Hyaluronans: ls Ctinicat Effectiveness - Crespine Gel

Hyaluronans: ls Ctinicat Effectiveness
Dependent on Motecular Weight?
Peter C. Vitanzo, Jr, MD, and Brian J. Sennett, MD
Abstract
The original rationale for viscosupplementation with
hyaluronans was fluid replacement, suggesting that the
most viscous materials {eg, those of highest molecular
weight [MW] would provide the most clinical benefits'
Howevei it has become clear that mechanisms of action
for osteoarthritis pain management are not only mechanical
but also biological. After intra-articular injection,
hyaluronans exert a range of biological actions within the
joint. Although high- and low- to mid-MW hyaluronans
't'rut not hyaluronans

Hyaluronans: ls Clinical Effectiveness Dependent on Molecular Weight?
in clinical efficacy, as shown by outcoraes such as pain
relief. Data from recent clinical studies carried out in
patients receiving a low- to mid-M.V/ product have suggested
that intra-articular hyaluronan therapy has diseasemodifying
activity and may alter progression of
osteoarthritis.2l-25 Jhess issues are discussed in more detail
later in this reyiew
Pnoposro Mecuaursrrrs 0F AcroN
OF HYÂLURONAN
Hyaluronans are responsible for elastoviscosity of synovial
fluid in joints, and elastoviscosity is higher with high-MV/
hyaluronans. This elastoviscosity is shear-dependent,
meaning that the mechanical behavior of hyaluronan
changes with the shear force applied and the speed of flow:
Hyaluronan soiutions behave as viscous fluids when external
forces move them at low speeds but behave as elastic
bodies when subjected to high rates of shear or frequency.
Therefore, hyaluronans are effective as lubricants under
conditions of siow movement but are effective as shock
absorbers when movement is rapid.a
Clearly, this elastoviscous behavior will determine clinical
activity to a large extent, but the pharmacokinetic profile
of hyal*ronans does not support a purely mechanical
role for these substances. During movement, hyaluronan
flows into ihe lymphatic system of the joint capsule, rnovcs
info fhe general circulation, and is ultimately taken up by
the }iver, where it is degraded to water and carbon dioxide.26
Duration of the therapeutic effect of hyaluronan, as
noted in clinical studies, is also not consistent with a pure-
1y mechanical role, as treafnent benef,ts can persist for
several months after a course of injections is completed,
whereas the elimination halflife of hyaluronan in the joint
is measured in hours to days.27,28
Of additional interest is the reported increase in the elastoviscous
properlies of synovial fluid after exogenous
administration of hyaluronic acid. Mensitieri and
Ambrosio2e examined synovial fluid from the knee joints
of patients subjected to arthrocentesis or treatment with
hyaluronan compounds of MWs ranging from 500 kDa to
730 kDa. The elastoviscosity of synovial fluid from
patients who received hyaluronan was increased relative to
pretreatment values, as well as to values in patients who
had undergone arlhrocentesis. This increase in elastoviscosity
was present 1 week after administration, well after
the exogenous product would have been expected to have
been cleared from thejoint space, suggesting that the elastoviscosity
of knee synovial fluid benefits from increases
422 The American Journal of Orthopedicso
in both hyaluronic acid concentration and MW
Other potential pharmacologic mechanisms may be
receptor-mediated. These mechanisms include inhibition
of inflammatory mediators, inhibition of phagocytic cell
function, stimulation of cartilage matrix synthesis, and
decreased degradation of carlilage 6stria. 17'30-39
These and other potential mechanisms, and their association
with MW, investigated primarily in vitro, are summarrzed
n Tâble IL17,30-39 Results from in vitro evaluation of
the biological actions of hyaluronaas show that both highand
low- to mid-MW hyaluronans are more or less active
depending on the specific effect examined. For example,
mid-MW hyaluronans (500*999 kDa) appear to inhibit
apoptosis more,35 offer more protection against cartilage
1oss,17 sn6 suppress synovial cell proliferation moreoll'17
whereas high-MW hyaluronans are more effective in
inhibiting prostaglandin EZ and arachidonic acid activities.37'38
Both agents are similar with regard to effects on
leukocyte chemotaxis.3o Although an "ideal" MW rangr
should be related to the biological profile that will optimizàt
clinical benefit for the patient, it is not well understood
which of these actions are most meaningful regarding clinical
benefits, such as pain relief or potential structure (disease)
modification. Potential disease-modifying activities
demonstrated in animal models of osteoarthritis (Table I)
may provide a more meaningful picture of the benefit of
low- to mid-MW hyaluronans. Even in this case, however,
the activity may be distinct from the outcome of pain relief.
