ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 1, pp. 1-18 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 1, pp. 3-21.
1
REVIEW
4-Methylumbelliferone, an Inhibitor
of Hyaluronan Synthase, Prevents the Development
of Oncological, Inflammatory, Degenerative,
and Autoimmune Diseases
Viktoriya V. Fedorova
1
, Alexandra Tsitrina
2
, Noreen Halimani
1
,
and Yuri V. Kotelevtsev
1,a
*
1
Skolkovo Institute of Science and Technology, 121205 Moscow, Russia
2
Ben-Gurion University of the Negev, 8410501 Be’er Sheva, Israel
a
e-mail: y.kotelevtsev@skoltech.ru
Received September 24, 2024
Revised November 24, 2024
Accepted December 8, 2024
Abstract— Hyaluronic acid (HA) is the main structure-forming polymer of the extracellular matrix. HA metab-
olism plays an important role in intercellular interaction in healthy organism and in various pathologies.
HA is synthesized by hyaluronan synthase (HAS); mammals have three highly homologous isoforms of this
enzyme: HAS1, HAS2, and HAS3. No highly specific competitive inhibitors of HASs have been described so
far. 4-Methylumbelliferone (4-MU), a natural coumarin compound, is commonly used to inhibit HA synthesis
in vivo and in cell cultures. The review is focused on the molecular mechanisms underlying the therapeutic
effects of 4-MU and discusses results of 4-MU application in tissue cultures and animal disease models, as well
as in first clinical trials of this compound. It was found that along with receptors and transcription factors,
one of the pharmacological targets of 4-MU is HAS2, which is most common isoform of HAS. Moreover, it is
inhibition of HA synthesis that underlies the pharmacological effects of 4-MU in oncological, autoimmune,
degenerative, and hypercompensated regenerative processes (fibrosis, scar formation). New clinical drugs
based on specific HAS2 inhibitors will be the first-in-class compounds to treat a wide range of diseases.
DOI: 10.1134/S0006297924603459
Keywords: 4-methylumbelliferone, Odeston, Hymecromone, hyaluronan synthase inhibition, hyaluronan synthase
Abbreviations: HA, hyaluronic acid; ECM, extracellular matrix; 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbellif-
erone beta-D-glucuronide; HAS, hyaluronan synthase.
* To whom correspondence should be addressed.
“The most fruitful basis of the discovery of a new drug is to start with an old one”
Sir James Black, Nobel Prize Laureate 1988
INTRODUCTION
Development of new drugs in the post-genomic
era is based on detailed knowledge of signaling path-
ways and key effectors or pharmacological targets
(enzymes, receptors, and transcription factors). At the
same time, physiologically active substances still play
an important role in the identification and validation
of pharmacological targets. Numerous experimental
data have confirmed the therapeutic effect of the
natural coumarin compound 4-methylumbelliferone
(4-MU) in animal models of oncological, autoimmune,
degenerative, and hyperproliferative diseases. This
review is focused on the studies on the validation of
hyaluronan synthase (HAS) as the main 4-MU phar-
macological target, which is an essential step in the
development first-in-class drugs, namely, inhibitors of
hyaluronic acid (HA) synthesis.
FEDOROVA et al.2
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
THE ROLE OF EXTRACELLULAR MATRIX
IN NORMAL AND PATHOLOGICAL STATES
Extracellular matrix (ECM), which forms the basis
of connective tissue, is a highly organized interstitial
structure that ensures mechanical integrity and cell–
cell interaction.
ECM consists of polymeric carbohydrates glycos-
aminoglycans (GAGs), proteins (mainly, fibrillar), and
proteoglycans (PGs). ECM is a barrier and, at the same
time, a depot for peptide hormones and cytokines.
It also directly generates chemical and mechanical
signals essential for the maintenance of tissue homeo-
stasis. Pathological processes typical of many systemic
diseases lead to the ECM rearrangement and chang-
es in its structure, which eventually contributes to
changes in the tissue architecture and results in the
development of diseases, such as fibrosis, osteoarthri-
tis, and cancer [1, 2].
