[Short title + Author Name - P&H title] 31 (2023) 101831

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Saudi Pharmaceutical Journal

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Original article

Integrated genomic network analysis revealed potential of a druggable

target for hemorrhoid treatment
Wirawan Adikusuma a, b, *, Firdayani Firdayani b, Lalu Muhammad Irham c,
Darmawi Darmawi d, g, Muhammad Yulis Hamidy e, Baiq Leny Nopitasari a, Soraya Soraya f,
Nurul Azizah f
a
Departement of Pharmacy, University of Muhammadiyah Mataram, Mataram, Indonesia
b
Research Center for Vaccine and Drugs, National Research and Innovation Agency (BRIN), South Tangerang, Indonesia
c
Faculty of Pharmacy, Universitas Ahmad Dahlan, Yogyakarta, Indonesia
d
Department of Histology, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia
e
Department of Pharmacology, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia
f
Master Program in Biomedical Sciences, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia
g
Graduate School in Biomedical Sciences, Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia

A R T I C L E I N F O A B S T R A C T

Keywords: Hemorrhoids are a prevalent medical condition that necessitates effective treatment options. The current options
Drug repositioning for treatment consist of oral medications, topical applications, or surgery, yet a scarcity of highly effective drugs
Genomic network analysis still exists. Genetic markers provide promising avenues for investigating the treatment of hemorrhoids, as they
Hemorrhoids
may reveal intricate biological mechanisms and targeted drug therapies, ultimately enhancing more precise
Sapropterin
treatment tailored to the patient. This study aims to identify new drug candidates for treating hemorrhoids
through a meticulous bioinformatics approach and integrated with genomic network analysis. After extracting 21
druggable target genes using DrugBank from 293 genes connected to hemorrhoids, 87 possible drugs were
selected. Three of these drugs (ketamine, methylene blue, and fulvestrant) hold potential in addressing issues
associated with hemorrhoids and have been supported by clinical or preclinical studies. Eighty-four compounds
present new therapeutic possibilities for managing hemorrhoids. We highlight that our findings indicate that
NOX1 and NOS3 genes are promising biomarkers, with NOS3 gaining significance owing to its robust systemic
functional annotations. Sapropterin, an existing drug, is closely associated with NOS3, providing a clear target for
biomarker-driven interventions. This study illustrates the potential of combining genomic network analysis with
bioinformatics to repurpose drugs for treating hemorrhoids. Subsequent research will explore the mechanisms for
utilizing NOS3 targeting in the treatment of hemorrhoids.

1. Introduction dilation, vascular thrombosis, collagen degradation, fibroelastic tissue

transformation, and structural disruption of subepithelial anal muscles
Hemorrhoids, a prevalent medical condition, are characterized by (Lohsiriwat 2012). An exhaustive analysis of surgical specimens from
inflammation and swelling of blood vessels encircling the rectal and anal hemorrhoidectomy patients revealed a significant inflammatory
regions. This ailment arises from heightened vascular pressure induced response impacting vascular walls and connective tissue (Lohsiriwat
by factors such as pregnancy, aging, and persistent constipation 2015).
(Sardiñas et al., 2016; Sun and Migaly, 2016). While not life- Determining the prevalence of hemorrhoids remains intricate due to
threatening, hemorrhoids result in discomforting symptoms, including numerous asymptomatic patients avoiding medical consultation.
pain, itching, bleeding, and thrombosis (Sanchez and Chinn 2011). Remarkably, research involving colorectal cancer screening identified
Pathological changes linked to hemorrhoids encompass abnormal vein hemorrhoid prevalence in 39 % of participants, with 55 % being

Peer review under responsibility of King Saud University.