In a review, Ghosh and Guidolinls concluded that, based on
overall preclinical and clinical data, low- to mid-MW
hyaluronans used clinically for osteoarthritis may indeed
have a more beneficial sffucture- or disease-modifying pro-
Gl-
RruroNsHrps BETwEEN MoLEcULAR WETcHT
OF HYALURONAN AND ETTIcncy
In the United States, hyaluronan products marketed for
treatment of osteoarthriiis of the knee include Hyalgar
(sodium hyaluronate; sanofi-aventis, Bridgewater, NJ),v
Synvisc (hylan G-F 2A; Genzyme Corporation,
Cambridge, Mass), Supartz (sodium hyaluronate; Smith &
Nephew, Memphis, Tenn), Euflexxa (sodium hyaluronate;
Ferring Pharmaceuticals, Suffem, NY), and Orthovisc
(high-MW hyaluronan; DePuy Mitek, Raynham, Mass).
Characteristics of these preparations are summarized in
Table III.
Molecular Weight and Half-Life
Exogenous hyaluronan'begins to leave the joint within 2
hours of injection, though some remains up to 3 days (this
is particularly true of high-MW products). Synvisc remains
up to 3 days,a0 whereas Hyalgan,al Supafiz,az Euflexxa,43
and Orthoviscaa remain less than 24 hours. Although the
high-MW hylan B in Synvisc has a half-life of some
weeks,26 it is biologically inert (not in soiution to engage
receptors). Results from a study involving 1311-rad'olabeling
showed that hyaluronan is eliminated from the human

Asari et altt
Ghosh et all2
Ghosh et all3
Adamla
Ghosh et alls
Yoshimi et all6 Rabbit
Kikuchi et al7 Rabbit
Shimizu et alrT Rabbit
Table l. Preclinical Studies of lntra-Articular Hyaluronan {HA}
Preparations in Animal Models of Osteoarthritis {OA)*
OA lnduction Method
ACLT, then HA x 5 wk (starting 4 wk PO)
UTMM, then HA or saline x 5 wk
(stading 4 mo PO)
UTMM, then HA or saline x 5 wk
(starting 4 mo PO)
TBLM, then HA or saline x 5 wk
(starting 4 mo PO and after SF aspiration)
PM, then HA or saline immediately
PO Zx/wk x 2-4 wk
Seikagaku (840 kDa or
2300 kDa)
Seikagaku (840 kDa);
Nippon Roussel
(2000 kDa)
Seikagaku (BaO kDa);
Nippon Roussel
(2000 kDa)
Seikagaku (840 kDa
or 2300 kDa)
Female sheeo TBLM, then HA or saline x 5 wk Fidia (50&-730 kDa);
(starting 4 mo PO and after SF aspiration) Pharmacia-Upjohn
{s00M900 kDa)
ACLT, then single-dose HA or saline Hikarl (2020 kDa
or 950 kDa)
Seikagaku (840 kDa
or 1900 kDa)
P. C, Mtanzo and B. J, Sennett
Pathology { significantly
more with 840 kDa than
with 2300 kDa. Synovial
penetration of 840 kDa >
2300 kDa
Loading of operated joints I
and radiologic scores I with
HAs vs saline. Histologic
scores for MTPC worse with
2000 kDa
PG synthesis in MIPC of
operated joints ! with HA vs
placebo, but PG release I
SF HA MW (determined by
multiangle laser light-scattering
photometry) 1 at 5 wk
PO with both HAs, but
mean t with 840 kDa >
2300 kDa
SF storage modulus and
viscosity at 5 wk PO t vs
saline with both HAs,
lmprovement with 500-730 kDa
> 3000-6900 kDa
Both HAs protected against
histologic cartilage loss.