HA is a linear polymer consisting of D-glucuronic
acid and D-N-acetylglucosamine residues connected by
alternating β-1,4- and β-1,3-glycosidic bonds; it is the
main ECM component by weight. HA homeostasis is
maintained through the synthetic activity of HAS en-
zymes, decomposition by hyaluronidases, and chemical
degradation mainly via the action of reactive oxygen
species. There are three HAS isoforms. HAS1 is ac-
tive during embryogenesis. HAS2 is the main isoform
both in embryogenesis and in most tissues during the
postnatal period; it synthesizes high-molecular-weight
(HMW) HA fraction with a weight of 1000-6000 kDa;
HAS3 synthesizes low-molecular-weight (LMW) forms
of HA weighing less than 250 kDa. HMW HA usually
has the anti-inflammatory, antiangiogenic, and an-
ti-cancer properties. On the contrary, LMW fractions
of HA exhibit proinflammatory and proangiogenic ef-
fects and promote cell adhesion. Although these prop-
erties of HA have been well established, the mech-
anisms underlying them are poorly understood and
need further exploration [3].
Human body also has various types of hyaluro-
nidases (HYALs) that cleave HA. The most thoroughly
characterized of them are HYAL1 and HYAL2. HYAL2
degrades HA into the fragments approximately 50
monomers in length (~20kDa), while HYAL1 hydrolyz-
es HA into tetrasaccharides (~1600Da), which undergo
further degradation in the lysosomes [4]. Pathological
processes, such as disruption of HA metabolism, can-
cer, tissue damage, and inflammation, can change this
balance, thus increasing the concentration of LMW
HA. There is a large body of evidence indicating HA
involvement in the chronic inflammation character-
istic of type 2 diabetes, liver cirrhosis, asthma, and
cancer progression and metastasis. Thus, HA pro-
motes adhesion and motility of metastatic melanoma
cells [5], enhances motility of pancreatic [6] and pros-
tate cancer cells [7], hinders drug delivery to tumors
[8-10], promotes drug resistance [11], stimulates cell
division [12], and acts as an immune regulatory fac-
tor [13]. Upregulated HA synthesis in the tumor stro-
ma is a negative prognostic factor [14-18].
The blood level of HA is a marker of liver fibro-
sis. In a fibrotic liver, HA is synthesized by fibroblasts
originated from activated stellate cells. Normally, stel-
late cells do not express HAS2 (the main enzyme that
produces HA in adult tissues) and do not synthesize
HA. Liver damage leads to the production of TGF-β,
which triggers transdifferentiation of stellate cells
into myofibroblasts and dramatically increases HAS2
expression in them [19]. HA accumulation in the pa-
renchyma results in the activation of Notch1 signaling
pathway in stellate cells, leading to their activation,
increased synthesis of the ECM, and development of
fibrosis [20]. Therefore, HAS2 and HAS3 are important
pharmacological targets in the treatment of diseases
associated with pathological activation of HA synthe-
sis, in particular, liver fibrosis.
Elucidation of molecular mechanisms of HA syn-
thesis by mammalian HASs has become important in
the context of the targeted search for their specific in-
hibitors that can be used as drugs. These mechanisms
have been discussed in most detail in the review by
DeAngelis and Zimmer [21]. Within a few years af-
ter the discovery of bacterial HAS in Streptococcus
pyogenes (SpHAS), three isoforms of vertebrate en-
zyme (HAS1-3) and viral HAS (CvHAS of Paramecium
bursaria Chlorella virus-1, PBCV-1) have been identi-
fied. CvHAS is similar to the vertebrate enzymes in
the overall architecture of the cytoplasmic domain
containing the active site and transmembrane (TM)
domains, with two TM helices at the N-terminus and
four at the C-terminus. All these enzymes belong to
class I glycosyltransferases, but vertebrate HASs and
viral CvHAS add sugars to the nonreducing end of the
growing HA chain (Fig.1), whereas SpHAS adds sugars
to the reducing end.
