. Hyaluronic Acid, Potassium Hyaluronate, and Sodium Hyaluronate / Beckeret al 41

Clinical Assessment of Safety decrease was concentration dependent. There was no significant change in the amount of hyaluronic Dermal Absorption acid or CD44 expression in either type of skin at any concentration. The authors concluded that hyaluro Autoradiography was used to detect the dermal nic acid does not penetrate the skin. penetration of hyaluronic acid, in the form of H]hyaluronan, using human skin.’ The authors 45 Natural Occurrence/Distribution made 2 applications of 56.3 mg and 56.4 mg of {3H]hyaluronic acid gel in a 1.8-cm area of 1 fore Some hyaluronic acid accumulates in the spleen, 2 given). This was arm (number of subjects not lymph nodes, and bone 188marrow. An adult human repeated3 12 hours later. The same was done to the male has hyaluronic acid entering general circulation opposing[ forearm with nonradioactive hyaluronic at 10 to 100 mg/24 hours.’ 89 acid gel. Seven hours after the second application, According to Fraser et al,87 hyaluronic acid is the skin was swabbed as previously described, and naturally present throughout the human body, samples were taken from the treated and control particularly in the eyes and connective tissues. It is areas with a diagnostic trephine of 3-mm diameter. produced in the peripheral tissues where most of the Adherent subcutaneous fat was removed before turnover takes place in situ. A small amount is fixation. 3H activity removed before fixation was carried by lymph to the lymph nodes where it is measured in each case. The density of the grains was metabolized.’ The kidneys extract about 10% less intense, but there was still similar aggregation of 8890but excrete only 1% to 2% in 188190urine. layer, epidermis, and clear 87 grains in the keratinized Some cells, such as chondrocytes in cartilage, penetration activity to the deeper dermis with con ”actively synthesize and catabolize hyaluronic acid centration at the level and just beneath the epidermis throughout the lifetime of the’ tissue. The authors The authors 4 when examined autoradiographically. estimated that almost a third of the total hyaluronic concluded that hyaluronic acid penetrates normal acid in the human body is metabolically removed and epidermis to accumulate at least briefly in the dermis replaced during an average day. before its disposal and degradation via known meta bolic pathways. The transit is rapid, and because Metabolism there is no inward movement of extracellular fluid at this point, its passage must be mediated by extra- In humans, hyaluronic acid has a half-life of 2.5 to cellular diffusion, active transport through the cells, 5.5 minutes in blood. The mean amount of hyaluro or combinations thereof. nic acid in blood is 30 to 40 j.tg/L.’ From the 899192 liver where Laugier et 87al’ studied the dermal penetration blood, it is mostly taken up by the of hyaluronic acid and hyaluronidase using human catabolism takes place in the” endothelial cells in the and synthetic skin. Synthetic skin and skin excised from the cadaver of a 42-year-old woman were used. Hyaluronic acid breaks down into acetate and Hyaluronidase was obtained from bacterium S hya 93lactate.’ Hyaluronicacidisbroken downbyhyaluroni luroniliticus and purified. Applications of 40 .tL dases, 3-glucuronidase,and 3-N-acetylglucosaminidase, volumes of hyaluronidase and hyaluronic acid (at or by exposure to oxygen-freeradicals. Reactive 0.001, 0.010, and 0.100 mg/mL for synthetic skin oxygenspecies produced by keratinocytes are proba and 0.01, 0.10, and 1.00 mg/mL for human skin sam blyinvolvedin the catabolism of epidermal hyaluronic ples) were placed on the skin surface. Samples were 98acid.’ incubated for 24 hours in a humidified 5% 2CO The level of hyaluronic acid in the blood is atmosphere. A punch biopsy was sampled from each increased in people with liver cirrhosis where uptake experimental area and immediately placed into an and degradation are 9920impaired.’ This is also acid-alcohol-formalin solution. Samples were fixed seen in rheumatoid arthritis89 and 202scleroderma.’ for 2 hours for the synthetic skin and 12 hours for the The half-life of hyaluronic acid99 in cartilage is cadaver skin. The amount of hyaluronic acid and normally I to 3 weeks.” Reticuloendothelial cells lin lymphatics4 actively remove almost 90% of the CD44 receptors (the cell surface protein that recog ing the ’ nizes hyaluronic acid) in human and synthetic skin hyaluronic acid before the remainder’ reaches the decreased in those treated with hyaluronidase. The vascular system. The half-life of hyaluronic acid is 42 International Journal of Toxicology/ Vol. 28, No. 4S, July/August 2009

12 hours in the skin. The cells in the dermis actively formation of a reservoir of drug in the epidermis, synthesize more hyaluronic acid than they catabo which was confirmed using autoradiography. lize, much of 205 which escapes only to be rapidly cap Lin and Maibach demonstrated that hyaluronic tured by receptors on reticuloendothelial cells in acid delivered twice the diclofenac to the epidermis lymph nodes and liver, which internalize them for over 24 hours compared with an aqueous control and subsequent catabolism in lysosomes. sodium carboxymethyl 207cellulose. Similar effects Injected hyaluronic acid and its derivatives have been found with ibuprofen, clindamycin phos undergo local 203degradation. The metabolites are phate, and 208212cyclosporin. then further catabolized by the liver into carbon diox The development of Solaraze (PharmaDerm, ide and water. Melville, NY),3% diclofenac in 2.5% hyaluronic acid gel, is used for the treatment of actinic keratosis 213 Effect on Penetration of Other Chemicals The authors stated that hyaluronic acid enhanced the partitioning of diclofenac into human skin and its Hyaluronic acid facilitates penetration of other sub retention and localization in the epidermis when stances through the human stratum corneum compared with an aqueous control, other glycosami because a hydrated epidermis is more 204permeable. noglycans (ie, chondroitin sulphate), and commonly The effects of hyaluronic acid on the in vitro diffu used pharmaceutically acceptable gelling agents (ie, sion and deposition of diclofenac within the skin sodium carboxymethyl cellulose) at either molar or were 205tested. Human skin samples were obtained rheologically equivalent concentrations. directly after abdominoplasties and surgery from male and female donors aged 30 to 50 years. Split Dermal thickness or epidermal sheet sections were used Irritation rather than full-thickness skin. The fat layer was A “negative” result of a closed skin patch test of hya removed. The skin was placed on a cork dissection luronic acid produced by fermentation was reported. board, and the epidermis was gently teased off the No details were 31provided. dermis using forceps and mounted in a Franz cell. The receptor compartment was filled with previ Immunogenicity ously sonicated Sørensen’s buffer (pH 7.0). The area of exposed skin was 2.27 2cm The applied solution General was either C-labeled diclofenac. (0.75 MBq/mg) mixed with Sørensen’s4 buffer or C-labeled diclofe Hyaluronic acid binds to monocytes and lympho cytes in inflammatory nac (0.75 MBq/mg)‘ mixed with H]hyaluronic acid diseases such as ulcerative 4 colitis and reportedly participates (4.9 Mbq/mg). As a function of time‘ after application in other inflamma tory conditions of the labeled solution, 0.5 3mL samples were as well such as rheumatoid arthritis, scleroderma, and psoriasis. 14-218 Increased removed from the sampling port[ in the receptor 202 blood chamber and replaced with pre-equilibrated buffer. levels of hyaluronic2 acid have been reported in patients Diffusion of C-labeled diclofenac in buffer with 219sepsis. Physical activity, which enhances the lymph drainage,’ also leads to a tempo reached 10% in 124 hours, while it took C-labeled 14 rary increase in the serum hyaluronic acid level. diclofenac in the ‘hyaluronic acid formula over a 217 week to reach the same level. The buffer solution Hyaluronic Acid permeated the epidermal sheet relatively rapidly in the first 100 hours so that approximately 30% was In tests for the stimulatory function of hyaluronic in the receptor chamber, then leveled off. The hya acid on polymorphonuclear leukocytes (PMNs), luronic acid solution maintained a steadier rate over 5 to 10 mg of hyaluronic acid was subcutaneously the week of the experiment, and at 100 hours, only injected into 6 healthy subjects and 10 persons with about 3% of the diclofenac had diffused through to decreased resistance to bacterial infections and the receptor chamber. Approximately 20% of the impaired phagocytic 220activity. Heparinized venous solution had diffused through the epidermal sheet blood was collected every or every other day, and the by the end of the week-long 206experiment. Brown phagocytic rate of PMN was measured. PMNs were et al showed that hyaluronic acid minimized the per stimulated in all healthy subjects. The stimulation cutaneous absorption of diclofenac, indicating the was evident 1 day after injection, maximized after 2 fever tion immunocompromised increased serum-opsonized ties increased chemiluminescence late found for papillae fragmented inflammatory stimulating