Nonsignificant trend toward
greater efficacy with 2020
kDa
Cartilage histology
suggested grealer
protection with 840 kDa
than with 1900 kDa or saline
Proiective effect in cartilage
and synovium: higher MW
> MW 500-730 kDa.
Suppression of synovial cell
proliferation: 840 kDa >
larger HAs.
"lnvestigations shown are those in which HAs with differing l\4ws were compared directly. Compounds were âdministered intra-afiicularly in all studies. MW indicales
molecular weighi; ACLI anterior cruciate ligament transeclion; PO, postoperatively; UTMM, unilateral total medial meniscectomy; MTPC, medial tibial plâteau cartilages;
PG, proteoglycan; TBLM, total bilaleral lateral meniscectomy; SF, synoviâl fluid; PM, partial meniscectomy.
knee joint in 3 distinct phases (half-lives of 1.5 hours, 1.5
days, and 4 weeks) fitting a 3-erponent function.a5 In this
study, conducted with a high-MW-nonanimal stabilized
hyaluronic acid product (NASHA), it was suggested that
the rapid initial phase was linked to elimination of low-
MW fragments and the second phase to elimination of
high-MW labeled hyaluronan. During the slow third phase,
the daily decline in radioactivity was comparable to that in
the urine, indicating a slow release of hyaluronan or degradation
products from the ge1 in &e knee and subsequent
excretion by the kidneys. The possibility of uptake of
NASHA and its products by the synovial layer and
ACLT, then HA x 5 wk (starting 4 wk PO) Fidiâ (500 kDa);
Seikagaku (840 kDa);
Pharmacia {3600 kDa);
Biomairix {6000 kDa)
popliteal lyrnph nodes was also postulated.as Results from
another study showed increased clearance of hyaluronan
with osteoarthritis but no increase in normal joints.a0 The
elimination half-life of hyaluronan increased to 23.5 hours
as osteoarthritis developed but fell ta l'1.4 hours by the
time the disease was fully established.
lflolecutar Weight and Tissue Penetration
The discrepancy between retention in thejoint and duration
of clinical effecl may be attributable, at least in part, to
enhanced penetration of low- to mid-MV/ hyaluronans into
the extracellular matrices of the synoyial tissues and per-
September 2006 423

Hyaiuronans: ls Clinical Effectiveness Dependent on Molecular Weight?
!1. Molecular Weight and Effect" on BiologicalActivity of Hyaluronan
Biological Activity Low {6000)
haps the cartilage. There is some evidence-from a canine
osteoarthritis model evaluating increased expression of
prostaglandin E2, thickening of synovial lining layers, and
vacuolar changes in lining cells, together with fluorescein
staining of hyaluronan-that low- to mid-M\Y hyaluronans
(ie, 840 kDa) penetrale diseased tissues more effectively
than high-MW hyaluronans (ie, 230û kDa). Pathologic
changes suggested more accessibility of synovial tssues to
the 840-kDa product.ll However, there remains no direct
evidence that this phenomenon correlates with or explains
a clinical benefit.
lrtolecular l#eight and Elastoviscosity
Increasing the mean MW of hyaluronan clearly increases
the elastoviscous properties of the solution.a Although this
result would be advantageous if the mechanism of pain
relief were only mechanical, it seems counterintuitive
when biological mechanisms are involved. It does not fo1low
that côllulâr activation of the precisely regulated
hyaluronans and other associated pathways would occur in
response to a higher concentration and higher than normal
M'W of hyaluronan in the knee. Within the cartilage matrix,
struçtural propeflies of the exffacelluiar rnatrix ars deter-
424 The American Journal of Orthopedics@
Genzyme Corporation
B0% hylan A (6000),
rnined by aggregates of endogenous hyaluronan and proteoglycans
that include aggrecan and link proteins.4T
Hyaluronan MW seems to be determined by the binding of
elongating nascent hyaluronan to a receptor. Saturation of
these receptors may stall elongation of the nascent hyaluro- V
***r*r/
{€,q! {{}*ll
gK{*l
XûJlsd
DePuy Mitek
1 000-2900
HA
d
i:CELL
MOLICI Lt3
fening Pharmaceuticd
2400-3600
Figure. In vitro synthesis of hyaluronan by synovial fibroblasts
is inlluenced by eoncentration and molecular weight (MW) of
êxogenous hyaluronan {HA). The signal for synthesis is provided
by HA in the MW range of 500 lo 4000 kDa in a concentration-dependent
manner. Figure adapted with permission from
Smith MM, Ghosh P. The synthesis ol hyaluronic acid by
human synovial fibroblasts is influenced by the nature of the
hyaluronate in the extracellular environment. Rheumatol lnt.