Vertebrate HASs, CvHAS, and SpHAS have the
glycosyltransferase domain of the second type (GT-2),
which allows to incorporate both uridine 5′-diphos-
phoglucuronic acid (UDP-GlcA) and uridine 5′-disphos-
phate N-acetylglucosamine (UDP-GlcNAc). The three-di-
mensional structure of CvHAS has been determined by
electron cryomicroscopy [22]. 4-MU, which significant-
ly reduces expression of HAS2/HAS3 [1], is a widely
used and the only well-characterized inhibitor of HA
synthesis that is known under commercial names of
Hymecromone and Odeston. It has been approved for
the clinical application in Europe and Asia and is rou-
tinely used as a hepatoprotector, antispasmodic, and
choleretic in biliary dyskinesia. In Italy, this drug has
been approved by the Italian Medicines Agency (AIC
no.02130002) and is sold under the name Cantabiline.
THERAPEUTIC PROPERTIES OF 4-METHYLUMBELLIFERONE 3
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Fig. 1. HA synthesis by HAS enzymes. UDP-GlcNAc, uridine diphosphate N-acetylglucosamine; GlcA, glucuronic acid.
Fig. 2. Proposed mechanism of 4-MU effect on HA synthesis. a) Normal pathway of HA synthesis. b) UDP substitution by
4-MU resulting in the suppression of HA synthesis by HAS (from Nagyetal. [26]).
THE MECHANISM OF 4-MU ACTION
ON HYALURONIC ACID SYNTHESIS
There is no evidence of competitive inhibition or
even direct interaction of 4-MU with HAS. 4-MU does
not affect the enzymatic activity of the solubilized
HAS [23]. The most common hypothesis is that 4-MU
acts a competitive substrate for uridine 5′-diphos-
phate-glucuronosyltransferase (UGT), thus depleting
the cellular pool of uridine 5′-UDP-GlcA utilized in HA
synthesis [24-26] (Fig. 2).
However, this hypothesis has not been con-
firmed experimentally. As an evidence against it, it
was shown that 4-MU does not affect the synthesis
of other glycosaminoglycans, which utilizes the same
monomers as the synthesis of HA. In addition, couma-
rins with alkylated 7-hydroxy group, which cannot be
the substrates for UGT, were still found to inhibit HA
synthesis with a high efficiency in vitro [27]. 4-MU has
been shown to reduce the expression level of HAS2
mRNA [25, 28, 29] and simultaneously upregulate ex-
pression of Hyal1 gene [30]. It also reduced the levels
of phosphorylase and uridine 5′-diphosphate glucose
dehydrogenase [31]. It still remains unknown how HA
synthesis is regulated at the transcriptional level and
Fig. 3. Mechanisms of HA synthesis inhibition by 4-MU:
competition with HA precursor UDP-GlcA; inhibition of HAS2
gene expression; indirect inhibition of HASs (from Vitale
et al. [1]).
FEDOROVA et al.4
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
whether involved mechanisms are selective for these
particular mRNAs (Fig. 3).
As has been shown by our group and other re-
searchers [32, 33], 4-MU has multiple targets not di-
rectly related to HA metabolism. It is possible that a
decreased HA accumulation observed in vitro studies
is the cumulative effect of several parallel processes,
including possible substrate depletion, downregula-
tion of HAS2 expression (as experimentally demon-
strated), and activation of Hyal1 expression [30]. HAS2
expression is also known to be regulated by nuclear
receptors, in particular, glucocorticoid receptor. Thus,
expression of HAS2 was almost completely suppressed
by dexamethasone [34]. Cells exposed to 4-MU demon-
strated alterations in the cell cycle and p53 signaling
cascade [35, 36].