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and

synovitis

saline

5

described

AEs

Sodium

knee buffered hemolytic

study

days

and C3

scores

treatment,

AE control

treatment; of are of saline

and

knee chloride

Treatment

withdrawal

after

hyaluronic

sodium

of

(not

The

were the either acid

there

were

and

AEs from

to

4

summarized

of

(hyaluronic

received

was (n

as S group.

period.

an

(OA).

Hyaluronate

of

evaluate

subjects

pain,

treatment-related patients injection

(placebo

in immunization.

sera

groups,

acid safety defined),

milcllmoderate. =

in

sodium and

these on

reported

injection

3 was

seen

treatment activity.

as leading

arthralgia,

60 both

(0.9%; hyaluronate

detail;

347). the

Three in

being showed sufferers

respectively. and

1

treatment

The

The

no

mg

acid attributable evaluation

AEs

events in

reported

pain,

their

the

did

a

(2.9%)

groups

group).

respectively.

significant in

6

chloride

acid

in /

plasma

all

pH

hyaluronic

A

majority other

by

No such unrelated

control of Becker

to most

both

group

not

subcutaneous

Table

(>70%)

(7 saline

of

effectiveness

stiffness,

a total

non-animal- 227

were

7;

reported

group),

of

conversion

withdrawal,

significant

which

in in have

trials

decreased finish

The

worsening

Ten groups

trials

treatment n

included et

common

AEs samples

OA

patients

the

the

patients = and

vehicle

of Of 11

consid

to

of

al

differ

group)

num been of

to

172)

were

acid (the

were

hya was AEs and and

AEs hya The 513 and was

the

and

43

the

the

the

14

by in 44 International Journal of Toxicology / Vol. 28, No. 4S, July/August 2009

Table 11. United States and International Placebo-Controlled Trials Evaluating the Safety of Hyalgan, Snyvisc, and 3Supartz 1 2 No. of Study Country: Regimen Patients Results Hyalgana studies Carrabba et ’at29 Italy: placebo- and arthrocentesis-controlled, 1, 3, 100 All regimens well tolerated; AEs mild and or 5 weekly injections with a 6-month follow-up transient; no serious AEs Bragantini et at292 Italy: saline-controlled, 3 weekly injections 55 Well tolerated; no serious AEs Grecomoro et at293 Italy: vehicle-controlled, 3 weekly injections 36 Well tolerated; no reported AEs Dixon et al294 UK: 0.2 mg sodium hyaluronate—controlled, up to 63 Well tolerated; 3 reports of local joint reaction 11 injections given at 1, 2, 3, 5, 7, 9, 11, 15, 19, in Hyalgan patients and 23 weeks Dougados et at295 France: vehicle-controlled, 4 weekly injections 110 Well tolerated; no serious AEs; = control Henderson et a1296 UK: saline-controlled, 5 weekly injections 91 More poorly tolerated than placebo; local pain/swelling in 47% Hyalgan vs 22% placebo patients Corrado et a1297 Italy: buffered saline-controlled, 5 weekly injections 40 Decreased inflammatory effusion in Hyalgan patients Listrat et at298 France: no injection-controlled, 3 courses of 3 39 Well tolerated; 8 reports of injection pain weekly injections over 1 year limited to moment/few moment of injection Altman and US: saline- and nonsteroidal anti-inflammatory 456 Gastrointestinal AEs higher in naproxen 299Moskowitz drug—controlled, 5 weekly Hyalgan injections group; local injection site pain higher in Hyalgan vs control Huskisson and UK: saline-controlled, 5 weekly injections 100 Well tolerated; = control; injection site reac 30Donnelly tions similar in placebo and active groups Synvisc’ studies Wobig° et Germany: placebo-controlled, 3 weekly injections a130 110 No serious AEs; only local AEs FDAS ’ 3 Premarket US: arthrocentesis-controlled, 5 weekly injections 94 No significant differences in numbers or types Approval Applica- of AEs between Synvisc and control tion Study #5 Supartzc studies Puhl et al302 Germany: vehicle-controlled, 5 weekly injections 195 Well tolerated; no clinical abnormalities Dahlberg et al303 Sweden: vehicle-controlled, 5 weekly injections 52 Well tolerated; no serious AEs; similar injection-site pain with Supartz and control Lohmander et a1304 Sweden: vehicle-controlled, 5 weekly injections 240 Well tolerated; no serious AEs; greater sever ity of injection-site AEs in control patients (P .041) Wu et a1305 Republic of China: saline-controlled, 5 weekly 90 Well tolerated; no clinical abnormalities injections AE, adverse event. a Hyalgan - sodium hyaluronate. Fidia Pharmaceutical Corporation, Washington, DC. Approved for marketing in US in May 1997. Molecular weight, 500-730 kDa; 1% protein. b Synvisc - Hylan G-F 20. Sodium hyaluronate chemically crosslinked with formaldehyde and vinylsulfone to increase molecular weight. Biomatrix mc, Ridgefield, New Jersey. Approved for marketing in US in August 1997. 80% Molecular weight, 6000 kDa; 20% molecular weight, indeterminate; 1%protein. Supartz - sodium hyaluronate. Seikagaku Corporation, Tokyo, Japan. Approved for marketing in US in January 2001. Molecular weight, 620-1710 kDa; 1% protein.

Hyaluronic acid was tested for effectiveness at baseline and 6, 12, and 24 months. Monitoring and safety as a treatment for OA of the knee. for possible treatment-related undesirable or AEs Seventy-six patients (92 knees) with moderate to was carried out at each clinical assessment. severe OA received injections of 20 mg of sodium Seventy-two percent achieved a >50% improve hyaluronate into the knee joint (intra-articular) at ment for 1 year or longer. No systemic effects weekly intervals for 5 224weeks. There were no were noted during the follow-up period. The AEs placebos. Clinical assessments were carried out were minor and infrequent. They included brief postinjection

site, acid injections

underwent pressive symptoms evidence prior mise, chicken 5

open in fracture Previous This no and scopic tion approximately patients No inflammatory performed normal No on aspirate count fewer Gram cultures scopic of shaver 6 knees (hylan femoral 20. and case phous, ing giant-cell 5 cally

special

patients

and

months

2.