* Corresponding author at: Departement of Pharmacy, University of Muhammadiyah Mataram, Mataram, Indonesia.
E-mail address: [email protected] (W. Adikusuma).

https://doi.org/10.1016/j.jsps.2023.101831
Received 19 August 2023; Accepted 14 October 2023
Available online 20 October 2023
1319-0164/© 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
W. Adikusuma et al. Saudi Pharmaceutical Journal 31 (2023) 101831

asymptomatic (Riss et al., 2012). This underscores the need for inno­ 2. Methods
vative therapeutic avenues. Hemorrhoids predominantly affect in­
dividuals aged 45 to 65 and correlate with conditions that elevate 2.1. Fetching hemorrhoid-associated genes
pressure on the hemorrhoidal venous plexus, such as straining during
defecation due to constipation (Chong and Bartolo 2008; Riss et al., We conducted an extensive inquiry utilizing three authoritative da­
2012). Additional factors, including obesity, pregnancy, chronic diar­ tabases renowned for their comprehensive genetic and disease-related
rhea, anal intercourse, cirrhosis with ascites, pelvic floor dysfunction, data, accessed on July 12, 2023, to systematically assemble a compre­
and a low-fiber diet, contribute to hemorrhoid development (Chong and hensive repertoire of genes implicated in the pathogenesis of hemor­
Bartolo 2008; Jacobs 2014). In addition, genetic factors may influence rhoids. These databases encompassed the GWAS Catalog (https://www.
the presence of haemorrhoidal disease. According to research published ebi.ac.uk/gwas/home) (Sollis et al., 2023), the Comparative Tox­
in 2020, there are a total of 102 risk factors for hemorrhoids in the icogenomics Database (https://ctdbase.org/) (Davis et al., 2023), and
human genome. This research suggests that hemorrhoids are influenced the Open Targets Platform (https://www.targetvalidation.org/) (Ochoa
by genetics (Zheng et al., 2021). et al., 2023). Our search methodology entailed querying these re­
Given the substantial prevalence of hemorrhoids and the potential positories using the term “hemorrhoids” to extract pertinent genetic
for discomfort, pain, and bleeding (Mott et al., 2018), the urgency to insights. A stringent filtering threshold was applied for the Open Targets
advance effective treatments becomes paramount. Present strategies Platform, exclusively retaining genes with score values surpassing 0.3.
encompass adjustments in lifestyle, such as heightened dietary fiber This rigorous criterion ensured the inclusion of genetic information with
intake, as well as utilization of oral medications and topical in­ heightened relevance and confidence. Following the meticulous imple­
terventions like creams and ointments (Lohsiriwat 2012; Sun and mentation of these filtration parameters, the final compendium of
Migaly, 2016). However, the efficacy of these measures might be hemorrhoid-associated genes was curated by eliminating duplicative
limited, offering only transient relief. Consequently, the quest for entries. This amalgamation is a valuable and reliable resource for sub­
innovative and potent solutions for hemorrhoid management is keenly sequent explorations and investigations into the genetic underpinnings
anticipated. The integration of genetic markers represents a pioneering of hemorrhoids and its associated disorders.
leap in the realm of hemorrhoid therapeutic exploration. Exploiting
these genetic markers provides us with the means to unravel the intri­
cate biological mechanisms underpinning hemorrhoids and precisely 2.2. Prioritizing biological risk genes in hemorrhoid susceptibility
identify drug targets. In contrast to the predominantly time-intensive
and costly trial-and-error approach characteristic of many drug discov­ Applying a scoring system, five distinct functional annotations were
ery and development endeavors, a more systematic and rational meth­ integrated into the filter for genes associated with hemorrhoids. The
odology can be embraced to circumvent these challenges. One avenue criteria were as follows: (1) Knockout mouse phenotype (KOMP) - Gene
that shows great promise involves harnessing the power of bioinfor­ contribution to specific mouse phenotypic diseases was assessed through
matics to forecast potential targets and novel drug candidates for hem­ Mammalian Phenotype Ontology (MP) from WebGestalt (2019),
orrhoid treatment. A comprehensive depiction of the study workflow is considering a False Discovery Rate (FDR) q-value < 0.05 as significant
illustrated in Fig. 1, offering a detailed overview of the sequential steps (Liao et al., 2019); (2) Kyoto Encyclopedia of Genes and Genomes
undertaken in this innovative research pursuit. (KEGG) - Molecular pathways were identified using KEGG (Kanehisa
and Goto 2000) within WebGestalt, with a significance threshold set at
q-value < 0.05 (Liao et al., 2019); (3) Gene Ontology (GO) – Utilizing
biological process (BP), cellular component (CC), and molecular func­
tion (MF) categories, distinct biological functions related to hemor­
rhoids were identified. A GO enrichment analysis from WebGestalt was

Fig. 1. An illustration of the bioinformatics-guided drug repositioning process for hemorrhoids. This figure was created with BioRender.com.