EFFECT OF 4-MU ON VARIOUS TYPES
OF CANCER AND AUTOIMMUNE
AND INFLAMMATORY PROCESSES
4-MU multiple processes associated with tumor
progression, such as migration, proliferation, and in-
vasion of cancer cell and angiogenesis, as well as in-
fluences cells of tumor microenvironment (fibroblasts,
endothelial and immune cells). Cancer development
involves rapid changes in the structure and composi-
tion of the ECM (whose main component is HA), which
requires creation of new drugs capable of changing
the properties of the ECM. This approach is promis-
ing in the treatment of various types of cancer, and
4-MU has already been approved for the use in this
new capacity.
Using a mouse model of carbon tetrachloride-in-
duced liver fibrosis, we demonstrated that formation
of collagen fibers is preceded by HA synthesis along
the boundaries of liver lobules. 4-MU inhibited HA
synthesis and significantly decreased formation of col-
lagen fibers around hepatic lobules [30]. In our recent
study, siRNA-mediated knockdown of the HAS2 gene
reproduced the effect of 4-MU on several signaling
pathways and transcription of some key genes, result-
ing in suppression of liver fibrosis [37].
The use 4-MU in the treatment of brain cancer is
especially interesting. The ECM of malignant gliomas
and glioblastomas is characterized by an increased HA
content; HA stimulates adhesive and invasive process-
es of tumor cells [38]. 4-MU is a small molecule ca-
pable of passing through the blood-brain barrier and
inhibiting the synthesis of HA, which has promoted
studies on its possible application in the treatment
for gliomas and glioblastomas. Thus, in mouse mod-
els, high doses of 4-MU reduced HA synthesis, reduced
proliferation and migration of glioblastoma cells, and
stimulated their apoptosis [39-41]. As shown in in vitro
and in vivo experiments, 4-MU reduced prolifera-
tion of glioma cells by regulating autophagy [42].
Chistyakov et al. [43] demonstrated that 4-MU inhibited
the inflammatory response of astrocytes. Oral adminis-
tration of 4-MU in mice caused a significant decrease
in the HA content in the spinal cord and brain, re-
duction in synaptic stability, and reactivation of neu-
roplasticity, which resulted in improved memory [44].
The data on preclinical studies on 4-MU applica-
tion for the treatment of various diseases are given
in Table 1.
THE PROSPECTS OF COMBINED THERAPY
WITH 4-MU. THE EFFECT OF 4-MU
ON THE TUMOR PHYSICAL BARRIER.
THE USE OF 4-MU AS A HYALURONIC ACID
SYNTHESIS INHIBITOR
HA-rich ECM forms a biological barrier of the
tumor microenvironment. This barrier regulates the
activity of immune effectors [13, 98], prevents drug
diffusion [99], hinders the adsorption of transgenic
vectors in gene therapy [100], and plays an import-
ant role in the acquisition of resistance to anticancer
drugs [1, 11, 101].
The possibility of changing the properties of tu-
mor microenvironment in order to improve the result
of antitumor therapy has been actively investigated.
The pathological tumor microenvironment is charac-
terized by hypoxia and high interstitial fluid pressure,
leading to tumor progression and resistance to treat-
ment [102]. Increased interstitial pressure is consid-
ered to be the most important barrier for efficient
drug distribution within the tumor. The reasons for
the increased interstitial pressure in the tumor are
numerous and include extensive intratumor vascular
network, insufficient development of lymphatic ves-
sels, changes in the ECM components, and pressure
created by constantly dividing tumor cells [103, 104].
An increased HA content in tissues surrounding the
tumor contributes to the increase in the ECM volume
and, as a result, increases in the pressure inside the
tumor [105, 106]. Such high HA content in the tumor
microenvironment forms a physical barrier that re-
stricts the access of monoclonal antibodies and immune
cells to the tumor tissue, which is one of the mech-
anisms of tumor resistance to immunotherapy [107].
Due to the ability to inhibit the synthesis of
HA, 4-MU was suggested for the adjuvant therapy in
combination with the primary anticancer therapy.