1987:7:113-122
\
tAS 1&ô c f*t{ s,Xlô8 l&t
1 I
J

nan chains, resulting in an intracellular response that
affects both hyaluronan and proteogiycan synthesis.
Although an efficacy advantage has beea claimed for
products of a MW similar to that of normal, healthy synovial
fluid, analysis of the clinical evidence has not shown
a correlation between MW and degree of improvement or
duration of efficacy.as
Molecular Weight and Receptor Binding
It is thought that hyaluronan concentration is monitored by
synovial fibroblasts and that homeostasis is maintained
through specific cell-surface receptors. These receptors
have also been shown in vitro to be activated by exogenous
hyaluronan, with maximal responses being elicired by
exogenous hyaluronans of certain size ranges.36 In one
study, investigators found the most marked response by
synovial fibroblasts from an osteoarthritic joint exposed io
hyaluronan of MW >5t0 kDa, whereas smaller molecules
had little or no effect.36 The investigators also found that a
Jirigh-MW hyaluronan (4700 kDa) was less effecrive in
stimulating synthesis than a compound of MW 3800 kDa.
They thought this finding indicates that synovial fibroblasts
do not increase synthesis of endogenous hyaluronans in the
presence of functionally acceptable (ie, very high MW or
high concentration) hyaluronans.
Study results have shown that hyaluronan binding at the
cell surface is a complex interplay of multivalent binding
events affected by the size of the multivalent hyaluronan
ligand, the density of cell-surface CD44, and the CD44
activation state.4e The most significant feature dislinguishing
CD44 from other hyaluronaa-binding proteins is that,
with CD44, binding to hyaluronan takes place at the cell
surface, where multiple, closely arrayed CD44 receptor
molecules interact with the highly multivalent repeating
disaccharide chain of hyaluronan. The actual afËnity of a
single CD44*hyaluronan binding domain for hyaluronan is
likely to be very low. Thus, binding of a CD44-positive cell
to a soluble hyaluronan molecule must involve multiple
s/vveuç receptor*ligaad interactions, dependent on the relative
valency of the native hyaluronan molecule. In high-
MW hyaluronan polymers, each molecule interacts with
more than one receptor*binding site, and this increases the
likelihood that the molecule will remain bound to the
receptor, thereby increasing the receptor aftnity (avidity)
of the molecule. Beyond a certain limit, howevet, very
large hyaluronan molecules will be less efficient in engaging
multiple receptors because of steric trindrance-hence
the bimodal nature of many biological activities. This suggests
that the maximal response would be produced by
hyaluronans within a specific size range (neither too big
nor too small). The existence of isoforms of the hyaluronan
receptors may also contribute to differential binding.
However, these ideas remain speculative at this time, and the
exact mechanism of receptor binding is not known. Either
the response may be proportional to the number of receptors
bound, or it may be an "all-or-nothing" phenomenon that
depends on the simultaneous binding of 2 or more recep-
P. C. Vitanzo and B. J. Sennett
tors.18 In addition, biological activation is also balanced with
"bioavailabiiity," the ability to penetrate iato diseased tissues.
Again, larger hyaluronan molecules may be less ef[cient
in penetrating into the synovium (see Figure).tt
ClrNlcal Ernclcy
There have been several reviews1,5O-sz and meta-analyses53'54
on the clinical efficacy of intra-articular hyaluronans
in the treatment of pain associated with osteoarthritis-including,
most recently, publication by the Cochrane
Collaboration of the most comprehensive meta-analysis of
this class.5s However, there have been no reports of any
well-controlled, randomized clinical studies directly comparing
hyaluronans across a wide range of MWs
(500-6000 kDa). Several clinical studies have compared 2
or more hyaluronans of differing MWs. Wobig and col-
1eagues56 reported on a study fhat was said to compare
hylan G-F 20 (MVi =>60û0 kDa) with sodium hyaluronate
(MV/ = 800 kDa) and find the high-MW hyaluronan superior.