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conducted, with a significance threshold of q < 0.05 (Liao et al., 2019). 3.2. Prioritization of genes through functional annotation
Genes scoring greater than or equal to 2 were classified as biological
hemorrhoid-risk genes. Higher annotation scores corresponded to genes We meticulously executed a rigorous functional annotation process
exerting a more pronounced biological impact on hemorrhoid patho­ to identify genes associated with the biological risk of hemorrhoids. To
genesis, termed as “biological risk genes.” prioritize genes for potential drug discovery within the confines of this
study, we adopted a scoring system previously employed and docu­
2.3. Mapping drug targets for hemorrhoids using DrugBank mented in various research publications (Irham et al., 2020; Adikusuma
et al., 2021; Adikusuma et al., 2022; Afief et al., 2022; Zazuli et al.,
During this phase, we employed DrugBank (data released on January 2022; Adikusuma et al., 2023). The ensuing description presents the
4, 2023) to associate biological risk genes linked to hemorrhoids with outcomes of the scoring process across five functional annotations: 1)
potential candidate drugs. DrugBank serves as an online repository of­ genes prioritized via KOMP (n = 22); 2) genes prioritized through KEGG
fering comprehensive insights into drugs and their corresponding gene (n = 22); 3) genes prioritized based on BP (n = 67); 4) genes prioritized
targets, serving as a valuable resource for bioinformatics and chem­ based on CC (n = 24); and 5) genes prioritized based on MF (n = 33).
informatics applications in drug discovery across clinical medical do­ Comprehensive visual representations of the score distribution for each
mains. Notably, DrugBank supports in silico drug target identification, criterion are depicted in Fig. 2A–C and Table S2. Ultimately, a total of
drug design, docking or screening, prediction of drug metabolism and 38 genes, exemplifying characteristics indicative of a biological hem­
interactions, and facilitates general pharmaceutical education (Wishart orrhoid risk, met the established criteria by attaining scores of 2 or
et al., 2018). A range of parameters guided our database queries, higher. A more thorough examination of the gene scores revealed the
including drugs demonstrating pharmacological activity, proven human preeminent status of two genes, NOX1 and NOS3, both of which sur­
efficacy, approved annotations, involvement in clinical trials, or desig­ passed a score of 3, as elucidated in Fig. 2A. This comprehensive
nation as experimental agents. Additionally, each identified drug un­ approach to gene prioritization scores not only facilitates the identifi­
derwent verification on ClinicalTrials.gov (https://clinicaltrials.gov/; cation of pivotal candidate genes for subsequent investigation but also
accessed on July 12, 2023) to ascertain its ongoing clinical investigation unveils notable targets, such as NOX1 and NOS3, which hold consider­
status for hemorrhoids or alternative medical conditions. able promise within the realm of hemorrhoid research.
Moreover, the outcomes stemming from the five distinct functional
2.4. Statistical analyses annotations encapsulate the realms of KOMP, KEGG, BP, CC, and MF.
Notably, the KEGG analysis strategically identified the major pathway as
Statistical analyses were carried out using R (version 4.2.1). Over- “Fluid shear stress and atherosclerosis,” furnishing a pivotal molecular
representation analysis (ORA), encompassing KOMP, GO, and KEGG context that underscores prospective mechanistic associations between
pathway enrichment analysis, was conducted utilizing the WebGestalt hemorrhoids and this specific pathway. Delving into the KOMP domain,
2019 R package (Liao et al., 2019). Visualization of KOMP, GO, and the findings intriguingly underscored a pronounced correlation with
KEGG results was accomplished using the ggplot2 package (R v4.2.1) “abnormal wound healing.” This revelation implies a nuanced connec­
