Hyaluronidase Review - MP Am SB

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Biochemistry

HYALURONIDASE: 2021’S AGING ENZYME – AN ETHICAL


DISCUSSION - Review
Andrea Monsalve 1, Sofía Bárcenas 2 and Mónica Porcell 3
1
Ingeniería química; andreamoch@unisabana.edu.co
2
Ingeniería química; paulabagi@unisabana.edu.co
3
Ingeniería química; monicapogo@unisabana.edu.co

Abstract
Aging is a natural process that every human being goes through. The decrease in the concentration of hyaluronic acid in
the body over the years is one of the main factors to which the onset of physical effects such as wrinkles is attributed.
This decrease is mainly due to the action of hyaluronidase (HYAL) as an enzyme that breaks down hyaluronic acid
(HA). The use of HA dermal fillers was implemented as a tool to combat or delay this effect; however, this solution is
not permanent because it is well received for human body and as the HA and HYAL are naturally synthetized for the
body. In the present review we evaluate the enzymatic activity of HYAL in the presence and absence of inhibitors with
the objective of determined the best inhibitor until now, as well as the extraction methods, sources of origin and the
ethical implications involved in the extraction, application in industries and inhibition processes. It was determined that
Vitamin C is the best inhibitor found so far, however, the inhibitory part of it is still unknown. It was also determined
that inhibitors analyzed have in common the presence of carbonyl groups or aromatic rings in their structure and that the
highest inhibition percentage is attributed to inhibitors with origin on natural sources.

Key Words: Aging, degradation, ethics, hyaluronidase, hyaluronic acid, inhibition, vitamin C.

Introduction of macromolecules, consisting of proteins,


The skin extracellular matrix (ECM) plays a proteoglycans and glycoproteins that confer the
fundamental role in the protection, nutrition, and structural properties of cells and tissues [2]. Among one
regulation of various cellular mechanisms, activating or of the main components of the extracellular matrix are
deactivating the processes of cell growth, cell death, glycosaminoglycans (GAGs), with hyaluronic acid
adhesion, invasion, gene expression and cell (HA) being the only GAG that is not covalently bound
differentiation [1]. It is composed of a complex network to a protein to form a proteoglycan [3]. It is a high