However, it was subsequently noted thaT the original
study was a 4-arm trial that also included another sodium
hyaluronate (MW = 2300 kDa) and a control group of a
degraded, nonelastoviscous hylan G-F 20 preparation.sT In
the total analysis, it was shown that none of the hyaluroîans
was statistically different from the control group. A
retrospective clinical practice reporl compared hylan G-F
20 with sodium hyaluronate (MW = 615 kDa) and found
that, for both products, a1l patients repofied a statistical
improvemenl in pain after treatment.s8 The report further
suggested fhat, compared with sodium hyaluronate, hylan
G-F 20 had a superior response in many parameters.
However, it was subsequently reported thar significant limits
in the scope and design of the study called its conclusions
into question.se
Three recent prospective studies from clinical practice
also compared the clinical efficacy of hylan G-F 20 (MW
-6000 kDa) with that of sodium hyaluronate (MW
500-73û kDa): Brown and colleagues60 (N = 54) reported
no evidence for a benefit of either product over the other,
and Garcia6l (N = 51) and Richardson and colleagues62 (N
= 90) reported in 2 separate studies that the short-term efficacy
of hylan G-F 20 (MW = 6000 kDa) and sodium
hyaluronate (MV/ 5û0*730 kDa) could not be distinguished.
However, a1l 3 reports documeated a product-specific
pseudoseptic reactioa to hylan G-F 20-also termed
severe acute inflammatory reaction {SAIR). None of these
studies reported any SAIRs to the sodium hyaluronate
injections. Incidence of SAIRs in these reports ranged kom
5.3Vo ta -24.77a of the patients injected. Similarly,
Hamburger and colleagues63 reported that in clinical practice
study af 171 patients, 5 weekly injections of sodium
hyaluronate O,{W 500*730 kDa) had a mean duration of
benefit of 447 days, whereas 3 weekly injections ofhylan
G-F 20 had a mean duration of 370 days (P = .018 in favor
of sodium hyaluronate). F*rthermore, incidence of SAIRs
was 16.3Vo with hylan G-F 20 versus 07o for sodium
hyaluronate (P

Hyaluronans: ls Clinical Ëffectiveness Dependent on Molecular Weight?
Karlsson and colleagues6a recently reported on a multicenîer,
randomized, saline-controlled study comparing the
clinical efficacy of hylan G-F 20 (MW * 6û00 kDa) or
sodium hyaluronate (MW - 890 kDa) with that of an intraarticular
saline control. Patienis with knee osteoarthritis
treated with injection of hyaluronan or saline showed clinical
improvement during the frst 26 weeks of trealment,
though nei*rer hyaluronan preparalion demonstrated any
significant difference from t}le saline control. Collecrively,
the limited clinical trial evidence suggests {hat there is no
defînitive difference in efficacy (sympiomatic relief)
between hyaluronans ranging in MV/ from 5Û0 kDa to
6000 kD4 though there seem to be clear differences in
safety profiles and other clinically relevant benefils (eg,
disease-modifuing) in several studies.
Kirshner and Marshall compared the safety and effectiveness
of sodium hyaluronate (MW 24æ-3600-kDa) prepared
by biological fermentation wilh those of avian-derived
hyaluronan ftylan G-F 20 (MW * 6û00 kDa). The effectiveness
was not inferior aad lhere was a significantly higher
incidence of post-injection effusions in hylan G-F 20.65
This conclusion was reiterated in the American College
of Rbeumatology 2000 treatment guidelines,o6 in which it
was recommended that intra-articular hyaluronan therapy
be considered a possible therapy for knee osteoarthritis:
"To date, differences in clinical efficacy between these
preparations fHyalgan and Synvisc] as a function of
molecular weight have not been demonstrated." In agreement,
the Cochrane meta-analysis concluded that no evidence
would lead one ts conclude that hyaiuronan products
differ in pain-relieving efficacy, though it was
acknowledged that few well-designed, head-to-head studies
have been conducted.5s
426 The American Journal of Orthopedics@
there have been several case reports of patients having a
SAIR to hylan G-F 2A ard subsequently being treated with
sodium hyaluronate with good clinical results and without
further sequelae,Tl adding further support for a hylan G-F
20*specific reaction.