Using various models, it has been shown that the use
of 4-MU in a combined therapy for various types of
cancer increased the treatment efficacy, reduced the
toxicity of antitumor drugs, and helped to overcome
emerging chemoresistance (Table 2).
THERAPEUTIC PROPERTIES OF 4-METHYLUMBELLIFERONE 5
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Table 1. Preclinical studies on the application of 4-MU in the treatment of various diseases
Organ/system Studied disease Year Type of investigation Reference
Inflammation
acute respiratory distress syndrome (ARDS)
2013 in vitro [45]
2015 in vitro [46]
allergic inflammation 2022 in vitro [47]
allergic rhinitis 2022 in vitro/in vivo [48]
inflammation 2022 in vitro [49]
Head and neck oral squamous cell carcinomas 2022 in vitro [50]
Bile ducts
biliary dyskinesia 1984 in vivo [51]
biliary colic 1995 in vivo [52]
Immune response
Graves’ orbitopathy 2020 in vitro [53]
transplant rejection 2021 in vitro/in vivo [54]
autoimmune response to transplanted
islets of Langerhans
2020 in vitro/in vivo [55]
acute lung allograft rejection 2021 in vitro/in vivo [56]
Bone marrow chronic myeloid leukemia
2013 in vitro [57]
2016 in vitro [58]
2017 in vitro [59]
Lungs
pleural mesothelioma 2017 in vitro/in vivo [60]
pulmonary fibrosis,
pulmonary hypertension
2017 in vivo [61]
Mammary glands breast cancer
2019 in vitro [62]
2022 in vitro [63]
Bladder bladder cancer 2017 in vitro/in vivo [64]
Peripheral
nervous system
malignant peripheral nerve sheath tumor 2017 in vitro/in vivo [65]
Liver
hepatocellular carcinoma
2012 in vitro/in vivo [66]
2015 in vitro/in vivo [67]
2019 in vitro/in vivo [29]
2021 in vitro/in vivo [68]
2022 in vitro/in vivo [69]
liver metastasis of malignant melanoma 2005 in vitro/in vivo [70]
liver fibrosis 2019 in vivo [30]
steatohepatitis 2021 in vitro/in vivo [71]
FEDOROVA et al.6
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Table 1 (cont.)
Organ/system Studied disease Year Type of investigation Reference
Pancreas
pancreatic cancer
2006 in vitro/in vivo [72]
2016 in vitro/in vivo [73]
2017 in vitro/in vivo [74]
2018 in vitro/in vivo [75, 76]
pancreatic ductal adenocarcinoma 2019 in vitro [77, 78]
Kidneys
renal cell carcinoma 2013 in vitro [79]
kidney ischemia-reperfusion injury 2013 in vivo [80]
metastatic renal cell carcinoma 2020 in vitro [81]
diabetic nephropathy 2021 in vivo [82]
advanced renal cell carcinoma 2022 in vitro/in vivo [83]
Prostate prostate cancer
2010 in vitro [84]
2015 in vitro/in vivo [85]
Connective tissue fibrosarcoma
2017 in vitro [86]
2019 in vitro [87]
2020 in vitro [88]
2021 in vitro [89]
Large intestine colorectal carcinoma 2015 in vitro/in vivo [90]
Central nervous
system
glioblastoma
2021 in vitro [39, 40]
2022 in vitro/in vivo [91]
Endometrium endometriosis
2016 in vitro/in vivo [92]
2020 in vitro [93]
2023 in vivo [94]
Ovaries ovarian cancer
2014 in vitro [95]
2019 in vitro/
in vivo [96]
2020 in vitro [97]
Table 2. Preclinical studies on the treatment of various types of cancer using combinations of anticancer drugs
and 4-MU
Type of cancer Main treatment Type of study Year Reference
Hepatocellular
carcinoma
immunotherapy:
IL-12-encoding adenovirus (AdIL-12)
in vitro 2018 [111]
Glioblastoma temozolomide in vitro 2023 [41]
THERAPEUTIC PROPERTIES OF 4-METHYLUMBELLIFERONE 7
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Table 2 (cont.)