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molecular weight non-sulfated polysaccharide enzymes. Since these fillers are not permanent and HA
composed of repeating polymeric disaccharides of D- is degraded by them after 18 months [13]. Currently
glucuronic acid and N-acetyl-D-glucosamine linked by several types of inhibitors have been developed from
alternating β (1-3) and β (1-4) bonds, present especially plant and fruit extracts, however, all of them are
in connective tissues, but also in nervous tissue and characterized by very low yields, around 37% - 50%
epithelia [4]. It is an extremely hydrophilic molecule, [14]. However, vitamin C has been one of the inhibitors
which gives it a high hydration capacity, contributing to that has shown a higher yield (60-80%), as well as a
the viscoelastic properties of the skin, providing tissues higher inhibitory activity on HYAL, presenting
with resistance to mechanical stress and lubrication [5]. inhibition percentages of up to 97% [15, 16]. It is
50% of HA present in the body is found in the dermis, thought that its inhibition is due to its antioxidant
ranging from concentrations of 0.5 mg/g to 1 mg/g [6]. activity, although it is not totally attributed to this cause,
Unfortunately, the presence of this polysaccharide in the since there are other antioxidants that have not been able
body decreases progressively over the years because its to inhibit it or have inhibited it minimally, such as
degradation is necessary for the permeabilization of quercetin [17].
ECM, generating the decrease of moisture and laxity in
the skin, promoting skin aging [7]. The present review aims to study the enzymatic
activity of hyaluronidase in the presence and absence of
One of the main degradative agents of this acid inhibitors and its capacity to degrade hyaluronic acid.
are hyaluronidases (HYAL). In humans, six types of Additionally, it explains the ways of obtaining
HYALs have been identified: HYAL-1, -2, -3, -4, PH- hyaluronidase, its sources of origin and the ethical
20 and NYALP1 [8]. Enzymes capable of releasing the implications of the extraction process and its application
oligosaccharides present in HA by breaking down in different industries.
hyaluronic acid into monosaccharides by cleaving at
their glycosidic bonds [9]. This generates multiple Aging and antiaging
effects on tissues, decreasing the high viscosity and Aging is a physiological phenomenon defined
lubricating quality of hyaluronic acid. To prevent these as a progressive failure of metabolic processes [18] but
effects, cosmetic injection of HA dermal fillers has despite this, it is called a natural process in humans [19].
emerged, being one of the main strategies used for the Aging is caused by different theories, among them the
prevention of aging [10]. In addition, these enzymes are free radical theory, the neuroendocrine theory, the
causative of various diseases, it has been shown that telomerase aging theory, the wear and tear theory, the
their concentration in the body can be affected by rate of life theory, the accumulation of waste products
diseases such as Alzheimer's disease and dementia. theory, the cross-linking theory, the immunological
Nevertheless, HYALs can be employed in medical theory, the error and repair theory and the order of
practice to increase tissue absorption of various drugs, disorder theory [20, 21]. The common denominator of
as they act as a "spreading factor" facilitating the these theories lies in the presence of molecules such as
diffusion of different substances injected free radicals, telomeres, and enzymes such as
subcutaneously, such as anesthesia and antiviral hyaluronidase, accompanied by external factors such as
vaccines [11, 12]. exposure to x-rays and UV rays, resulting in premature
aging that is phenotypically evident in wrinkles and skin
Despite this innovative tool, multiple findings blemishes [22].
have been made to find the right inhibitor for these
Hyaluronic acid and hyaluronidase related tumor growth [10]. A recent colorectal cancer
Hyaluronidase is a natural enzyme, produced by investigation showed the presence of different types of
humans, capable of locally degrading hyaluronic acid, hyaluronidase (Hyal-1, -2, -3 and PH-20) especially in
specifically its β1-4 bond between hyaluronic acid and N- advanced cancer stages; each type of hyaluronidase was
acetylglucosamine [23]. In the aesthetic and present in different tissue distribution which allowed the
dermatological industry, it is beneficially used in identification of different cancer isoforms at certain
treatments against incorrect hyaluronic acid filler stages [27].
injection [24]. Hyaluronic acid (HA) is a
glycosaminoglycan (GAG), not bound to a core protein, Hyal-1 is found within different cell types
naturally synthesized by the human being that fulfills partially compartmentalized in vesicles that traffic in a
important functions in the skin such as hydration and manner different from clathrin or caveolin endosomal
anti-aging effect. HA production in the skin decreases and in the lysosome. The presence of this enzyme has
with age and exposure to solar radiation, affecting the been attributed to the capacity of metastasis in some
transport of ions and macromolecules in the types of cancer, especially in colorectal, genitourinary,
extracellular space due to decreased hydration [23]. ovarian, breast, pancreatic and lung cancers; moreover,
Almost proportionally to the decrease in HA there is it has not been found in its normal structure but its
evidence of an increase in hyaluronidase, which is capacity to generate variants called Hyal1-v1, Hyal1-v3
involved in aging processes due to the release of and Hyal3-v1 has been evidenced [28]. By performing
cytokines by keratinocytes which degrade the three- different studies on Hyal-2 it has been possible to
dimensional network of fibers of the dermis and identify that it becomes more present in premalignant
phenotypically are evidenced by wrinkles, skin and malignant melanomas but when the cancer is in an
blemishes and loss of elasticity [25]. Hyaluronidase is advanced stage the concentration of this isoenzyme
found naturally in the dermis, which is divided into decreases considerably as the body develops specific
layers, specifically it is found in the epidermis and is antibodies to Hyal-2, for example the Hyal2ex2-3
compartmentalized and especially present in the basal variant was associated with early gastric tumor lines and
and spinosum stratum inside the cells and in the spaces Hyal-2 was seen present in many stages related to
between them. In the dermo-epidermal junction only astrocytoma tumor growth [29, 30, 31]. The Hyal-3, -4
hyaluronic acid is found and from there the molecule and PH-20 isoenzymes are categorized as the three least
migrates to the extracellular space and to the upper studied members of the hyaluronidase family, of which
layers of the skin, enriching keratinocytes and in the it has been determined that Hyal-3 is not anchored to the
dermis the highest amount of hyaluronic acid is found plasma membrane of the cell as Hyal-4 and PH-20 are,
and at the highest concentration because it is and of the latter two only PH-20 shows similar activity
synthesized specifically in the plasma membrane of the to hyaluronidase, but specifically in the testis and does
fibroplasts, very present in this layer of the dermis [26]. not change tumor cell lines. Although the relationship
of these isoenzymes with cancer has not been deeply
In addition to aging, hyaluronidase has also studied, their presence has been identified in the phases
been found to be present in cancer and non-cancer of tumor growth and in remote metastasis [28, 32].