(Wickham 2009). The Venn diagram, illustrating the intersection of five tion between the identified genetic factors and the intricate processes of
functional annotations, was generated using the dedicated R package (R tissue healing that could play a role in hemorrhoidal development. In the
v4.2.1) (Chen and Boutros 2011). Moreover, a chord diagram generated sphere of BP, a pervasive engagement was noted in the “regulation of
by the circlize package in R (Gu et al., 2014) and an alluvial diagram cell death,” unveiling a central biological facet that likely contributes to
generated by the RAWGraphs visualization program were used to the intricate interplay of genetic elements and the manifestation of
visualize the hemorrhoid candidates drugs (Mauri et al., 2017). hemorrhoids. Simultaneously, the MF analysis cast light on a pivotal
molecular mechanism, specifically “cofactor binding,” signaling poten­
3. Results tial interactions that play an integral role in the underlying biological
processes associated with hemorrhoids. Furthermore, the CC explora­
3.1. Gene identification associated with hemorrhoids tion underscored the prominence of the “plasma membrane protein
complex,” highlighting the significance of this specific cellular structure
We meticulously gathered genes linked to hemorrhoids by system­ in the context of gene associations related to hemorrhoids.
atically extracting data from three prominent genomic databases: GWAS To visually encapsulate the significance of the KOMP, KEGG, and GO
Catalog, Comparative Toxicogenomics Database, and the Open Targets enrichment analyses (BP, MF, CC), we present Fig. 3A–E. This figure
Platform. These databases are renowned for their extensive genetic and functions as a succinct summary, vividly illustrating the enriched
disease-related information, making them ideal sources for our investi­ functions within each category. It accentuates the pivotal KOMP, KEGG
gation. Through precise queries employing hemorrhoid-specific search pathways, key biological processes, cellular components, and molecular
terms, we retrieved a diverse array of genetic details pertinent to hem­ functions interwoven with the identified genes, offering an inclusive
orrhoidal conditions. Following a rigorous process of data curation and snapshot of their functional roles. Detailed results for all five functional
the elimination of redundancies, we established a comprehensive annotations are comprehensively outlined in Table S3, thereby serving
compilation of 293 genes that are associated with hemorrhoids. These as a comprehensive resource for further exploration and investigation.
carefully documented genes are meticulously cataloged in Table S1,
furnishing researchers with a valuable and meticulously curated 3.3. Comprehensive analysis for drug repositioning
resource. This compilation serves as an indispensable foundation for in-
depth explorations and inquiries into the genetic intricacies of hemor­ To discern potential therapeutic candidates for hemorrhoids, we
rhoids and its correlated maladies. This curated gene assemblage not embarked on a process of aligning genes associated with biological
only aids in unraveling the molecular underpinnings of hemorrhoids but hemorrhoid risk to relevant drugs within the DrugBank database. This
also holds the potential to uncover novel therapeutic targets. Further­ investigative approach sought to ascertain whether genes culled from
more, it contributes significantly to enhancing our comprehension of the the profiles of biologically risky genes could serve as pharmacologically
genetic factors that influence the initiation and progression of viable targets for approved drugs. This comprehensive endeavor
hemorrhoid-associated conditions. culminated in the identification of a total of 87 drugs, all of which
exhibited targeting interactions with 21 distinct hemorrhoid risk genes.
Consequently, these drugs hold significant promise as prospective can­
didates for the therapeutic management of hemorrhoids, as meticulously