Another

abnormalities. a

patient

routine

patient

warmth

This

numerous

rare and that

peaked

capsular

injections

specimen

revealed

inflamed reduction or

Routine

staining. than

shavings <10

underwent and

9 G-F

debridement,

C-reactive

or pink

medications. in was

were a

condyle, managed

of surgical

stains its

developed

headache,

resulted

months

of

that

of

history

surgical

egg in

had

000/mm3 preserved

1 reaction

had

after approach

infection,

S preoperative

20). in

OA.

at

derivatives

found,

patient pain,

fluid-like

4

hylan

negative.

neutrophils

medications,

tissue.

the I

similar

products.

received 4

synovium

areas histological All

a

knees;

of

from trap

to evidence

and

for

Specimens

No

to

fever

last

procedures

shaved

after

in of knee

protein Each

corticosteroids

the

2

4 procedures

total

minor

5 within

and

patient

G-F and

weeks

aspirates

connected

the long-term internal

and

microorganisms in of

to

surrounding the Soft-tissue

(<10.0

days

one

No

evidence

injection histological

no

or

pain

Two

material following

final

the

was

10%

a histiocytic

patient

no

knee

Two collection

and nausea.

bone

erythematous evaluation, latter

arthroscopic

frank of

20 patient

series

per level.

bruising

at

and

48

following

evaluation

but

knees

had organisms examination

with

were

peripheral buffered

taken.22’

injection

adipose x

2 viscosupplementation on

fixation patients had

high-power

hours

because

arthroplasty

fragments,

gradually

procedures

purulence use

of

months Hyaluronic Knee

a relief

had

to l09/L).

of were

of

sections

viscosupplementa the

nonsteroidal history

collected

had underwent to

a

immunocompro

findings.

and

3

at acellular, the

of

of hyaluronic

white

the

after pain,

final knee

intra-articular

and

formalin. tissue aspriation

the

was of

noted.

tibial

an were of

had had of immunosup Six

debridement

of

reaction.

leukocytosis arthroscopic

foreign-body

There

and

hyaluronic

knee

resolved

of

of

persistent

a hylan

from allergy

Acid,

blood

injection injection. meniscus, injection. field, all

or

knees

included

minimal. swelling,

received

revealed

ongoing

with

between

patellar

contain seen plateau, negative

Chroni

arthro

arthro

cloudy

one

Use

had

joint.

amor

anti-

were

Potassium

Four

each

acid

was G-F

and

cell

use

All

in

on

to

in

at

of

a

results. lular observed hyaluronic granulomatous dase

able linked with proximity Q-Med Thirty-nine injected stabilized

connective spheres

vomiting, also There 21 mized procedure.

hyaluronic cases. 3

white dermal tion lip Duranti

26-68). from placed prepare acid (n needles.

transient. experienced ing, at

3 1, and

complained,

Hyaluronate,

studies.

days 2,

Vesicoureteral

augmentation

the

were

Tissue

digestion. material

treated Gel,

therapy

(Restylane;

4, tenderness, swelling 11)

the

vesicoureteral

women None

were

studies dextran

AB

treatment

implant

into are and

submucosally

No

and

et

under

the

or

study.

Common

treated

to

which

hyaluronic

In and

Patients

The

acid,

al24

research

acid. tissue,

extensive

gradually

Augmentation

subjects

the 8

of

This a

14

birefringent

skin.

of the

resolved

and was

immediate

the

particularly

months

with

total case

None

inflammation. these

polarized

and

treated

most

wrinkles, abdominal

suspended

Patients urinary

of ureteral

which

consists

Sodium

derived

mid-dermis

stained

first

with

and

discomfort,

There

site.

which

hyaluronic

complication

antiseptic a

lasted

of

followed

on

problems

of

Reflux

were

commonly follow-up

mean

requiring surrounded

recontouring. acid reflux

in

spontaneously. after

study,

170

158 antibiotic disappeared

the Most

their

tract

Hyaluronate AEs

were

orifice.

light the

were

with

from

folds,

provided

of

crystalline

after

treated

5

to

pain bone

patients

in

the subjects age

urinary

microspheres

Treatment

days. product 2

that

events using infections

(VUR).15 examined them

microscopy.

alcian

solutions acid

be

edema,

bacterial

were

12.5%

a

(n patients

injection. acne

of

lip

Dextranimer reported

fragments

following

gel

resolved used =

further by

prophylaxis.

were

36.8

with

gel

Thirteen

a

augmentation,

/

All with

after 4) attributed

27- for

bladder

blue,

in

scars,

lasted the

Becker

bulking

of

(34

Deflux

material

One for

were

and

patients

2 for

years

localized

were at

sources)

had Deflux,

12 or total nonanimal,

body’s

facial

hyaluroni

were

Hyaluronic

nonrando

Deflux

cases) augmenta a

time

The

as of

revealed

injection

et stain

erythema children erythema

30-gauge

less

excluded

in

implants months.

in

nausea,

patients

Inject

used micro-

(range, well al effect.

in cross-

to

acel intra

close

bruis They

0

both

were

own

was

than and

who

for

and the was

the

and

45

is

as

to

of 46 International Journal of Toxicology/ Vol.28, No. 4S, July/August2009 an intermittent swelling of the implanted material. A test on the use of stabilized hyaluronic acid for There were 7 cases of erythema, S of edema, 5 of dis dermal augmentation was 41reported. This was an comfort, 3 of tenderness, 3 of bruising, 1 of itching, open-label, 12-month study conducted at 6 sites. and 1 of pain. There was no clinical evidence of major Investigators were all experienced plastic surgeons systemic side effects nor of acute or chronic hypersen and dermatologists. A total of 216 patients were sitivity. No blood chemistry data were available. enrolled in the study, 191 female and 25 male, These authors carried out a histological study on between 23 and 76 years old. All patients received 5 volunteers (aged 26-54 years) for 52 24weeks. Prior at least I treatment, 30% received a second injection, to inclusion in the study, they were examined for skin and 17% received a third. The mean total volume of diseases and double-tested for possible sensitivity to hyaluronic acid injected was 0.32 mL, ranging up to hyaluronic acid gel and collagen. The treatment con 1.60 mL. A total of 177 patients completed the sisted of spot injections of 0.05 mL of each product 1-year study. Two of those who did not complete the at 4 sites alternating between the products on the study left because of dissatisfaction with their treat volar surface of the left and right forearm. Each per ment and 3 because of localized discomfort or reac son received a total of 8 implants. Biopsies were tions associated with treatments. The authors taken at weeks 4, 12, 24, and 52 after a physical eva stated that reactions were as expected and included luation of the sites. The biopsies were examined transient and mild erythema, itching, swelling, and blind by 2 experienced pathologists who reached a pain. Related or probably related adverse reactions consensus on each biopsy with respect to inflamma occurred in less than 2% of all treatments and tion, foreign body reaction, and fibrosis. Hyaluronic included persistent erythema, acne papule forma acid implants maintained their spot size between the tion, and ecchymotic changes (blood from ruptured 12th and 24th week. At week 52, 4 of the implants blood vessels leaking into subcutaneous tissue). No were still clearly visible under the skin. Staining for antigenic or immunogenic responses were observed. hyaluronic acid revealed the presence of such mate A prospective study on the use of hyaluronic acid rial but with a significantly more watery appearance (Restylane) for lip tissue augmentation was than at earlier biopsies. All of the specimens were 54reported.’ This hyaluronic acid product was free of fibrosis and severe foreign body reaction but injected into the upper lips of 192 women aged 24 often presented a slight inflammatory reaction. to 77 years (average age, 46 years). Of these patients, There were no differences in the presence of cells 88% had previously received collagen treatments. around each of the implants over time throughout Two percent of the patients were allergic or had some the 52 weeks of observation. adverse reactions to collagen. One patient had The use of hyaluronic acid (Restylane; stabilized Hashimoto’s thyroid disease, and 1 had rheumatoid hyaluronic acid), for tissue augmentation, was arthritis. All patients received an initial treatment. 226tested. One hundred thirteen patients (106 Second and third treatments were at the mutual dis females and 7 males) were recruited, each receiving cretion of the patient and the investigator. Treat treatment in up to 3 sites, including glabellar lines, ments were spread over 4 to 6 weeks. All patients nasolabial folds, mouth angle wrinkles, and other were anesthetized with lidocaine cream 5%, and facial lines. A total of 285 sites were treated. All endobuccal anesthesia of the troncular type was patients were monitored for at least 30 minutes after administered to the most sensitive patients (2% adre treatment for erythema, swelling, local pain, redness, naline/lidocaine). Each patient received between 0.7 itching, and tenderness. All patients were evaluated and 1.1 mL of hyaluronic acid. Swelling was noted in at weeks 0, 1, 12, and 26. Twenty patients were ran 86% of the patients during the first 24 hours and was domly chosen to come back after 52 weeks for addi noticeable at 5 days in 14% and 1% at 10 days. Red tional assessment. Additional injections were given ness was noted during the first 24 hours in 52% of to 66% of the 113 patients who were deemed to be the patients and in 36% of the patients the following in need of a “touch up.” Nineteen (6.6%) of the sites day, and 12% on the third day. There was 1 case of a showed redness, red spots, and/or swelling. Four delayed effect. At the fifth week, 1 woman experi sites (1.4%) developed dark areas. Three of these enced inflammation that disappeared within 10 days developed at week 1 and one at week 2. One patient with no treatment. The authors speculated that her reported slight pain at week 2. All of these events trip to Africa with extra exposure to the sun in the were resolved within 1 week. fourth week may have been the cause. protein were combs.227 Restylane 2 techniques reactions contain ranged lane patients. tered injection.