3
in saliva and this process was not associated with
Hyaluronidase has also been beneficially used presence of any bacteria because it was isolated from
in human ocular surgery as an anesthesia potentiator, microorganisms that could affect the study [38].
and in cosmetic surgery as a treatment for wrong
hyaluronic acid injection, but more frequently and Obtention, quantification and purification
safely in cosmetic surgery because surgeon and Hyaluronidases have been identified in
esthetician have more experience in this stage plus the eukaryotes and prokaryotes as they can be found in
numerous investigations about this topic. Its use in many of our tissues. For example, in the kidney, spleen,
human ocular surgery has still been investigated so it skin, liver, placenta and testes [25]. Also, in venoms of
can be advantageously used [33, 34, 35]. However, this snakes, lizards, bees, fish, scorpions or body liquids as
enzyme has shown negatively effects in pig vocal cord sperm, tear liquid and blood. It can be found in some
involvement causing dehydration and decreasing its bacteria like fungi or invertebrate animals as leech or
tangent moduli in the kinetic study from Ꜫ=0.3 when the crustaceans [39]. The samples vary on molecular
vocal cord is correctly hydrated to Ꜫ=0.15 when it is weight, substrate specificity, optimal conditions, and
dehydrated because of presence of hyaluronidase [36]. catalytic mechanism, depending on the source of origin
Sleep apnea is another topic that hyaluronidase has (Fig.1) [40]. Therefore, these extractions can be
affect suggesting that an increase of Hyal-1 levels and classified in three different groups. First, the
an accelerated degradation of the High Molecular hyaluronate 4-glycanohydrolases are responsible of
Weight Hyaluronic Acid (HMW-HA) caused for the degrading hyaluronan by the breach of beta-1,4-
enzyme or for alterations in the hyaluronan metabolism glycosidic bond, with tetra-saccharides as main product
increase levels of chronic hypoxia and inflammatory [41]. These enzymes can also depolymerize chondroitin
process generating sleep apnea in patients [37]. and dermatan sulfate [42]. They can be found in
Alzheimer and dementia are also related with increase lysosomes, testicles, and bee venom. The second type
of hyaluronidase levels in the body; as mentioned before are the 3-glycanohydrolases, found in the salivary
hyaluronidase is extremely related with aging and high glands of invertebrate animals as leech or crustaceans.
levels of hyaluronidase can make human body to get These enzymes hydrolyze the beta-1,3-glycosidic bond
older in least time and allows the development of some of sugar fragments in glucuronic acid, producing tetra-
mental diseases and even in renal and vascular risk saccharides [43]. The third group are hyaluronate
factors [11], same thing has happened with collagen lyases, commonly known as microbial hyaluronidases.
degradation. Collagen can be stored in human dentin They produce an unsaturated disaccharide by an
and considering that hyaluronidase can be present in elimination reaction and can be isolated from
saliva, a study showed that collagen was degraded for microorganisms as Micrococcus, Streptococcus,
the action of increased concentrations of hyaluronidase Clostridium and Streptomyces [41].
Fig 1. Classification of hyaluronidases [39]

