2018.degradation Hialuronidase

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May 2018 548 Volume 17 • Issue 5
Copyright © 2018 ORIGINAL ARTICLES Journal of Drugs in Dermatology
SPECIAL TOPIC
Degradation of Hylauronic Acid Fillers Using Hyaluronidase

in an In Vivo Model
Rodrigo Moraes Ferraz MD,a Ulrika Sandkvist MSc,b and Björn Lundgren PhDb
a
Rodrigo Ferraz Dermatologia, Belo Horizonte, Brazil
b
Galderma, Uppsala, Sweden
ABSTRACT
Introduction: Soft tissue fillers manufactured with hyaluronic acid (HA) dominate the filler market around the world and the fact that
HA can be dissolved using hyaluronidase contributes to its popularity. Degradation of cross-linked HA products can be performed in situ
and access to hyaluronidase is therefore essential for health care professionals (HCP) to perform safe filler treatments. The aim of the
present study was to develop an in vivo model where hyaluronidase degradation of HA fillers can be studied in a standardized manner
and then secondly, to explore the degradation of marketed HA fillers with different product characteristics.
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Methods: Intradermal injections of HA fillers were performed and the injection sites were treated with hyaluronidase. The degradation
was evaluated by measuring the heights of the injection site bumps during 5-7 days and with histology at day 7 post injection.
Results: The results showed that there was a correlation between the hyaluronidase dose and HA filler degradation. The onset of the
degradation was fast for all products and the products were easily degraded.
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Discussion: This standardized animal model proved efficient in the study of in vivo degradation of HA fillers using injected hyaluroni-
dase where products manufactured with different technologies were evaluated.
J Drugs Dermatol. 2018;17(5):548-553.
INTRODUCTION
H
istorically, many different agents like paraffin, silicon, will then either be further degraded locally or drained from the
and collagen have been used for aesthetic purposes tissue via the lymphatic system. Local degradation in the skin
to shape and restore the face and body. However, where HA was synthesized is one of the turnover pathways
since more than 20 years, cross-linked hyaluronic acid (HA) but the majority of the HA fragments leave the tissue with the
filler products are used for correction of wrinkles and lines and lymph and is cleared by endothelial cells in the lymph nodes.
today HA products dominate the soft tissue filler market. What remains after passage through the nodes will reach the
circulation and finally be degraded by the liver.3
HA is a naturally occurring molecule present in all tissues and a
major component of the extracellular matrix (ECM) of the skin. Since hyaluronidase can be used to degrade HA fillers, access
The HA molecules consist of long un-branched chains of alter- to hyaluronidase has become an essential part of the HCP’s
nating units of D-glucuronic acid and N-acetyl-D-glucosamine. toolbox to perform safe filler treatments. Unwanted effects
This linear polysaccharide can reach a size of 6 to 8 MDa.1 In such as overcorrection or nodules may be resolved using hy-
the body, there is a constant turnover of HA and the half-life aluronidase and in case of serious adverse events (AE) such
in skin is less than one day.2 In order to extend the residence as intravascular injection, hyaluronidase can be used as a res-
time for the HA based filler products, cross-links are introduced cue therapy. This is a major advantage of HA over other soft
to stabilize the HA molecules into a 3D network and thereby tissue filler materials such as poly-L-lactic acid (PLLA), calcium
prolonging the duration from days to months or years. The high hydroxyapatite (CaHA) and autologous fat.
water-binding capacity of HA together with an excellent safety
profile makes it an ideal material for soft tissue filler products. However, there is a common perception among HCPs that
some HA products are more difficult to degrade than others,
Degradation or de-polymerization of endogenous HA involves but standardized studies where in vivo degradation was stud-
primarily enzymatic degradation by hyaluronidases but also ied are lacking. In vitro tests (performed at the analytical lab of
degradation by free radicals. In degradation of the HA chains Galderma but not published) have shown that cross-linked HA
by hyaluronidases, HA fragments are created as the glycosidic products can be degraded with hyaluronidase. The modification
bonds between the disaccharides are hydrolyzed. In order for of the HA molecules that takes place in the cross-linking step
the large HA molecules to be released from the ECM the poly- does not seem to hinder the degradation with hyaluronidase of
mer needs to be at least partially degraded. The HA fragments the studied products. When comparing degradation behavior of
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549
Journal of Drugs in Dermatology R. Moraes Ferraz, U. Sandkvist, B. Lundgren