at These oped erythema. patients injection injections luation included

form inflammation 180 oped erythematous. Aliquots reduce tion, 24 on dermally tested from form reactions

resolved Hylaform biopsy tions

fat. after which biopsy changes

and 1

sources

patient,

the

A

These

weeks. of The

5

The

without treated,

lower

and

these 6

an

appeared

and mildly

study

with

the the

developed of time patients

from

was on

to

the and

varying

from

showed

after

abscess

No

(0.1 in into existed.

upper in

confined

authors 1

sites

for

8

Repeated

with

one Hylaform

produced and authors the

(extracted Restylane

The

with

Delayed

before

dermis original

areas induration

consistent

and

2 weeks

therefore Three

this

an

of

25

abnormal

inflammatory 438 of

prior outside tissue

the mL)

to

at

patient’s

1

injections

patients

in

between injection

an

Resolution authors

dermis

Restylane) to

the had

intradermal, patient

report.

amounts 12

in

Hylaform

the

were with

Patients forearm

a

discernable

performed

of

also after

3

showed 75

abscess

to this

patients

skin the

weeks.

patients

augmentation

treatments inflammatory normal

inflammatory

caused use

from

both of by injection

the not

years.

a

the Hylaform

with

and

firm,

skin

nasolabial

showed

nasolabial

theoretic

the performed had

Four

the

procedure. with S testing. stated

microbiologic

study

other

of

skin.

had (677

dermis of and fillers

rooster of

developed inflammation.

a

nodules in

actinic reactions

extracted injection

was

no tender, The

and reactions

required moderate 709

hyaluronic

epidermis

retrospective

patients

hyaluronin-associated

skin

hyaluronic

horizontal

1 delayed

were 7

sites.

Four tissue

women

reaction.

elastic than

that

were

and

Hyaluronic

patient.

weeks.

were

The

56

filler

achieved

risk

area

patients

and

combs) region.

reactions were

damage.

reaction.227 reactions

of

a to

referred

edematous,

271 of

They

Also,

both

induration

mild at were

performed

patient’s

for augmentation injected

from the

about (3 was

in 3-mm

subcutaneous the

inflammatory treatment

degeneration,

and infiltration

reactions.227

All

engineering

some

conducted:

with

with

These

Both

acid

with

3

sensitivity

On

tunneling

Restylane

reacted

Acid,

acid

5

products all

transient

skin observed adminis

reactions (0.42%).

32

in patients. 3

The

patients

for rooster

ranging

6

Resty

palpa devel of

punch

devel

men) weeks

Hyla

other

intra from

major

Hyla

6

reac ages

Potassium

tests

eva

and

and

and

The

the

mid

on

to to

in

of

body lymphocytes macrophages nophils nent

the ufacturer tissue hypersensitivity induration that 222 edema, no 2000, of erythema, temporary lems reports

ial a cases

patients; authors ber of authors the used luronic

augmentation cians. syringes sensitivity evaluated of and resolved Sixteen weeks ticosteroid injection delayed vascular of before cases

Skin

Hyaluronate,

few

vascular

reports

infection, reticular,

these

The

262

subcutaneous

“treatment”

Physicians of

adverse

out

in giant

1

or

testing

resolved of of there

days augmentation

lasted

preloaded

A

of resolving the

000

later,

data were

stated

multiple

acid

considered

delayed

of

cases tenderness,

injection

were

resolved reactions.

injection.

there with

with

of

total localized

in cells.

swelling,

at

of palpable

were supply

after

lower

cream.

144

were

livedoid collected

was

reactions

a

this

treatments.

2 containing the and

produced and

and

systemic

not

but

that

a

months, hyaluronic sold were

were

in

several

is

within

of

000

number Organizing

streptococcal reactions, abscess.

injection

transplant without

not doses reported syringes

Sodium

survey.

144

dermis.

acneiform fat.

plasma

seen, from

within

European the

site

the

One I

4320

granulomatous

These

by

pain,

likely

lumpiness.

Most

possible

were

recommended

reactions immediate

surveyed.229

case

reported

injection by

results

possibility

symptoms reported,

necrosis

applied

the

excessive evacuations.

abscess by 2

resolving

hemosiderin

and was

The Hyaluronate

A

Q-Med

any

patients

3 itching, sold,

All

These

secondary

were

2 weeks. examined.228

cells

between

appeared acid

of

Q-Medical

total

swelling,

company

site,

fibrosis weeks,

cases

deduced and

countries author

there

changes

sequela may treatments

infection.

these

of

adverse to

not

was

gel,

Most observed,

of in with

in

reactions. Esthetics,

site

including

the

use

cystic with

of

included the or

be

discoloration,

A

There

lasted were

34

reactions,

1999.

by

the

were

1 by was

reported

/ attributed

for 1997

doses anaphylaxes.

appeared

nose

skewed to

of

total 6

There

Becker

erythema,

pigment. lasted

to

same and/or

from

cases of few

inflammation,

the

extended

the

the

for

reactions

that

glabelar

product.

months

lesions.

most

compression

There

use

They

these

treated

these

There

there no for

were

to number

author. which

and

use

soft-tissue

of

scattered not

the person.

the localized

Fourteen

et of

redness,

use 6

were

because in

scarring. an

foreign

and

2

promi

bacter

al

weeks, 12344 found

2

of hyper

were

physi

Eosi prob man

2001.