Type of cancer Main treatment Type of study Year Reference
Malignant pleural
mesothelioma
trametinib in vitro/in vivo 2017 [60]
Colorectal
carcinoma
cyclophosphamide
with immunotherapy (AdIL-12)
in vitro/in vivo 2015 [90]
Melanoma vemurafenib in vitro 2021 [109]
Esophageal squa-
mous cell carcinoma
dichloroacetic acid in vitro/in vivo 2019 [108]
Oral squamous cell
carcinoma
radiotherapy in vitro 2022 [50]
Renal cell
carcinoma
sorafenib in vitro 2013 [79]
Advanced renal cell
carcinoma
sorafenib in vitro/in vivo 2022 [83]
Pancreatic cancer 5-fluorouracil in vitro/in vivo 2018 [76]
Pancreatic cancer gemcitabine in vitro/in vivo 2006 [72]
Ovarian cancer carboplatin in vitro/in vivo 2019 [96]
Bladder urothelial
carcinoma
cisplatin or doxorubicin in vivo 2019 [110]
Fibrosarcoma radiotherapy
in vitro 2021 [89]
in vitro 2019 [87]
in vitro 2017 [86]
Chronic myeloid
leukemia
imatinib in vitro 2017 [59]
Chronic myeloid
leukemia
doxorubicin in vitro 2016 [58]
According to the data on the use of 4-MU as an
addition to the main therapy, 4-MU enhanced the ra-
diosensitivity of radiation-resistant cells in oral squa-
mous cell carcinoma [50] and fibrosarcoma [86-89].
Inrenal cell carcinoma, sorafenib in combination with
4-MU inhibited more efficiently proliferation and in-
vasion of cancer cells, suppressed capillary formation,
and induces apoptosis of tumor and endothelial cells
[79, 83]. 4-MU increased the efficacy of 5-fluorouracil
[68] and gemcitabine [72] against pancreatic cancer,
inhibited cell proliferation, and decreased the size of
primary tumors and metastases, as well as promot-
ed survival of affected animals. 4-MU increased the
sensitivity of glioblastoma cells to temozolomide by
enhancing the cytotoxic effect of the drug [41]. 4-MU
exacerbated the cytotoxic effect of carboplatin on che-
moresistant ovarian cancer cells [96]. A combined use
of dichloroacetate and 4-MU in a model of esopha-
geal squamous cell carcinoma promoted apoptosis of
cancer cells and inhibited tumor growth [108]. 4-MU
increased the sensitivity of myeloid leukemia cells to
doxorubicin [58] and promoted their senescence [59].
A combination of vemurafenib with 4-MU reduced the
survival of melanoma cells more efficiently compared
to vemurafenib monotherapy [109]. 4-MU enhanced
the chemosensitivity of bladder urothelial carcinoma
cells to doxorubicin and cisplatin [110]. 4-MU signifi-
cantly reduced the interstitial tumor pressure and im-
proved perfusion, thus ensuring more efficient expres-
sion of the adenovirus transgene in the IL-12 (AdIL-12)
immunotherapy of colorectal cancer [90]. In a liver
cancer model, a combination of 4-MU with AdIL-12
FEDOROVA et al.8
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
led to a more pronounced inhibition of tumor growth
and increased survival of mice compared to AdIL-12
monotherapy [111].
THE USE OF 4-MU AS A HEPATOPROTECTOR
AND CHOLESTATIC AGENT TO REDUCE
THE HEPATOTOXICITY OF PRIMARY THERAPY
Immune checkpoint inhibitors, cytokines, and
antibodies against these proteins are used as immu-
nomodulators to enhance the body immune response
to tumors and chronic inflammation foci in rheuma-
toid, autoimmune, and inflammatory diseases [112,
113]. These drugs have successfully passed clinical
trials and have been approved for the use in clinical
practice by the European and American drug agen-
cies [114]. However, up to 17% patients receiving such
immunotherapy suffer from complications associated
with the damage of liver and bile duct [115-119].