May 2018 • Volume 17 • Issue 5
HA products in vitro it is essential to use a strict and standard- the OBTTM technology which creates a softer gel structure that
ized procedure. For example, an important aspect is the ability distributes more in the tissue for contouring and expression.
of the enzyme to directly access the HA. If the enzyme is mixed The non-cross-linked HA solution (20 mg/mL) was manufac-
with the HA filler or if the enzyme is applied on the surface of tured specifically as a comparison to the cross-linked products
a gel drop, the degradation profile will be different. Other as- to evaluate how the cross-linking affected the degradation.
pects like pH and temperature will also affect the activity of the
enzyme. Overall, this demonstrates the importance to design The hyaluronidase used in this study was Hylenex® recombi-
and carry out degradation studies with a strict and standardized nant (150 units/mL, Halozyme Therapeutics). Hylenex (HYL) is a
protocol to be able to have relevant results. Furthermore, as ready to use formulation of human recombinant hyaluronidase
the degradation behavior in vitro might not be predictive of the intended for subcutaneous fluid administration and for disper-
behavior in vivo, relevant studies to evaluate this are needed. sion and absorption of injected drugs in the tissue. HYL is also
widely used to reverse complications after HA filler treatments.4
In the present study, an in vivo animal model was used to evalu-
ate the degradation behavior of several commercially available The study was performed in two phases where the first phase
cross-linked HA products in comparison to a non-cross-linked aimed to evaluate several protocols with hyaluronidase treat-
HA solution. The model was designed with emphasis to stan-
dardize the injection technique regarding volumes, depth and Do Not Copy
ment on a cross-linked HA product (RES). The degradation
efficiency of each protocol was evaluated, and based on the re-
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placement and the injections were performed by a trained op- sults, the most effective protocol was chosen and modified and
erator who also measured the bump heights with a caliper. The used on all products tested in the second phase of the study.
study design aimed to mimic AEs like nodules and bumps rath-
er than complicated vascular occlusion or necrosis. However, In both phases, intradermal bolus injections of HA products
this article does not intend to give clinical guidance for treating (100 µl) were performed on the back of rabbits. The injections
filler related complications with hyaluronidase. formed defined elevated skin bumps. The height of the bumps
were measured daily during the study. Following the filler in-
MATERIALS AND METHODS jection, hyaluronidase (HYL) was injected approximately one
This study was performed on rabbits (New England White) in hour after the HA product injection and repeated with one-hour
accordance with ISO 10993-2 Biological Evaluation of Medical intervals according to the hyaluronidase protocols described in
Devices- Part 2: Animal Welfare Requirements (2006), and after Table 1 and Table 2. Injections of hyaluronidase were performed
approval of the ethical committee at the test lab. The first phase perpendicular to the skin into the center of the filler implant at
of the study included four animals and the second phase in- a slow injection speed. Non-hyaluronidase-treated sites were
cluded eight animals. All animals received 10 injections each on also included to follow the natural development of the bump
the back with HA products. sizes during the study.
The marketed HA products included in the study are all cross- In the first phase, four different hyaluronidase protocols were
linked and with a HA concentration of 20 mg/mL. Restylane® evaluated, see Table 1, on intradermal skin bumps of a cross-
(RES), Restylane® Lyft (RESLyft), and Restylane® Skinboosters linked HA filler product (RES). The different protocols aimed to
Vital (RESVital) are manufactured with the patented NASHATM evaluate whether the number of hyaluronidase treatments or
technology in which defined and homogenous gel particles are the dose was most efficient in macroscopic removal of intra-
created. The NASHA technology is designed for lifting and pre- dermal bumps. In protocols A-C, the hyaluronidase injections
cision with targeted product integration for a more pronounced were performed on day 0. In protocol D, the hyaluronidase
lifting capacity. Restylane® Refyne (RESRef) is manufactured with treatment was performed on day 0 and then repeated again on
TABLE 1.
Description of the Different Hyaluronidase Protocols in the First Phase of the Study
Protocol A Protocol B Protocol C Protocol D
Volume of filler product 100 µl 100 µl 100 µl 100 µl
Injected volume for each injection 150 µL 100 µL 150 µL 100 µL
Hyaluronidase Number of injections 4 4 2 4

treatment Days of injections D0 D0 D0 D0* + D4*
Total hyaluronidase dose 90 60 45 60
*2 injections per day