a weeks

being num

were

were

soft-

intra

cases Two into

area

rare

after hya

and

The and

out cor

and

was

the

few

47

In

of 18

3

prognosis. 251 ’ 252

have

hyaluronic

polysaccharide

tasizing

the

angiogenic

molecular hyaluronic This

there can formed

to

and

gested

tumor. 24

and

luronic found

as

growth growth

derived acquired

shown have

interfere drug

has also

lates breast,

Increased

hal

substance been

acid; mulation postulated

acid

tumor

Elevated

48

produce

did

metastasizing

Mesothelioma

If

Increased

Increased The

LMW

cells

urine

Cancer

International

true

circulated

on

presumably

glycoprotein

cell-survival

resistance

been

been

disturbances

the

is

the

found

in

epidermal,

that progression

factor

factors. 239 ’ 24 °

and

renal

acid

rests

cell

levels

growth

that

1-245

the

hyperviscosity

may

of

levels

cells

von

with

of

weight

of

hyaluronic

hyaluronic

around

concentration

acid.

activity

to

acid. 249 ’ 25 °

HASA.

shown atherosclerotic

division

ovarian

Wilm’s

Wilm’s

shown

serum

stimulating

and

Another

to

angiogenesis. 230 ’ 23 ’

of

tumors. 247 ’ 248

result

can

be

amounts

of

Willebrand’s

nephroblastomas

in

hyaluronic

of

platelet

is

in

in

Journal

the

increase

factor,

hyaluronic

predictors

power

(2

The

vitro

signaling

basic

hyaluronic

activate

is

induce

Other

has

of

them

drug-sensitive

fetal

could

Through

of

in

to

and

tumor

x

from

cancer

and

tumor

synthesized

fetal

to

a

patients

acid

factor,

acid

blood

106

been

are

patients

be

of

of

High

fibroblast,

of

in

iow

function,

in

and cancer

blood.

attachment

inhibit

activity”

be

the

Toxicology

of

Diseases

hyaluronic

Da),

production

kidney

patients

hyaluronic

seen

patients,

acid

other

an

and

(3-12

cardiovascular

the

the

in

occurs

tumors. 231 ’ 233 ’ 234

of

acid

disease. 238

shown

the

HASA,

hyaluronic

molecular

which coagulation

acid

influence

hyaluronic

it

the

suffering

malignancy

indicator

as

growth

reportedly

serum. 247 ’ 248

to

These

levels

migration,

underlying

is

who

in

by

disaccharide

cancer

in

produce tumor

are

retains

cells

Tumor

(HASA)

have

serum

and and

with

neoplas

/

known

when

to

they

was

Wilm’s

the

Vol.

the

as

acid

associated

acid

responded

have

of

medium. 232

in

authors well

transforming

of

a

the

bone

from

reported. 246

weight, It

to

fetal

called

28,

tumor cells.’ 84 ’ 237

acid

acid

of

halo

the mimicking

hyaluronic

growth. 236

disease. 235

is hyaluronic

the

have stimulates

the

cells

synthesis,

tic

of

has

in

that

patients,

an

cause

elevated

as

produce

platelet-

No.

patients

of

a

Wilm’s

metas

gastric,

This

kidney epithe

stimu

ability serum

If

trans

levels

its

units)

of

blood

“hya effect

poor

been

been

cells

high

4S,

have

sug

with

this

the

the

the

sti

to

of

is

July/August

with

them

growtn.

utilized of

Serum during imity

that of

myeloma nostic patients

serum demonstrate cant patients

conclusions.

metastatic of luronic

times

not

types for vides

are and

cell potential

scopic to

enhancer Anttilla

luronic

cases,

cancer

E

mesothelial

HASA negligible

studied

treatment

rosette

hyaluronic

the

facilitate

serum

studies

An

Cancer

Two

In

Hyaluronic

several

hyaluronic

it

been

growth

increase breast

i

to

been

2009

of

a

cases

to

was

factors.

hyaluronic

the

hyaluronic

mucosal

discussing

increase

in

fetal

176261

acid. 48 ’ 97 ’ 255257

periosteum

et

novel,

in

studies

with

correlates

cancer

with

acid)

target

forming

by were

Wilm’s

hyaluronic

50

of

correlated

caused al

level

involvement. 255 of

noted

culture,

hyaluronic

had

tumor

have

reports

cancer

of

cells.

Patients

development

While

any

reported

Wilm’s

tumor

acid

metastases,

patients

in

breast

However,

cancers

any

independent

noticed,

acid,

accumulation

acid

for

of resection. 77

have

of

have

serum

decreased

normal

correlation

in

tumors. 17 ’

T

acid

acid

by

excision

that

the

This

collagen

hyaluronic

therapy. 234

residual

or

a

mesothelioma

Delpech

lymphocytes

and

with

in

significantly

progression

Laurent

tumor

specifically

myeloma

few

cancer

acid

with

been

joints. 260

that

relationship

is

was

facilitate

acid,

which

with

hyaluronic

is

and Increased

both

values;

26 to

of

patients,

Ponting

an

in

tumor

and

surgery

stromal

sampled

peptides

any

screened

and

no

serum

without

tumor

with

et

prognostic

researchers

and

benign

in

et

patients

promote

groups

analogous

patients

acid

activity

may

prognostic

Hyaluronic

tissue

a1 258

The al

mesothelioma,

particular

the

growth

contrast

assayed

in

inhibited

and,

arrive

a

stated

while

et

aggressiveness. 254

cell

may

levels

acid

hyaluronan

vitro. 253

that

cells

number between

levels. 252

high

from

same

and

be

found

al 259

concluded

for

and

breast

formation

with

in as

lines

stimulate

with

especially

and

the

surrounds

marker

their

at

serum

values that, a

an

situation

serum

have

after

close

speculated

57

such,

properties

compared to

value.

could

the

acid

tissue

a powerful

different

multiple

increase

majority

of

In

produce

motility

women

various

lesions

tumors

signifi

normal

except

some

When

endo

active

prog

prox

many

there

from

(hya

have

used

own

hya

pro

that

and

not

are

the

in

or

to a HyaluronicAcid, Potassium Hyaluronate, and Sodium Hyaluronate / Beckeret at 49