Based on the amino acid sequence, the glucosamine. They have been studied because
hyaluronidases can also be classified in two main hyaluronate lyases are consider a factor that facilitate
families, from eukaryotes or prokaryotes. spreading of bacteria in host tissues. Human infection
Hyaluronidases from eukaryotes are mainly obtain from by S. agalactiae is one of the major causes of serios
mammals or bovine testicles [44]. Several diseases as meningitis or septicemia [47]. That´s why
hyaluronidase’s samples can be studied inside the the design and development of inhibitors of this enzyme
mammalian category. Human testicular hyaluronidase become important.
can be found because is required for sperm penetration
through oocyte´s cell layer. It is known as PH-20 protein The extraction of hyaluronidase is considered a
or SPAM 1 (sperm adhesion molecule 1). Within these very expensive and difficult process, as the source,
samples, there were two hyaluronidase-like found with cellular tissue, must be alive and fresh [48]. It has been
about 40% identical amino acid sequence, Hyal-1, and proved that hyaluronidase extraction from frozen
Hyal-2. They differ on their function inside the body. testicles or seminal plasma is not recommended nor
Hyal-1 produces oligosaccharides, while Hyal-2 possible, as it inactivates rapidly upon release or during
degrades high molecular weight HA [45]. They have the extraction [49]. One of the extraction methods
been widely studied as Hyal-1, as it is believed they are applied is the Hahn´s method in testicles obtained as
an inactivated suppressor gene in many tobacco-related soon as possible after dead. Also, high pressure
lung tumors. Hyal-2 was found to promote tumor cell homogenization has been studied, but is not
cycling. On the other hand, bovine testicular recommended as it demands thermal, chemical, and
hyaluronidase (BTH) breaks beta-1,4-glycoside bonds other potential disnaturing factors. Centrifugation and
of hyaluronan, acting as endo-glucanohydrolase [46]. precipitation are one of the most common methods.
Usually, homogenizing the source with sodium acetate
The second family are the samples from buffer, NaCl and phenylmethanesulfonylfluoride,
prokaryotes as Streptococcus pneumoniae and S. centrifugated at 26000 g for 75 min recovering the
agalactiae. Both enzymes break beta-1,4-glycosidic supernatant. Then subjected to an affinity
bond between D-glucuronic acid and N-acetyl-D- chromatography column [50]. Quantified with Lowry´s

5
method using bovine serum albumin as standard [51]. compounds with commercial potential in the medicine
The most common purification route consists of and aesthetic industry [53].
(NH4)2SO4 fractionation separation of insoluble
protein, Column I. Sephadex G-100, Column II. DEAE- The hydroalcoholic extract of the Piper
cellulose and Column III. Sephadex G-75 [50]. Peltatum plant was also evaluated as an inhibitor against
bovine hyaluronidase and extracted from the venom of
Inhibition two types of snakes (Naja naja atra and B. atrox) by
Due to all the processes in which hyaluronidase performing different tests with respect to the
in involved, its inhibition has been extensively studied. compounds of the extract (flavonoids, phenols, and
Hyaluronidase inhibitors are described as a potent alkaloids). All compounds showed an inhibition higher
regulator capable of maintaining hyaluronic acid than 50% with bovine hyaluronidase, between 4.69 and
homeostasis, which in turn could serve as anti- 23.05% with Bothrops atrox venom and between 14.59
inflammatory, antioxidant, antimicrobial and even and 40.17% for Naja naja atra venom and from these
anticancer agents, and provide a deeply insight into the results it could be concluded that low inhibitor
functioning of hyaluronidase-type enzymes in terms of concentrations result in high inhibition percentages and
hyaluronic acid metabolism [52]. that the inhibitory activity is given by the complex of all
the compounds of the extract and not as a singularity
Some of the inhibitors that have been identified [54].
are phlorotannin, alginate and fucoidan extracted from
two types of Sargassum seaweeds and from the Eisenia Another study evaluated the inhibitory activity
arborea thallus, with a maximum anti-hyaluronidase of seven acylated iridoid glycosides extracted from
percentage of 37.67% with the phlorotannin extracted Picrorhiza kurroa rhizomes on hyaluronidase and
from a leaf of S. tennerimum, also showing antioxidant determined the chemical structure of each of them (Fig.
activity with anti-aging potential. The study showed that 2), showing a maximum inhibition of 61.8% by the
increased anti-hyaluronidase activity was associated Picrorhizaoside E with a concentration of 100µM of
with decreased production of N-acetylglucosamine, inhibitor [55].
concluding that the 3 inhibitors are natural origin