Depending on the severity of complications, the
treatment for hepatotoxicity might include cessation
of therapy with immune checkpoint inhibitors. Cor-
ticosteroids and immunosuppression (in more se-
vere cases) can be recommended as well [120-123].
Ursodeoxycholic acid (UDCA) is used to improve the
liver function in the case of cholestatic hepatotoxicity,
when corticosteroids are ineffective [124-127]. UDCA
has the hepatoprotective and choleretic effects and
is considered as the treatment standard for choles-
tatic liver diseases with the autoimmune component
(primary biliary cholangitis, primary sclerosing chol-
angitis) [128-130]. To our knowledge, there are no
reports on the effect of 4-MU on the risk of hepato-
toxicity development in response to immunotherapy.
However, its established cholestatic and hepatoprotec-
tive properties make 4-MU a promising agent for such
studies.
The data accumulated strongly suggest the need
for the clinical trials of 4-MU as an agent for the adju-
vant/additional antitumor therapy that would reduce
the HA content, modify the ECM and tumor micro-
environment, decrease interstitial pressure, improve
tumor perfusion, facilitate drug access, and produce
hepatoprotective and cholestatic effects, thus decreas-
ing the risks of hepatotoxicity during immunotherapy.
TOPICAL APPLICATION OF 4-MU
TO PREVENT FORMATION OF STRETCH MARKS,
SCARS, KELOID SCARS, SUNBURNS,
AND HYPOPIGMENTATION FOCI
Topical application of 4-MU leads to efficient in-
hibition of HA synthesis in the skin [131]. 4-MU has
been shown to prevent keratinocyte activation and to
reduce epidermal hyperproliferation [132] and migra-
tion rate of keloid keratinocytes, thus decreasing the
likelihood of keloid scar formation [133].
4-MU enhances the processes of melanogenesis,
which makes it a promising agent in the treatment
of skin conditions associated with hypopigmenta-
tion, as well as a cosmetic product to provide natural
tan [134].
METABOLISM OF 4-MU.
TOXICITY AND SAFETY FOR HUMANS
Like all coumarins, 4-MU is poorly soluble in wa-
ter. It is a nonpolar molecule and therefore, can easily
pass through the lipid barrier in the intestine. It is
almost completely absorbed upon oral administration
and is excreted in urine and bile [26]. The methyl
group at position 4 ensures low toxicity of 4-MU by
preventing its metabolism to coumarin 3,4-epoxide
by cytochrome P450, as well as weak anticoagulation
properties compared to other coumarins, such as
dicoumarol and warfarin [135].
When ingested, 4-MU is very rapidly and almost
completely metabolized to 4-methylumbelliferone be-
ta-D-glucuronide (4-MUG) in the liver and small intes-
tine, which until recently, has limited its use in the
treatment of bile ducts only [1, 136-138]. Less than 3%
of orally administered 4-MU remains unchanged at
the systemic level, while intravenous administration
of 4-MU provides 10-30 times higher concentration of
this compound in the blood [26, 139]. The half-life of
orally administered 4-MU is only 28 min for humans
and 3 min for mice [140, 141]. At the same time, the
median concentration of 4-MUG in the plasma is more
than 3000 times higher than the concentration of
4-MU [26, 141], i.e., most of 4-MU is present as 4-MUG
in a body. However, despite its low bioavailability and
short half-life, orally taken 4-MU efficiently inhibits
HA synthesis. 4-MUG was proven to be as efficient as
4-MU in inhibiting HA synthesis; moreover, it is hydro-
lyzed back to 4-MU inside the cells [137]. Therefore,
to evaluate the pharmacodynamics of 4-MU, it is nec-
essary to take into account the effect of its metabo-
lite 4-MUG. These data suggest that 4-MU can be used
for the treatment of diseases beyond the biliary tract.
For example, as a small nonpolar molecule, 4-MU is
able to cross the blood-brain barrier and inhibit pro-
liferation of glioma cell [42].