Antibodies to hylan have been noted in the sera of some
patients.Tl In addition, the immunologic response may
progress to more serious conditions such as granulomaious
reactions. Cases of hylan G-F 20-related granulomatous
reactions have been reported in patients who had knee
osteoarthritis treaied with intra-articular injections of this
agent.71 The reactions take the form of chronic inflarnmation
of the perisynovial area and surrounding tissues and
are shown by histologic evaluation to include histiocytic
and foreign body giant cells. A recent report also characterized
this response as a pseudosatcama.T2 A recent animal
study comparing the biocompatibility of sodium
hyaluronate and saline with hylan G-F 20 has confirmed
these clinical observations.T3 After intradermal injection in
guinea pigs and intramuscular injection in rabbits, hylan G- V
F 20 induced definitive macroscopic changes in guinea
pigs by day 14 and in rabbits by day 28. Severe granulomatous
inflammation in guinea pigs and acute inflammation
with minimal infiltration of macrophages and foreign
body giant cel1s in rabbits were seen on histologic assessment.
Furtbermore, speciflc antibodies against hylan G-F
20 were demonstrated in guinea pigs by passive cutaneous
anaphylaxis, and substantial deposits of immunoglobulin G
on hylan G-F 20 were evident by immunohistochemistry.T3
Couct-ustoNs
Data on intra-articular hyaluronan therapy for osteoarthritis
pain-which have been accumulating for almost 2
ReurroNsHrp BETwEEN MoLEcULAR WEtcHT
AND TOLERABILITY
decades-have shown rhat the original (1980s) hyporhesis
of a link between hyaluroaan MW and duration of efficacy
is not bome out by precliaical and clinical evidence. It is
As a class, the hyaluronans have a well-documented toler- instead likely that the mechanism of action of hyaluronan
ability profile, with no known systemic effects and few is based on several probable biological activities mediated
contraindications or drug interactions.6T The most cornmon through receptor-based pharmacologic mechanisms that go V
side effect aoted ia most clinical studies has boen injection- beyond the physical, mechanical, and elastoviscous propersite
pain. Although any intra-articular injection can elicit an ties of these products.
inflammatory response, a clinically distinct reaction known Current evidence suggesls that there is an optimal MW
as pseudosepsis or SAIRs has emerged after hyaluronan range for induction of various biological activities. In pri-
injeclions. Although it is not clear whether pseudoseptic marily in vitro analyses, some of these activities are
reactions can occur with any hyaluronan, al1 published induced more effectively by high-MW hyaluronans, where-
reports to date have been linked to hylan G-F 20 (Synvisc). as others are induced by low- to mid-MW hyaluronans. In
Whereas the manufacturing practices for all hyaluronan contrast, preclinical studies evaluating joint-structure out-
products have similar quantitative specifications for the comes in animal models of osteoarlhritis indicate that low-
amount of allowable proteins within the products (generalto mid-MW hyaluronans may have more potential for disly

P, C. Vitanzo and B. J. Sennett
disease progressiol, which has been described for low- to
mid-MW sodium byaluronate (500-730 kDa).22'2s'7+'ts
When risk-benef,t assessment is used in making treatment
decisions, lack of evidence for a superior clinical benefit
from use of high-MW hyaluronans versus low- to mid-MW
hyaluronans should be considered together with the finding
that cross-linked polymer products may be associated with
increased risk of adverse events in the knee, specifically
SAIRs and granulomas.
AurnoR AcxNowleDGMENTs AND
Drsclosunr STATEMENT
This study was supporled by a grar* from Sanofi-
Synthelabo. Dr. Vitanzo wishes to note that he is a consultant.
and lecturer for sanofi-aveatis and for Smith &
Nephew. Dr. Sennett wishes tû note that he has received a
grant from sanofi -aventis.
\'
V
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