550
TABLE 2. where the hyaluronidase treatment was performed both on day

Description of the Hyaluronidase Protocol in the Second Phase zero and day four, the decline was slower, but ended up almost
of the Study as low as for protocol A on day 7. None of the protocols com-
Protocol E pletely removed the skin bumps but there was a clear trend that
a higher dose gave both faster and more effective degradation.
Volume of test articles 100 µL
Protocol D had the same dose as protocol B but the injections
Injected volume for were split on two days and the mean bump size on day five was
150 µL
each injection
a little smaller. Based on the fact that none of the protocols in
D0
Number of injections 4 the first phase gave a complete degradation, a combination of
Hyaluronidase units 90 protocols A and D was chosen for the second phase of the study
Hyaluronidase (Protocol E; Table 2).
Injected volume for
treatment 100 µL
each injection
D4
Number of injections 3 Second Phase: Macroscopic Evaluation
In the second phase, four products were included of which
Hyaluronidase units 45
three were commercially available fillers, RESLyft, RESVital and
Total hyaluronidase units 135
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RESRef, and one was a non-cross-linked HA solution (20 mg/mL).
Penalties Apply
day 4. The total hyaluronidase dose ranged from 45 IE (proto- Injections of all products resulted in clearly defined skin bumps
col C) to 90 IE (protocol A). (Figure 2A), although the bump height of the HA solution sites
were lower than the heights of the cross-linked products (Figure
Following the first phase, protocol A was selected and modi- 3). It can be assumed that the lifting capacity of non-cross-linked
fied (protocol E), see Table 2 to be included in the second HA solution is lower than for cross-linked products resulting in
phase. A total dose of 135 IE hyaluronidase was given since a lower elevation of the skin level. The onset of the degradation
the degradation of the bumps was not fully complete in any was fast for all products; the day after the first hyaluronidase treat-
of the protocols in the first phase. In the second phase three ment, the skin bumps had flattened substantially (Figure 2 B).
commercial HA filler products (RESLyft, RESVital and RESRef) were
included together with one solution of non-cross-linked HA. The hyaluronidase treatment was continued on day four with
additional injections, and on day seven the bumps had almost
In the first phase, the degradation was followed macroscopi- completely disappeared. For the non-cross-linked HA solution,
cally by daily measurements of skin bump height whereas in the degradation was complete already on day one, both for the
the second phase histologic evaluation of the injection sites treated and the untreated sites. This was an anticipated result as
was also performed on day 7. the half-life of HA in skin tissue is less than one day.2 The heights
of the non-hyaluronidase-treated sites remained constant dur-
Histological processing of the injection sites was performed by ing the study for the three cross-linked HA products whereas
fixation in 10% buffered formalin, followed by dehydration in the sites where non-cross-linked HA solution was injected the
alcohol solutions of increasing concentration, clearance in xy- decline in height was almost as fast as for the hyaluronidase
lene, and embedding in paraffin. Central cross-sections (4-7 µm treated sites.
thickness) were prepared of each site and stained with Alcian
blue. The degradation evaluation was based on the qualitative A slight increase in bump height was seen for the cross-linked
and semi-quantitative histopathology evaluation of the degra- products on day two and three as compared to day one, and
dation, by comparison of the test sites (hyaluronidase-treated
sites) with the corresponding untreated sites (sites without hy-
FIGURE 1. Macroscopic evaluation of bump heights in the first phase
aluronidase treatment).
of the study.
3,5
Statistical Analysis
3
Each product was evaluated by measuring the heights of the injec- 2,5
Height (mm)
tion sites and comparing them to its respective non hyaluronidase 2