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Fig. 2. Systematic prioritization of candidate genes from hemorrhoid risk loci based on multi-criteria analysis. (A) Summary scores based on five criteria are dis­
played. Fulfilled criteria are represented by filled boxes. Genes with a score of ≥2 were identified as “biological hemorrhoid risk genes.” Refer to Table S2 for
comprehensive information. (B) The Venn diagram presents the prioritization criteria used to select the biological candidate gene from the hemorrhoid risk loci. The
distribution of gene scores is presented in the histogram. The figure displays 38 genes that have a total score of ≥2.

Fig. 3. The top 10 enriched analyses for five functional annotations were identified as follows: (A) Knockout mouse phenotype, (B) Kyoto encyclopedia of genes and
genomes, (C) Biological process, (D) Cellular component, (E) Molecular function.

detailed in Table S4. 2022), and methylene blue was supported by case series evidence for
Within this range of 87 drugs, a diverse landscape emerged: keta­ hemorrhoids (Baldiwala and Trivedi, 2022; Long et al., 2023), as shown
mine (NCT04248205) was under active clinical investigation for hem­ in Fig. 4. Notably, 84 of these compounds represented novel therapeutic
orrhoids, fulvestrant was supported by preclinical in vitro models avenues not previously explored in the treatment of hemorrhoids
demonstrating potential efficacy for hemorrhoids (Fanyu Meng et al., (Fig. 5). A particularly noteworthy discovery within our investigation

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Fig. 4. Biological links between hemorrhoid risk genes and drugs potentially useful for hemorrhoids.

was the identification of NOS3 overlapping sapropterin, which surfaced inadequately explored (Green et al., 2011; Webb 2012). Our study seeks
as an exceptionally promising target for hemorrhoid intervention. This to predict therapeutic targets and innovative drug candidates for the
assessment was further fortified by its attainment of a high systemic treatment of hemorrhoidal afflictions. Our methodology employs
score in functional annotations, affirming its potential relevance as a genomic networks and advanced bioinformatics analyses, leading to the
critical molecular player in hemorrhoidal pathogenesis. Collectively, development of detailed genomic networks that uncover the complex
our study serves as a compelling proof-of-principle, illustrating that molecular interactions involved in disease progression. These networks
genomic data not only facilitates the discovery of potential biomarkers incorporate hemorrhoid-associated genes from diverse databases,
for hemorrhoids but also propels the realm of drug repurposing for this providing valuable understanding of the genetic foundation of hemor­
debilitating affliction. This innovative synergy of genomic insights and rhoids, and proposing encouraging implications for the detection of
drug repurposing underscores a transformative paradigm that holds the feasible diagnostic biomarkers and improved therapeutic approaches.
potential to alleviate the burden of this ailment through targeted ther­ The detection of novel biomarkers is critical for classifying patients,
apeutic interventions. foretelling therapeutic responses, and evaluating prognosis (Santri et al.,
2022).
4. Discussion In our study identified 87 drugs that target 21 genes, of which 3 have
clinical and preclinical validation, demonstrating the effectiveness of
Advancements in genomic research have improved our understand­ our approach in guiding hemorrhoid treatment. Therefore, our findings
ing of the pathogenic mechanisms present in a variety of disorders, emphasize the value of implementing a combined approach that in­
including hemorrhoids. Genetic research aims to uncover the biological tegrates genomic networks and bioinformatics analyses, providing a
complexities of diseases, instead of merely identifying susceptibility robust scientific foundation to advance drug discovery efforts for hem­
genes, and then translating these findings into practical clinical appli­ orrhoids. Among the drugs identified, three have received support from
cations (Green et al., 2011). Although several genetic risk loci associated clinical and preclinical evidence for hemorrhoids. Ketamine, having
with various human phenotypes have been identified, the merging of completed phase IV clinical trials (NCT04248205), operates as an N-
genetic research insights into biologically relevant risk genes has been methyl-D-aspartate (NMDA) receptor antagonist and exhibits

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Fig. 5. Associations between biological risk genes for hemorrhoid and drugs that have been approved for other indications. For complete information, see Table S4.