258(controls). Hyaluronic acid was increased in the large primary residual tumor (>2 cm; P < .03) but not sera of metastatic patients compared with nonmeta correlated with high CD44 expression. High static patients (P < .0001) as well as the control sera amounts of hyaluronic acid in cancer cells were (P < .01). The difference was not related to the num associated with poor differentiation of the tumor ber of metastatic sites. Lower concentrations of hya (P < .002). Low levels of stromal hyaluronic acid luronic acid were observed in patients after 3 months were associated with early FIGO stage (P < .008) and who were responding to chemotherapy. The initial mucinous histological type (P < .05). concentrations of hyaluronic acid had no predictive The 5-year survival of the disease decreased with value. increasing stromal hyaluronic acid levels for both The level of hyaluronic acid in the sera of 238 overall (45% vs 39% vs 26%; P = .002) and women with breast cancer, measured by radiometric recurrence-free (66% vs 56% vs 40%; P = .008) sur assay, showed no increase when compared to 120 vival. High levels of stromal hyaluronic acid were healthy women 259(controls). Predictive properties more frequent (P = .0001) in metastatic lesions than of hyaluronic acid were examined for stage of dis in primary 234tumors. ease, lymph node involvement, tumor size, histology, Samples were collected from 143 human breast and presence of estrogen or progesterone receptors carcinoma 23patients. The localization and signal in the tumors. There was no correlation with any of intensity of hyaluronic acid were analyzed in the these parameters. paraffin-embedded’ tumor samples using a biotiny The cellular expression of hyaluronic acid in 215 lated hyaluronic acid—specificprobe. In the immedi stage I to IV gastric cancer patients was measured ate peritumoral stroma, hyaluronic acid signal was using a specific biotinylated 233probe. Of the tumors, moderately or strongly increased in 39% and 56% 93% were stained for hyaluronic acid in the paren of the cases, respectively. Normal ductal epithelium chyma, and all had hyaluronic acid in the stroma showed no hyaluronic acid, but in 57% of the inside and around the tumor. I-lyaluronicacid expres tumors, at least some of the carcinoma cells were sion was compared to clinical and histological hyaluronic acid positive. Hyaluronic acid in the features of the tumors. Strong staining intensity in the malignant cells was located on the plasma membrane tumor parenchyma was associated with deep tumor in 77 of 143 (54%) cases, in the cytoplasm in 65 of invasion and with mixed types of classifications 143 (45%) cases, and in the nucleus in 21 of 143 (diffuse, intestinal, mixed, unclassified). Hyaluronic (15%) cases. The intensity of the stromal hyaluronic acid—positive cells were associated with deep tumor acid signal and the presence of cell-associated hya invasion (P < .0001), nodal metastasis (P < .07; luronic acid were both related to poor differentiation F = 4.2), positive lymphatic invasion (P < .002), poor of the tumors (P < .003), axillary lymph node positiv differentiation grade (P < .006), and inferior prog ity (P < .015), and decreased survival of the patients. nosis in univariate survival analysis (P < .002 5) (but The levels of CD44 (hyaluronic acid receptor) not with multivariate analysis). and hyaluronic acid associated with disease progres The relationship between hyaluronic acid and sion and survival of cutaneous melanoma were stud epithelial ovarian cancer tumors was 234examined. ied.231 A series of 292 clinical stage I cutaneous Samples and histories were collected from 309 melanomas were analyzed by immunohistochemistry patients with adequate archival tumor material. using an anti-CD44H antibody (clone 2C5). Hya A biotinylated affinity probe specific for hyaluronic luronic acid was demonstrated histochemically using acid was applied to histological sections of the sam a biotinylated hyaluronic acid—specificaffinity probe ples and 45 matched metastatic lesions. The staining (bHABC). CD44 was positively associated with cel was scored for the percentage of area of strong hya lular hyaluronic acid. Decreasing levels of cancer luronic acid signal (peritumoral and intratumoral cell—associated CD44 and hyaluronic acid were stroma) as iow (<35%), moderate (35%-75%), or related to increasing Breslow thickness (P < .00005 high (>75%). and P < .00 1, respectively), increasing Clark level Levels of stromal hyaluronic acid were 95(31%) (P < .00005 and P < .000 05, respectively), and low, 116 (37%) moderate, and 98 (32%) high. The increasing pT category (P < .00005 and P < high stromal hyaluronic acid level was associated .000 05, respectively); this trend was evenly distribu with poor differentiation (P < .000 5), serous histolo ted within all 3 categories. Decreasing CD44 and gical type (P < .05), advanced stage (P < .03), and hyaluronic acid levels also associated with bleeding

and

depth

antibody. method

inhibitory

were

stained sera gical

lected

Hyaluronic luronic

hyaluronic in

sican tumors was

dermal

sican

nic

nic

and hyaluronic pared

CD44 skin.

differentiated

the cinomas

squamous TPA

.001), a scans). patients

as luronic

examined antigen

mesothelioma. 262

overall

CD44

mal disease

1 Stromal

125) (P

50

decrease

other

to the

=

The

presence

acid Paraffin-embedded

The

not

acid

hyaluronic

International

lymph-vascular

irregular

determined. 264

levels

signal

Less-differentiated

from

staining

.024

18

were

manifestations

on

of

from

blood

while

was

with

mesothelioma

or

survival

In

noted

acid

using

(P

acid

tumors.

(TPA),

uses

by

had

was

immunohistochemical

hyaluronic

(BCC),

The

samples.

carcinoma

in

myometrial

ELISA.

for

3

any

and

<

cell

acid

in

and

was

more

acid

acid

22

explored

patients,

the

increasing

the

vessels

.00005

of

frequently

synthase

59

of

or

all

expression

in normal

CD44

was

11

anti-HAS

SCC

expression

P

of

carcinomas

acid

present

hyaluronic

survival

or

hyaluronic

correlation

Journal

3

the

healthy

31

cancer

3

concentration endometrial

=

patients

connective

frequent signal

avidin-biotin-peroxidase

The

serum

the

recurrence-free

positive

serum

Correspondence

Historical

in

.011,

other

acid

intensity

of

cells.

Sera

and

samples

expression

space

stable

in

situ

progressed

invasion,

cancer

normal

clinicopathological

epidermis,

of

(HAS)

of

sections

in

serum

1,

were

present

antigen

of

on

markers

P

intensity

SCC

following

Toxicology

respectively)

marker

and

postmenopausal

types

a

over

In

carcinomas

2,

in

of

acid

<

around

endometrial

acid,

HAS

with

part

(SCC)

disease

involvement,

carcinoma

BCC

.01,

was

and

better

tissue

SCCs

tissues

showed

cancer

skin.

hyaluronic

levels

cancer samples

and

blood

of

1

and

of

was

of

expression

1

on

histological the

and

resembled

to

125

levels.

not

(tissue

respectively).

was

samples.

3

was

(according

samples,

the

114

survival.

hair

/

were

ISC

serum

Peritumoral

than the

cell

was

than

CD44. 263 Vol.

63

and

stroma of

clinicopatholo

between

determined

were

tissue

cancer correlated

markers.

profiles

related J3CCs

(ISC),

patients

an basal

and

not

all

had

months

follicles

28,

nuclei;

progress

followed

polypeptide

cells

anti-CD44

stained

carcinoma BCC.

either

and

enhanced

3

immuno

acid

related

variables.

recurrent

levels Hyaluro

but

complex

was

hyaluro

women.

No.

reduced

markers

of cell

but

around

antigen

normal

and

grade,

to

to

initial

Com

(P

well-

were Stro

Var

hya

4S,

with

with

hya

var Five

col

and

and and

this car

not

the

not

CT

for

by

35

by

of

of

to

<

July/August

the

epithelial poorly

hyaluronic luronic

acid cells.

nic

demonstrated moral

cytomas,

compared regard

samples nic

hyaluronic

tumor mal

Irrespective no bladder,

creas, tomas, moral, gland,

hyaluronic well-differentiated

acid—affinity

In

acid

histology.

selected collected

and tumors

unrelated

HAS increased

gical

higher

(P 210.6

Serum CD44 in

groups.

(99.5 HAS2-

1995

HAS

the

<

hyaluronic

acid

poorly

HAS2-

The

acid

in

areas

the

was

.000

1-positive

grade,

and

Epithelial

2 2009

caecum,

samples

poorly

the

areas

expression.

infiltrating thyroid,

to

ng/mL)

in

and

was ±

differentiated

acid

and

in

hyaluronic (P

amount

The

displayed

and

gangliogliomas,

for

grade

accomplished

in

after

1);

cell

the

with

with

the

2002.

to

well-differentiated

differentiated

Histochemical

the

showed

acid

acid 48.2

acid

and

measured. 265

of

in

3.

in

and

the

the

intratumoral

colon

serum

expression

probe.

of

differentiated

.0094)

surrounding

endometrial

moderate

surfaces

tumor

acid

surgery

benign

their

showed depth

group

CD44

prostate,

the

the

of

infiltrating

than

negative.

staining

staining

The of expression

HAS3-positive

cells all

presence

lymph-vascular

ng/mL;

breast,

intense

Serum

tumors

hyaluronic

tumors),

moderate

immediate stain;

associated

hyaluronic

acid

Twenty

tumors

origin,

the

epithelial

of

or

samples

in

than

expression

areas.

of

tumors from

negative

were

myometrial

by

to

HAS3-negative

of

in

tumors.

ovary),

in

thyroid,

the

well-differentiated

A

levels

benign

stomach,

the

hyaluronic

In

localization

P

accumulation

low

cancer

evaluated.

of

areas.

the

tissue,

a

breast, HASI

total

intratumoral

of

the

the

tumor

cancer

all

samples

Carcinomas

the

positive biotinylated

<

(astrocytoma,

tumors healthy

intratumoral

benign

hyaluronic

were

HAS2

acid

levels

vicinity

HAS

or

acid

surrounding

of

of

poorly

tumor

staining

were

cells

groups

of

.0001).