treated site. No statistical analysis of the data was conducted. 1,5
1
RESULTS 0,5
0
First Phase: Evaluation of Injection Protocols 0 1 2 3 4 5
A rapid decline in the mean bump height was seen from day Time period (days)
zero to day one for protocols A and B (Figure 1). In protocol D, Protocol A Protocol B Protocol C Protocol D
551
FIGURE 2. Elevated skin bumps after intradermal injection of HA filler FIGURE 3. Macroscopic evaluation of bump height in the second
products (A). The decrease in bump height was clearly visible the phase, expressed as mean ± stdev.
day after hyaluronidase treatment (B).
5
RESLyft
4
Height (mm)
3
0
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day7
Treated Non-treated
5
RESVital
4
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Height (mm)
3
2
Penalties Apply
1
0
Treated Non-treated
then followed by a decrease. Injection of the hyaluronidase so- 5

lution might in itself have added to the bump heights (0.6 mL RESRef
4
on day 0 and 0.3 mL on day 4) as the HA filler products have the
ability to absorb and retain water.
Height (mm)
Second Phase: Histological Evaluation 2
In order to evaluate the in vivo degradation by histology, the 1

area of filler product was assessed and compared for hyaluron-
idase-treated sites versus non-hyaluronidase-treated sites. The 0
cross-linked HA fillers appeared as blue foreign material in the
Treated Non-treated
histological slides, see arrows in Figure 4. For all cross-linked
products, the area occupied by filler product had decreased but 5
was still visible in the dermis of the test sites seven days after
HA solution
4
injection and subsequent hyaluronidase treatment (Figure 4).
The HA solution sites were completely resorbed on day seven,
Height (mm)
3
both in the hyaluronidase-treated and the non-hyaluronidase-
2
treated sites.
1
DISCUSSION
0
In this study, HA products manufactured with different tech-
nologies were evaluated regarding their degradation profiles
Treated Non-treated
in vivo using injected hyaluronidase. RES, RESLyft and RESVital
(NASHA) as well as RESRef (OBT) were all degraded in a fast
manner. Products with other manufacturing technologies were the ability to withstand exogenous hyaluronidase injection is
not evaluated in this study and hence, the in vivo degradation however not confirmed. In order for the injected hyaluronidase
patterns for such might be different. In other publications, re- to actually perform its missioned cleavage, the enzyme needs
searchers have suggested that HA concentration, particle size to be in direct contact with the HA chains. As seen in the his-
and degree of cross-linking are factors that would affect the tological pictures in Figure 4, the HA products are integrated
residence time of HA products.5 If these features also affect into the tissue as small islets of gel mixed with the collagen
552
FIGURE 4. Histology pictures (Alcian blue) of intradermal injections of HA products on day 7. (A) RESLyft non-treated, (B) RESLyft, hyaluronidase treated
(C) RESVital non-treated, (D) RESVital, hyaluronidase treated (E) RESRef non-treated, (F) RESRef, hyaluronidase treated (G) HA solution, non-treated,
(H) HA solution, hyaluronidase treated.
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bundles and other components of the ECM. As the hyaluroni- pharmaceuticals in tissue6 it could be assumed that the enzyme
dase is injected, the enzyme solution can either directly come itself will diffuse. Diffusion of the enzyme could probably also
across the gel islets or be trapped in the surrounding ECM tis- appear into and through the gel islets. This raises the question if
sue. As hyaluronidase is often used as a spreading factor for the gel islets are degraded from the outside and in or both from
553
the outside and from within. Looking at the gel islets in the his- hyaluronidase protocols for degradation of HA filler products.
tological slides in Figure 4 it seems as if the size of individual The conclusion was that a correlation between the hyaluroni-
islets are similar between the hyaluronidase treated and the dase dose and HA filler degradation was seen as measured by
non-hyaluronidase treated islets. The cleavage of the HA chains the decrease in bump height.
could in this case have been done both on the surface and also
within the individual gel particles. On the other hand, as HA is The onset of the degradation was fast for all products as the
degraded, the ability to attract and hold water increases. This skin bumps were almost macroscopically flat already the day