Fig 2. Determined structure of seven acylated iridoid glycosides used as inhibitors [55]
Vitamin C is an acid synthesized from glucose The inhibition of enzymes related to anti-aging
and galactose only by plants and some animals. It is has been necessary not only because of their
highly recognized for its many beneficial functions in counterproductive effects on human health but also as a
the human body as an antioxidant used in the treatment treatment for different diseases, for example, a study
of diseases ranging from the common cold to cancer that sought to inhibit enzymes related to anti-aging
[56], for this reason, the inhibition of hyaluronidase with processes found that proper inhibition should lower the
vitamin C has been a current subject of study. In a levels of captopril, perindopril and enalapril to control
related study, 6-O-acylated vitamin C derivatives have the development of diseases such as Alzheimer’s
shown capacity of inhibiting the enzyme but with disease [11] and as this example there are numerous
selectivity on bacterial lyases [57], however, in another studies of inhibitors applied in hyaluronidase that are
study the inhibition with vitamin C derivatives of bovine shown in table 1. Therefore, enzyme inhibition becomes
sperm hyaluronidase was applied and the maximum a challenge for today’s scientists and researchers. To
inhibition found was 97% with an inhibitor have accurate and certain results it is necessary to
concentration of 5%, making it to date the best inhibitor understand the kinetics of the enzyme, in this specific
of this enzyme [16]. case hyaluronidase, and its substrate, in this case
hyaluronic acid.

Table 1. Comparison of inhibitors and its effect in different origin hyaluronidase


Origin of Chemical Inhibitor
Inhibitor Concentration Reference
hyaluronidase definition Percentage
Bovine testes Phlorotannin from Leaf of Polyphenol 37.67% 22.405 µg/mg [53]
S. tennerimum
Bovine testes Flavonoids from Piper Flavonoid 69.75 – 72.78% 1000-6.25 mg/mL [54]
peltatum hydroalcoholic
extract
Venom from snake Flavonoids from Piper Flavonoid 14.59 – 40.17% 1000-6.25 mg/mL [54]
(Naja naja atra) peltatum hydroalcoholic
extract
Venom from snake Flavonoids from Piper Flavonoid 4.69 – 23.05% 1000-6.25 mg/mL [54]
(Bothrops atrox) peltatum hydroalcoholic
extract
Not specified Picrorhizaoside E from Acylated iridoid 61.8% 100 µM [55]
Picrorhiza kurroa glycoside
Bovine testes Vitamin C Antioxidant 97% 5% [16]
Bovine testes Gallic Acid Antioxidant 42.4% 5 µg/mL [58]
Bovine testes Sodium Alginate Polysaccharide 78% 22.3 µg/mL [59]
Bovine testes Oleanolic Acid Fatty acid 76.5% 219.2 µg/mL [60]
Bovine testes Quercetin Flavonoid 29% Not specified [17]
Snake venom Quercetin Flavonoid 8% Not specified [17]
Bovine testes Tranxanox Anti – allergic drug 85% 0.04 mM [61]
Bovine testes Indole carboxamides Synthetic molecule 2 – 6% 50 µM (pH 7) [62]