20

breast,

space

areas

was

stromal

which

Highly

patients was

there

present

samples 256

different gall

stomach,

fixed patients

invasion,

1-negative

levels.

tumors

and

acid

of

were

compared

showed

for

and

more

Samples

control

higher

differentiated

of

not

cells,

bladder,

of

samples

involvement.

were

stromal

(P

was

than

for

and

had

and

and

of

from HAS3

The

hyaluronic

acid

hyaluronic

hyaluronic

area.

levels

the

aggressive

malignant

graded

related

in

(418.4 and

=

(astrocy

hyaluro

hyaluro

between

frequent

studied,

intratu

salivary

stroma.

intense salivary

histolo

intense

tumors urinary

stained

origins

peritu

human

mostly

almost

tumor

in

.0

in

astro

group

stain.

group

levels

With

were

with

were

stro were

pan

were

hya

134)

the

the

as

± to HyaluronicAcid, Potassium Hyaluronate, and Sodium HyaluronateI Becker et at 51

Hyaluronic acid levels were measured by immu difficulty and was uneventful. Within 2 hours, the noradiometric techniques in 850 patients with inva patient developed rapidly progressive painful swel sive breast 266cancer. The mean follow-up time was ling of the knee. A large effusion developed in less 55.1 months. Cytosolic hyaluronic acid levels in than 24 hours and was evaluated in the emergency tumors ranged from 4 to 59 767 mg/mg protein; the room. He had a fever of 38.9°C (102°F) and com median was 4960 mg/mg protein. Hyaluronic acid plained of severe knee pain. Arthrocentesis yielded levels were higher in younger patients (P = .0001); 100 cc of clear yellow fluid. The cell count was the levels were also higher in premenopausal women 9600 leukocytes with 27% neutrophils, 38% mono compared with postmenopausal women (P = .00 1). cytes, 25% lymphocytes, and 16%eosinophils. There Hyaluronic acid levels were higher in ductal or lobu were no crystals. Gram stain revealed leukocytes but lar histological type than other histological types no bacteria. Cultures of syriovialfluid and peripheral (colloid, medullary, papillary) (P .00 1). No associ blood were negative at 72 hours. He was first treated ation was found between hyaluronic acid intratu with naproxen but returned to the emergency room moral levels and relapse-free survival and overall 24 hours later. He then received an intra-articular survival. High hyaluronic acid intratumoral levels injection of triamcinolone hexacetonide. The swel were associated with longer relapse-free survival ling and pain improved gradually over the next 5 days (P = .01) in the subgroup of patients with ductal his and was controlled with ibuprofen. The authors tological type tumors and those (P = .01) without stated that the temporal pattern, marked synovial any type of systemic adjuvant treatment. fluid eosinophilia, and response to corticosteroid injection were all consistent with an allergic reaction. Case Reports A 54-year-old white woman underwent treat The case of a 74-year-old man who was referred for ment for facial lines and wrinkles with modified hya worsening OA of the right knee was 267reported. Pre luronic acid gel.269 She was treated in April 1998 and vious treatments included arthroscopic debride November 1998 with only mild bruising and ment, physical therapy, and intra-articular steroid erythema at the injection sites, which resolved in injection. He declined total knee replacement. 1 to 2 days. In June 1999, she again had treatments. Radiographs showed advanced OA with faint linear Two weeks later, she developed acute, multiple, ten calcifications suggestive of chondrocalcinosis. Phys der red nodules within the treatment areas. Physical ical examination showed a mild varus deformity and examination showed multiple, discrete nodules mea anterior and medial joint tenderness. There was no suring 0.5 to 1 cm in diameter in various stages of joint effusion. Three intra-articular injections of hya development. Some nodules exuded a coagulated, luronic acid (Hylan G-F 20, Synvisc) were adminis yellow, stringy material and appeared to be seconda tered weekly for 3 weeks. The first 2 injections rily infected with frank pus. Other lesions were more were uncomplicated. One week after the third injec indurated, almost fibrotic, with significant erythema tion, the patient developed a large, painful right knee but still with intact overlyingskin. A culture detected effusion. Synovial fluid analysis revealed 5300 white no pathogenic bacteria. The patient was treated with blood cells and 680 red blood cells/tL. Gram stain minocycline and methyiprednisolone. Warm saline and cultures were negative. Enzyme-linked immuno compresses were also applied. The most purulent sorbent assay did not detect antibodies to Borrelia and fibrotic nodules were injected with triamcino borgdorferi. Microscopic analysis revealed multiple lone acetonide for symptomatic relief. The symptoms intracellular rhomboid crystals typical of pseudo- rapidly cleared. However, 2 weeks later, the patient gout. The patient underwent joint aspiration and con returned with recurrent inflammation. The same servative treatment, which improved his symptoms. treatment was repeated with resolution of her Adverse effects of intra-articular hyaluronic acid symptoms. (Hylan GF-20) injections to a 46-year-old male A 53-year-old woman was described with an exu patient with moderate medial compartment OA were dative reaction that increasingly turned granuloma 268reported. He was otherwise healthy and had no tous 2 days after the injection of hyualuronic acid history of allergy to food or drugs, including eggs or (Hylaform). The peak of the eczematous reaction 27 4 6 it after a chicken. Three injections 1 week apart were per was reached after to days, and healed formed. The third injection was performed without further° 4 to 5 days.

with

fluid.

aspiration was

crystals.

improved

tion, small

not years.

90 injection.

aspirated

GF-20) thritic

flexion from white

detectable persisted,

a within

paracetamol,

crystal

72% nuclear

pain the

was

fluid was

80000 38°C routine

rated,

310 knee.

and tions popliteal

genu

been

graphs ination

tions, pain.

inflammatory

injections

73-year-old

x

52

59-year-old

10 6 /L,

x

diminish

second

Two

90

uneventful.

Tnternational

x

red

given.

severe

the

the

with

was

The

surgically

of

1

granulocytes

cell

valgum.

There

She

Before

and

effusion

changes

yielding

0 6 /L.

7 1

(100.4°F).

injection,

was

showed

was

x

0 6 /L,

showed

Two

x

cells

cultures

hyaluronic

days. blood

white

reaction

respectively.

and

patients

aspirated

white

count

cyst,

spontaneously.

yielded

Before

and

1

local

physical

had for

10 6 /L

crystals.

injection, Before

0 6 1L

knee

After

was

woman

limited.

hours

detected.

with

sterile

and

examined.

woman

the

and

At

OA

blood

She

cell

drugs,

without 20

removed

been

5

and

Journal

cell

and

osteoarthritic

crepitus

had

inflammation

white

One

no

the

pain,

less

with in

were

mL

first

the the

remained

NSAIDs

50

were

with

mL

ice the

after

The

counts

with

therapy.

acid

underwent

effusion 2

and

and

count

She

with

reduced

a

third

cell

treated

than

Radiographs

swelling

she

weeks.

week

who

mL

first

of

No

first

injection,

steroid

sterile

swelling,

injection, slight

resulted

cells

of

negative.

of

Treatment

meniscocalcinosis.

no

the

reported. 27 ’

6

White

(Hylan reactions

a

with

clear

was

count

remained

synovial

Toxicology

developed

a

years

crystal

Before

injection,

of

was fluid

white

injection,

1

were

had

crystals.

or

later,

hyaluronic

long

injection,

mL

A

sterile

with

and The

with

to

inflammatory

knee

cultures

completely

reduced

steroid

changes

right

synovial

32

within signs

blood intra-articular

right

intra-articular

prior.

of

15

of

GF-20)

was

and

in

90

blood

history

no

was the

The

second

a

10

the

82%

600

nonsteroidal

the synovial

750

/

to

showed

after

fluid

with

second

effusion.

partial

Four

erythema.