means that the gel particles could retain their size during deg- after hyaluronidase treatment. However, histological evalu-
radation even if the concentration of HA in the implant islet is ation showed that only the non-cross-linked HA solution was
decreasing. If this is the case it would imply that gel particle size completely removed, both with and without hyaluronidase
does not affect the degradation considerably. This hypothesis is treatment. All other products were substantially degraded and
strengthened by the fact that there was no obvious difference cleared from the tissue but still visible in the histology sections
in the degradation between RESLyft and RESVital, two products although the reduced lifting capacity hindered the products
which differ only by their particle size. from elevating the skin from the base level. Products manu-
factured with both the NASHA and the OBT technologies were
Massage of the injection site bumps were not applied in this
study. This is often proposed in clinical treatment of AEs like Do Not Copy
easily degraded and cleared from the tissue using injected
hyaluronidase.
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nodules and bumps.7 Massage of the hyaluronidase treated
sites might facilitate the mixing and the dispersion of the en- DISCLOSURES
zyme with the HA products in the tissue. Rodrigo Ferraz has been a speaker for Galderma. Ulrika Sand-
kvist and Björn Lundgren are employees at Galderma.
It is also interesting to note that complete degradation of the
HA products was not achieved even with multiple injections of REFERENCES
hyaluronidase and a high total dose of 135 units. This non-com- 1. Stern R, Kogan G, Jedrzejas MJ, Soltés L. The many ways to cleave hyaluro-
nan. Biotechnol Adv. 2007;25(6):537-57.
plete degradation has also been seen in other studies.5,8 One 2. Laurent TC, Fraser JR. Hyaluronan. FASEB J. 1992;6(7):2397-404.
could argue that the gel might be degraded but not yet cleared 3. Lepperdinger Gün, Fehrer C, Reitinger S. Chemistry and Biology of Hyaluro-
nan. Elsevier Ltd; 2004;p:71-82.
from the tissue, but this is unlikely since the degradation and 4. Rao V, Chi S, Woodward J. J Drugs Dermatol. 2014 Sep;13(9):1053-6
clearance of HA solution was completely achieved, as seen 5. Juhász MLW, Levin MK, Marmur ES. Dermatol Surg 2017 May;28(3):838-
both in the macroscopic evaluation of bump size, see Figure 841.
6. Halozyme therapeutics. Hylenex recombinant (hyaluronidase human injec-
3 and microscopically at day 7 (Figure 4). However, complete tion): Highlights of prescribing information. Accessed January, 2015.
degradation might not be vital to treat AEs of lower severity. In 7. Rodrigues-Barata AR, Camacho-Martínez FM. J Drugs Dermatol.
2013;12(4):e59-62.
case of nodules, bumps, or overcorrection, a decrease in the lift- 8. Hwang E, Song YS. J Craniofac Surg. 2017;28(3):838-841.
ing capacity (most likely due to a decreased HA concentration
within the implant) could be enough to reverse an unaesthetic AUTHOR CORRESPONDENCE
appearance.
Ulrika Sandkvist MSc
E-mail:................……...................... ulrika.sandkvist@galderma.com
In this study, several injections of hyaluronidase were per-
formed into the HA filler implants. Looking at the results from
the first phase, protocol A and B with four injections of hyal-
uronidase gave substantially faster degradation as compared to
protocol C where only two injections were given. As discussed
earlier, the enzyme needs to be in contact with the HA product
to perform the cleavage and in theory several injections might
be favorable compared to a single injection of hyaluronidase
to renew the flow of enzyme around and through the implant.
With only a single injection, the enzyme might diffuse away
from the implant and therefore the desired degradation could
fail.
CONCLUSION
In this in vivo model, defined intradermal skin bumps were cre-
ated that could be measured and followed in a standardized
way. It was possible to evaluate the effectiveness of different

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Degradation of Hylauronic Acid Fillers Using Hyaluronidase

J Drugs Dermatol. 2018;17(5):548-553.

Hyaluronidase Number of injections 4 4 2 4

*2 injections per day

TABLE 2. where the hyaluronidase treatment was performed both on day

tion sites and comparing them to its respective non hyaluronidase 2

then followed by a decrease. Injection of the hyaluronidase so- 5

Second Phase: Histological Evaluation 2

In order to evaluate the in vivo degradation by histology, the 1

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