Kinetic necessary to analyze the kinetics of each of them to


Considering that there are different determine the similarity or differentiation between each
hyaluronidase origins, as mentioned before, it is hyaluronidase and its subtypes. The most used enzyme

7
in the different industries is bovine’s testicular origin, competitive behavior by Lineweaver-Burke
which in a study carried out showed to follow the linearization. Such behavior is attributed to the presence
Michaelis-Menten enzymatic kinetic using hyaluronic of syndecans, which are components present in high
acid as substrate and obtaining Km and Vmax values of concentrations in cell membranes and in pericular
2.23 mM and 19.85 U/mL, respectively [63]. Another matrix [65]. Another inhibition carried out with an
study reached the same kinetic model result, but the hydrangenol derivatives called BT-1 showed strong
values reported for Km and Vmax were 0.16 mM and inhibition activity but with this inhibitor the enzyme no
0.05 mM/min (Fig. 3), respectively, in this study the longer followed the Michaelis-Menten kinetic model,
analytical method used was fluorescence, considered and the reason could not be determined [66], however,
one of the most effective and sensible methods for this allowed to get to the conclusion of that natural
measurement enzymatic activity at present [64]. inhibitors showed a higher percentage of inhibition than
inhibitors get from drugs.
An inhibition applied to hyaluronidase with
heparin at near physiological conditions showed non-

Fig 3. Michaelis-Menten graph of hyaluronidase activity [64]

The enzymatic activity carried out on the showed that when the concentration of the inhibitor was
hyaluronidase obtained from scorpion venom did not higher the inhibitory activity tended to decrease [15].
showed a Michaelis-Menten kinetic model and could Plus, the enzymatic activity was measurement
not be attributed to any of the known models due to their spectrophotometrically at 490nm because it was used a
structural complexity. However, its inhibition made colorimetric method that develop an orange color and
with five different types of Brazilian antivenoms this one can absorb blue color that corresponds to a
extracted from animal fluids is shown in Fig. 4 and wavelength between 450 and 500nm [67].
Fig 4. Different hyaluronidase scorpion venoms enzymatic activity [48]

At last, but not least, the inhibition made with vitamin C inhibitory percentage is required. However, in other
in testicular bovine hyaluronidase could not be study of inhibition with different concentrations (6.22
attributed to any defined kinetic model, but showed ng/mL, 10.6 ng/mL and 0.2 µg/mL) of ascorbic acid, the
none inhibition at 1mM and a 50% inhibition of the enzymatic activity was defined by the model BTH and
bacterial type of enzymes [68], this allowed the it was useful to define the amino acid sequence
conclusion that higher inhibitor concentrations are alignment with the sequences of hyaluronidase so the
counterproductive and significantly reduced the mechanism of action could be thoroughly understood
inhibitory activity that’s the reason why concentration [69].
between 5 and 250 µM are the right ones if a higher

Fig 5. Different concentrations of ascorbic acid (6.22 ng/mL (▲), 10.6 ng/mL (●) and 0.2 µg/mL (■)) as inhibitor and its
influence in the enzymatic activity of hyaluronidase [69].