Baker’s

Vol.

x

a

severe

sterile

the

mL

effusion.

The

knee

and

aspirated.

knee

cell

identified,

Physical

intra-articular

small

she

second

first

synovial

intra-articular

x

10 6 /L

flexion,

4

polymorpho

and x cell

and

acid

fluid.

into

28,

patient

of

knee

5

I

of

hours

piroxicam,

10 6 /L

count

presented first

a

days

0 6 /L. contained

recovered

OA

was

fluid

days.

injection

left

injection

Pain

left

recovery count

synovial

No.

osteoar effusion

without

cyst

fever bilateral

synovial

without

the

(Hylan

and

Radio-

White

injec

had

exam

Knee

injec

was

Knee

injec

for

fluid

a

knee

after

aspi

with

knee 4S,

The

anti-

The

was

was

and

was

and

had

did

left

left

No

50

of

of

a 7

July/August

a

border with

horizontal

who

tests and

they

tized collagen A

body

effects sible

and

folds.

lesions, polymer The

(Restylane, and

folds,

ness for There

controlled

cosaminoglycan gel with

than after

days

and

and

developed able

order allergy

long-lasting

author rash luronic

reactions

fluid

treatment

found.

declined

biopsy

about

A

injected

A

A

A

Ten

surgical

have gel

estimate

mildly

hyaluronic

can

oral

bovine

on

prior

to

reactions.

soft-tissue

the

painful

patient

and

The

treatment

followed

reactions

2009

case case

granulomatous

before

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been

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hyaluronic

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patient removal

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ment 48

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the effects

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no

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cell

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persons

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cases and

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cell

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injected

000

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had

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reports

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healthy

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IgG-coated

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counts

1997

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1999.

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of Hyaluronate

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x

34

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/

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acid total discoloration,

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of a Hyaluronic Acid, Potassium Hyaluronate, and Sodium Hyaluronate / Becker et al 57 used in cosmetics applied to the skin should not be of 5. SheehanJK, Gardner KH, Atkins ED. Hyaluronic acid: a concern. double-helical structure in the presence of potassium at The CIR Expert Panel recognizes that there are low pH and found also with the cations ammonium, rub data gaps regarding use and concentration of these dium and caesium. J Molec Biol. l977;1 17:113-135. ingredients. However, the overall information avail 6. Tan SW, Johns MR, Greenfield PF. Hyalruonic acid: a able on the types of products in which these ingredi versatile biopolymer. AustJ Biotech. I 990;4:3 8-43. 7. Gottschalk TE, NcEwen GN Jr. International Cosmetic ents are used and at what concentrations indicate a Ingredient Dictionary and Handbook. Washington, DC; pattern of use, which was considered by the Expert CTFA; 2006. Panel in assessing safety. 8. Billek G, Billek D. Hyaluronic acid: the history of an active ingredient in cosmetics [in German]. Parfuem Kosmet. 1988;69:788-790; 792-795. Conclusion 9. Sutherland 1W. Novel and established applications of microbial polysaccharides. Trends Biotechnol. 1998; The CIR Expert Panel concluded that hyaluronic acid, 16:41-46. sodium hyaluronate, and potassium hyaluronate are 10. Laurent TC. Structure of Hyaluronic Acid. New York, safe as cosmetic ingredients in the practices of use and NY: Academic Press; 1970. concentrations as described in this safety assessment. 11. Ogston AG, The BiologicalFunctions ofthe Glycosamino glycans. New York, NY: Academic Press; 1970. 12. Comper WD, Laurent TC. Physiological function of con Financial Disclosure nective tissue polysaccharides. Phyil Rev. 1978;58:255. 13. Balazs EA, Band P. Hyaluronic acid: its structure and use. Cosmetics & Toiletries. 1984;99:65-72. The articles in this supplement were sponsored by 14. Morris ER, Reese DA, Welsh EJ. Conformation and the Cosmetic Ingredient Review. The Cosmetic dynamic interactions in hyaluronate solutions. J Molec Ingredient Review Program is financially supported Biol. 1980;138:383. by the Personal Care Products Council. 15. Cleland RL, Wang JL, Detweiler DM. Polyelectrolyte properties of sodium hyaluronate. 2: potentiometric titration of hyaluronic acid. Macromolecules J. 1982; Conflicts of Interest 15:386-395. 16. Balazs EA, Laurent TC. Viscosity function of hyaluronic No potential conflict of interest relevant to this arti acid as a polyelectrolyte.J Polym Sci. 195 l;6:665-667. cle was reported. F. Alan Andersen, PhD, and Lillian 17. Cleland RL. Comparison of polyelectrolyte behaviour of C. Becker are employed by the Cosmetic Ingredient hyaluronate with that of carboxymethyl cellulose. Biopo lymers. 1968;6:15l9-l529. Review. 18. Scott JE, Cummings C, Brass A, Chen Y. Secondary and tertiary structures of hyaluronan in aqueous solu tion, investigated by rotary shadowing-electron micro scopy and computer simulation. Biochem J. 1991 ;274: References 699-705. 19. Scott JE. Secondary and tertiary structures of hyaluro Unpublished sources cited in this report are available for nan in aqueous solution: some biological consequences. review from the Director, Cosmetic Ingredient Review, 1998. Available at: http://www.glucoforum.gr.jp/science/ 1101 17th St. Suite 412, Washington, DC 20036, USA. hyaluronan/HAO2/HAO2E.html. Accessed February 21, 1. Balazs EA, Laurent TC, Howe AF, Varga L. Irradiation of 2005. mucopolysaccharides with ultraviolet light and electrons. 20. Lower E. Heralding hyaluronic acid. Soap Peifum RadRes. 1959;ll:149. Cosmet. 1998;7l:41-42. 2. Laurent TC. Biochemistry of hyaluronan. Acta Otolaryn 21. Denlinger JL, Balazs EA. Replacement of the liquid vitr gol. 1987;Suppl 442:7-24. eous with sodium hyaluronate in monkeys. I: short-term 3. Laurent TC, Laurent UBG, Fraser JRE. Functions of hya evaluation. Exp Eye Res. 1980;3l:80-99. luronan. Ann Rheum Dis. 1995;54:429-432. 22. Milas M, Rinaudo M, Fouissac E. Molecular weight and 4. Hascall VC, Laurent TC. Hyaluronan: structure and rheotological measurements of sodium hyaluronate. physical properties. 1997. Available at: http://www. Cosmetics & Toiletries. l993;108:57-63. glycoforum.gr.ip/science/hyaluronan/HAO l/HAO 1E.html. 23. Cleland RL, Stoomiller AC, Roden L, Laurent TC. Par Accessed February 21, 2005. tial characterization of reaction products formed by the 58 International Journal of Toxicology/ Vol. 28, No. 4S, July/August 2009

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