effectiveness in reducing sensitization and postoperative pain. It offers Distinct protein expression patterns, with significantly higher levels
superior analgesia compared to placebo without affecting cognitive of NOS3 and NOS1 in hemorrhoidal tissues compared to rectal tissue
function, hence relieving post-hemorrhoidectomy pain. Fulvestrant, an (Lohsiriwat et al., 2020), suggest NOS3 as a potential important
estrogen receptor antagonist, has shown promise in preclinical in vitro biomarker for hemorrhoids. The elevated immunoreactivity of NOS3 in
models by inhibiting ERα, thereby reducing VEGF expression and miti­ the vascular endothelium of hemorrhoids indicates exposure of these
gating postoperative hemorrhoid edema (Fanyu Meng et al., 2022). blood vessels to increased levels of nitric oxide (NO), which is known for
Methylene blue, supported by case series evidence, demonstrates anti- its vasodilatory effects. The clinical significance of these results suggests
inflammatory effects by suppressing nitric oxide production, ulti­ potential therapeutic implications for the management of hemorrhoids
mately alleviating post-hemorrhoidectomy pain (Baldiwala and Trivedi, (Lohsiriwat et al., 2020). Considering the notable vascular dilation,
2022; Long et al., 2023). bleeding, and swelling associated with the condition, regulating NOS3
The investigation has identified the two most promising biomarkers activity provides an interesting avenue for intervention. Using NOS in­
among the biological hemorrhoid risk genes: These are NADPH oxidase hibitors to decrease NO production could provide a targeted approach to
1 (NOX1) and nitric oxide synthase 3 (NOS3). NOX1 has a pivotal role in reduce the exaggerated vasodilation and relieve the characteristic
generating reactive oxygen species (ROS), which are involved in the symptoms of hemorrhoids (Gokce et al., 2020).
pathogenesis of numerous diseases, such as cancer, constipation, dia­ Notably, among the top two potential biomarkers identified for
betes, neurodegenerative disorders, and inflammatory bowel disease hemorrhoids, it is essential to highlight that NOS3 stands out as a
(Valko et al., 2007, Schwerd et al., 2018; Hauck et al., 2019; Attri et al., druggable target, offering a unique avenue for therapeutic intervention.
2020; Wei et al., 2022). However, the involvement of NOX1 in the What sets NOS3 apart is its alignment with an existing drug, sapropterin,
pathogenesis of hemorrhoids has not been explicitly explained in the presenting an intriguing opportunity for innovative hemorrhoid ther­
current literature. According to our predictive analysis, NOX1 is iden­ apy. Sapropterin’s primary indication lies in the management of
tified as a strong candidate for potential biomarker status in the context hyperphenylalaninaemia (HPA) among patients afflicted with phenyl­
of hemorrhoids. The close interplay between NOX1, inflammation, and ketonuria (PKU) and/or tetrahydrobiopterin (BH4) deficiency, demon­
oxidative stress within the haemorrhoidal milieu highlights the possi­ strating remarkable responsiveness to this treatment regimen (Sanford
bility that NOX1 levels or activity could be indicative biomarkers. and Keating 2009; Dubois and Cohen 2010). The proven effectiveness of
Oxidative stress induced by ROS plays a significant role in initiating and sapropterin in modulating these physiological processes raises the
progressing various pathological conditions, including hemorrhoids prospect of its applicability to other conditions characterized by dysre­
(Kusumawati et al., 2022). Increased NOX1 expression or higher ROS gulated NO synthesis, such as hemorrhoids. Given that NOS3 is impli­
production could indicate the level of inflammation and oxidative stress cated in the regulation of NO production, and NO itself plays a critical
in tissues affected by hemorrhoids. Therefore, evaluating NOX1 levels role in vascular dynamics and tissue responses (Lohsiriwat et al., 2020;
dynamically might provide a better understanding of the severity of Gokce et al., 2020), sapropterin’s modulation of NOS3 activity could
hemorrhoids, facilitating informed treatment decisions. have profound implications for hemorrhoid therapy. By fine-tuning

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NOS3-mediated NO synthesis, sapropterin holds the potential to exert a Culture, Research, and Technology of Indonesia, Indonesia. (No. 3547/
favorable influence on vascular tone, inflammation, and tissue repair LL8/AL.04/2023).
within the hemorrhoidal microenvironment. However, while the ratio­
nale for repurposing sapropterin for hemorrhoid therapy appears Appendix A. Supplementary material
promising, rigorous research is imperative to validate its efficacy, safety,
and optimal dosing in this novel context. Clinical investigations Supplementary data to this article can be found online at https://doi.
exploring sapropterin’s impact on NOS3 activity within hemorrhoidal org/10.1016/j.jsps.2023.101831.
tissues, its potential to ameliorate vasodilation, and its overall influence
on symptom relief are vital steps toward unlocking its therapeutic po­ References
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