9
Ethics lower yields giving enough reasons for researchers and
As it was discussed earlier in the article, investigators on not to use them and allowing to
hyaluronidase inhibition and obtention have a lot of conclude the reasons why they are not commonly used
industrial and medical applications. It can be used for in the applications on industries [44, 61].
Alzheimer’s and cancer treatments, antiaging or even
for ocular surgery. However, it´s obtention must be from Most common use of hyaluronidase is a reversal
specific cellular tissues of animals, bacteria, or humans. agent in bad hyaluronic acid injection; at the beginning
To what extent is it valid to treat diseases knowing it is it was difficult to understand that injection of hyaluronic
cure may involve killing or mistreating animals? It is acid could had side effects as it is well received by the
true that some hyaluronidase extraction methods do not skin because it is naturally produced by the dermis but
involve animals, but studies show these alternatives are when the acid is injected in the deep dermis it starts
the ones with the lowest yields, from around 18% to creating masses capable of roll against the bone showing
23%. For industrial processes, the extractions are up physically as shadows and deformities and to reduces
optimized by using slaughtered animals with a time of the measure of the masses hyaluronidase is used but it
death lower than three hours, by removing bovine has to be carefully use because higher concentrations
testicles, snake tails, fangs removal or bee´s sting [39, can create side effects like predisposition to the
44]. It is important to know that the biggest yields found development of diseases related with tumors [70].
for extractions fluctuates from 78,7% to 82% [47, 48, Hyaluronidase cannot be injected in its purest form
49]. Nonetheless, the total amount extracted from a because it can interfere with skin hydration processes,
singular tissue is not enough for the market demand, with the appearance of wrinkles and spots and with
which implies the sacrifice of numerous organisms [42]. growth of tumors or masses, for that reason the
Thanks to the difficulties of source and extraction recommend treatment of wrong hyaluronic acid
procedure, the hyaluronidase is one of the most injection is a solution of hyaluronidase (70 U) and 0.5%
expensive enzymes found in the market. As the medical lidocaine with epinephrine (1.5 cc) in women of slim
implications are constantly growing, several studies build and average height between 30 and 40 years old
attempted to use frozen seminal fluids from humans [47] [71]; the injection of a solution of hyaluronidase as a
or optimize Streptococcus production [40] with no treatment is well received by the human body because
success. its activity gradually decrease for being involved in
processes like dilution, diffusion and deactivation [72].
Otherwise, as it was mentioned before,
inhibition is mostly made with compounds obtained Additionally, the hyaluronidase should be
from natural sources or its derivatives, so this aspect is injected following some security protocols to avoid side
the most ethical aspect when working with effects. These protocols are kind of dependent of the
hyaluronidase in different stages such as investigation surgeon or esthetician, but in general terms is important
or application in some industries like medical and to dosage according to the quantity of affected tissues to
aesthetics, however, this does not mean some inhibitors it be enough to allow a vessel that cannot longer
have origin on pharmaceutical drugs or on animals’ transport blood but not too much to foster an
fluids, nevertheless these are also the inhibitors with environment of side effects, so in a nutshell it should be