A typical regimen of 4-MU administration for an
adult is 900-2400 mg/day [26]. No mutagenic or geno-
toxic effects of 4-MU have been found [1, 142, 143].
Clinical trials in patients with chronic hepatitis B
and C (NCT00225537), healthy individuals, and pa-
tients with respiratory diseases (NCT02780752) [144]
have proven the safety of 4-MU (see Table 3).
THERAPEUTIC PROPERTIES OF 4-METHYLUMBELLIFERONE 9
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Table 3. Clinical trials on the use of 4-MU in the treatment of various diseases
Disease (study) Status Year
Reference/
Identifier clinicaltrials.gov
Interstitial lung diseases
(SOLID Study)
phase II;
recruitment of participants
has not started
2024 NCT06325696
Primary sclerosing cholangitis
phase II;
participants are being recruited
2022 NCT05295680
COVID-19 no information available 2022 NCT05386420
Pulmonary hypertension,
including interstitial lung diseases
(SATURN Study)
phase II; completed 2021 NCT05128929
Healthy participants; study
of 4-MU effect on HA synthesis
phase I; completed 2016 [144]/ NCT02780752
Biliary sludge stage 2 no information available 2016 [145]
Chronic hepatitis C virus
and hepatitis B virus
no information available 2005 NCT00225537
Biliary dyskinesia no information available 2005 [146]
Biliary dyskinesia no information available 2001 [147]
Biliary dyskinesia no information available 1995 [52]
Study of 4-MU bioavailability no information available 1993 [141]
Symptoms after bile duct surgery no information available 1988 [148]
Biliary dyskinesia after
cholecystectomy
no information available 1984 [51]
CONCLUSION
Despite numerous experimental studies demon-
strating the efficacy of 4-MU in various animal mod-
els of oncological, immune, and degenerative diseases,
the molecular mechanisms of its action remain hypo-
thetical. It was demonstrated (at least in the model of
liver fibrosis) that the knockdown of the gene encod-
ing HAS2 led not only to the suppression of fibrosis,
but also to changes in the transcriptome that were
similar to those observed upon oral administration of
4-MU [37]. At the same time, it cannot be excluded
that some of effects of 4-MU may be independent of
the HA synthesis inhibition [62, 149]. 4-MU may also
act through different mechanisms depending on the
type of cancer. However, taken together, the data on
the effectiveness of 4-MU prove the need for a detailed
study of its pharmacokinetic and pharmacodynamic
properties to develop the treatment regimen (admin-
istration route, doses affecting 4-MU bioavailability, in-
tervals between doses, and administration schedule).
The first toxicology studies have already been con-
ducted in phase I clinical trials [144], which allowed
to proceed to clinical studies of the drug effectiveness
(phaseIIa). This is a worldwide trend. Thus, it is cur-
rently planned to conduct clinical trials on the use
of 4-MU in the treatment of interstitial lung diseases
and cholangitis (see Table3).
The clinical trials of 4-MU include selection of
appropriate doses for particular pathologies and in-
vestigation of metabolite excretion rates and drug bio-
availability. Another important factor is development
of new dosage forms (for example, 4-MU-containing
nanoparticles) that will not only increase 4-MU bio-
availability, but will also lead to the patent protection
of a new drug.
Finally, if HAS is indeed the main pharmacologi-
cal target of 4-MU, development of new chemical com-
pounds using 3D models of HAS2/HAS3 and docking
of potential ligands with the help of artificial intelli-
gence will inevitably result in the creation of original,
first-in-class targeted drugs based on HAS inhibitors.
FEDOROVA et al.10
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Contributions. Yu.V.K. participated in the devel-
opment of review concept and selection of articles and
supervised experimental work of the authors present-
ed in this review; A.C. and N.Kh. provided key con-
clusions from their experimental articles, edited the
manuscript, and participated in its discussion; V.V.F.
conducted literature search and analysis and wrote
the original version of the manuscript.
Ethics approval and consent to participate.
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
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