10
a flooding of the zone but not an excess of the enzyme. only limited to the affected areas. The specialist should
The injection of appropriate concentration and doses of adjust the amount of HYAL to be injected according to
hyaluronidase depends on the surgeon experience and the type of procedure performed, as fillers with higher
comes along with some techniques such as the concentrations of HA will require more HYAL [78]. In
identification of low or high-volume events, source of addition, it should be specified that the patient has not
hyaluronidase and decision making about injection, that ingested prior to the intervention, any of the antagonists
can be intra-arterial or externally applied and the of these enzymes, such as anti-inflammatory agents,
consequences of each of them [73]. among which are indomethacin, dexamethasone, and
salicylates; drugs of vegetable origin (flavonoids and
Once the dermal procedure has been performed, antioxidants), antihistamines, mast cell stabilizers,
it is recommended that antibiotics be started for the first heparin, and Vitamin C [79].
two weeks [74]. In more cases, such as when large
amounts of hyaluronic acid filler must be removed, an Ischemia is one of the side effects of HA dermal
incision is recommended for drainage of the HA along fillers, it consists in the decrease of blood flow in the
with the enzyme [75]. When hyaluronidases fail to tissues, generating a decrease of oxygen and nutrients in
dissolve inflammatory nodules or long-lasting fillers, the affected area, which can cause difficulty in
steroids could be employed as an alternative [76]. For breathing, paralysis, or arrhythmia, depending on the
these cases, they should only be administered after the affected tissue or organ [80]. For this case it is necessary
process with antibiotics has been initiated, as these to act quickly, and it is recommended to administer
should only be applied when HYAL are not effective in HYAL within the first four hours after the intervention
removing these fillers, i.e., as a second option and only [81]. There have been cases of patients who experienced
and exclusively for inflammatory nodules or long- vision loss after being injected with these fillers, and
lasting fillers [74]. after instant HYAL administration, got recovered. [78].
DeLorenzi proposed a new treatment to avoid these
The procedure that is usually carried out when secondary effects, which involves dosing HYAL in
performing HA dermal filler correction with relation to the volume of the ischemic tissue. It consists
hyaluronidase relies on a very thorough analysis of the in performing repeated doses every hour to keep
area to be intervened. The area should be investigated hyaluronidase concentrations high in the affected area
with ultrasonography prior to any treatment to identify and thus optimize the degradation of injected HA [82].
the amount, depth and extent of HA injected [77]. This The figure 6 shows the diffusion of hyaluronidase
injection is expected to be as precise as possible and through the arterial wall.

11
Fig 6. Diffusion of hyaluronidase through the arterial wall to degrade hyaluronic acid filler. [73]

The rate at which HYAL diffuses through the the inhibitor functions as a “team player” in the
arterial wall depends on the concentration administered. inhibition. Additionally, it has not been possible to
From a clinical point of view, low concentrations are determine if the inhibition of hyaluronidase is carried
much less effective, whereas high concentrations of out for molecules with specific characteristics like
HYAL may result in higher intra-arterial concentrations solubility; an example of this is the comparison between
and, consequently, more efficient hydrolysis of HA oleanolic acid and vitamin C, the first one is an
[73]. amphipathic molecule and the second one is completely
Therefore, an exhaustive process must be done soluble in water, but both showed a high inhibition
before the injection to determine the concentration of percentage, not allowing to determine if only one kind
hyaluronidase to inject and avoid possible allergic of solubility could be used to inhibit the enzyme.
reactions and side effects that may occur as mentioned
before. Until the moment of this review, it is
recommended to continue investigating the inhibition of
Conclusion hyaluronidase now considering that it is involved in
It has been possible to identify different various treatments and diseases and study the inhibition
hyaluronidase inhibitors, some better than others, but from vitamin C derivatives, so it is possible to identify
unfortunately none of them follows the Michaelis – the real fraction that makes this compound such a good
Menten kinetic model making the inhibition more inhibitor.
difficult to understand. Among the best inhibitors, the
structural coincidence is that they all have a wide variety In ethical terms it is important to resalt that
of aromatic rings or carbonyl groups, and in general hyaluronidase is present in a lot of animals’ fluids, but
terms they have shown a decrease in the synthesis of N- its extraction is difficult to get because the tissue most
acetylglucosamine producing a higher percentage of be fresh and alive, so this includes an unethical method
inhibition. Plus, to date the best inhibitor found is of extraction because of animals’ exploitation. When
Vitamin C but unfortunately it is not known which of its hyaluronidase is taken from human fluids it is also
derivatives carries out the inhibitory activity or whether counterproductive because of the specific conditions of

12
the tissue, plus the yields obtained are not higher enough
to use it as an object of investigation or in industrial
applications even when purifications are made.

When it is about industrial applications is


important to consider that hyaluronidase can generate
allergies and side effects such as tumor growth when the
concentrations used are higher than the recommended
or when the enzyme is purer than the recommendation
of dermatologists. To avoid the treatments on skin with
hyaluronidase the hyaluronic acid filler most be
evaluated before injection as well as the concentrations
to be injected, this will depend on the patient’s
characteristics and the state of its skin.

13
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