Elucidating the Mechanisms Underlying Interindividual Differences in the Onset of Adverse Drug Reactions

Abstract

Idiosyncratic drug toxicities (IDTs) pose a significant challenge; they are marked by life-threatening adverse reactions that emerge aftermarket release and are influenced by intricate genetic and environmental variations. Recent genome-wide association studies have highlighted a strong correlation between specific human leukocyte antigen (HLA) polymorphisms and IDT onset. This review provides an overview of current research on HLA-mediated drug toxicities. In the last six years, HLA-transgenic (Tg) mice have been instrumental in advancing our understanding of these underlying mechanisms, uncovering systemic immune reactions that replicate human drug-induced immune stimulation. Additionally, the potential role of immune tolerance in shaping individual differences in adverse effects highlights its relevance to the interplay between HLA polymorphisms and IDTs. Although HLA-Tg mice offer valuable insights into systemic immune reactions, further exploration is essential to decipher the intricate interactions that lead to organ-specific adverse effects, especially in organs such as the skin or liver. Navigating the intricate interplay of HLA, which may potentially trigger intracellular immune responses, this review emphasizes the need for a holistic approach that integrates findings from both animal models and molecular/cellular investigations. The overarching goal is to enhance our comprehensive understanding of HLA-mediated IDTs and identify factors shaping individual variations in drug reactions. This review aims to facilitate the development of strategies to prevent severe adverse effects, address existing knowledge gaps, and provide guidance for future research initiatives in the field of HLA-mediated IDTs.

1. INTRODUCTION

Adverse drug reactions exhibit individual differences that are influenced by specific genetic and environmental variations, occasionally resulting in life-threatening situations. These side effects, referred to as idiosyncratic drug toxicities (IDTs), are potentially difficult to detect during drug development and often become evident only after widespread clinical use post-market release.¹⁾

Recent genome-wide association studies have suggested a strong relationship between the occurrence of IDTs and human leukocyte antigen (HLA) polymorphisms.²⁾ HLA, which corresponds to the major histocompatibility complex (MHC) observed in many animals, is broadly classified into class I antigens (e.g., HLA-A, HLA-B, and HLA-C) and class II antigens (e.g., HLA-DP, HLA-DQ, and HLA-DR).³⁾ These antigens play a vital role as membrane proteins, distinguishing self from non-self immunologically, particularly in interactions with T cells. However, specific HLA molecules may present drug epitopes, leading to T cell-mediated hypersensitivity reactions that are classified as type IV allergic reactions.^2,4) A notable example is the association between HLA-B*57:01 and the antiretroviral drug abacavir, where individuals carrying this HLA have an over 900 times higher odds ratio of developing hypersensitivity reactions to abacavir.^5,6) Abacavir binds to the peptide-binding groove of HLA-B*57:01, altering the presented peptide repertoire, which is considered an activation signal for CD8⁺ T cells.⁷⁾ Other representative example combinations include HLA-B*15:02 and carbamazepine-induced Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN),^8,9) HLA-B*58:01 and allopurinol-induced SJS/TEN,^10–12) and HLA-B*57:01 and flucloxacillin-induced liver injury.^13,14) Adverse drug reactions mediated by HLAs often manifest as notable dermatological and liver injuries.²⁾

Considering the risk of IDTs, the importance of HLA polymorphisms has been acknowledged; however, the precise mechanisms underlying the onset of these adverse effects remain unclear. One critical reason is the limited number of studies that have employed models capable of evaluating HLA-mediated drug-induced immune responses. A significant breakthrough in research has been the advent of HLA class I transgenic (Tg) mice in toxicity studies over the past six years.^15,16) This review provides an overview of the current state of research on drug toxicity using HLA-Tg mice and aims to share the challenges faced in this field. Furthermore, the distinctive features of HLA molecules related to toxicity from the perspective of their intracellular behavior are discussed in this review. Lastly, the prospects for future research in the field of HLA-mediated IDTs are outlined.

2. REPRODUCTION OF DRUG HYPERSENSITIVITY REACTIONS USING HLA-TG MICE

Tg mice carrying class I or II HLA molecules have been available for over 30 years.¹⁷⁾ Using these mouse models has played a crucial role in the discovery of HLA-specific antigens against viral infections and the replication of HLA-mediated autoimmune diseases, providing valuable insights for the development of therapeutic strategies.¹⁷⁾ In recent years, HLA class I Tg mice have been increasingly employed in the investigation of IDTs. The pioneering hypersensitivity model involving HLA-B*57:01-Tg mice and abacavir was initially reported in 2018 by our group,¹⁸⁾ and independently by Cardone et al.¹⁹⁾

Class I HLAs are expressed as heterodimers consisting of an α-chain and β₂-microglobulin (β₂m), and the presented peptides on this complex are recognized by CD8⁺ T cells.²⁰⁾ The α-chain comprises three extracellular domains (α₁, α₂, α₃), a transmembrane region, and an intracellular domain. Among these, the α₃ domain is particularly crucial for recognition by CD8⁺ T cells, and substituting the murine α₃ domain of MHC with that of HLA allows better recognition of HLA towards murine T cells.²¹⁾ Considering this, human-mouse chimeric HLA-B*57:01-Tg mice have been generated^18,19) (Fig. 1a). Furthermore, to stably express the introduced HLA on the cell surface, human β₂m has been linked to the HLA-B*57:01 through a polycistronic sequence using the 2A peptide sequence, which can achieve co-translational cleavage in the Tg mice.^22,23)

Fig. 1. Chimeric Human Leukocyte Antigen (HLA)-Transgenic (Tg) Mice and Drug-Induced Immune Reactions

(a) Schematic representation of the complex structure of the human-murine chimeric HLA class I-Tg mice. The α₃ domain of the HLA complex is substituted with the murine α₃ domain of MHC. TCR, T cell receptor. β₂m, β₂-microglobulin. (b) Effects of abacavir exposure on CD8⁺ T cells isolated from the lymph nodes of HLA-B*57:01-Tg mice. The percentage of bromodeoxyuridine (BrdU)-positive subpopulations among the CD8⁺ T cells is represented. Mice are treated with abacavir (25 mg/kg/d) for 3 consecutive days. Each bar represents the mean ± S.E. of three independent experiments using a total of 4 or 5 mice. LM, littermate mice (wild-type). ** p < 0.01, *** p < 0.001. N.S., not significant. Data modified from Susukida et al.¹⁸⁾

Cardone et al. conducted in vitro experiments using CD8⁺ T cells from drug-naive HLA-B*57:01-Tg mice.¹⁹⁾ The T cells were stimulated with abacavir for up to 5 d, and the cells released interleukin 2 (IL-2), interferon-gamma (IFN-γ), and granzyme B to the culture media. These phenomena were not evident in T cells derived from wild-type (WT) mice, suggesting that CD8⁺ T cells from HLA-introduced mice have sufficient potential to respond to abacavir stimulation. Abacavir was then administered to the HLA-B*57:01-Tg mice intraperitoneally and applied topically to the ears for up to 4 weeks; however, no visible skin hypersensitivities were observed.¹⁹⁾ The authors suggested the potential influence of immunosuppressive mechanisms and/or immunosuppressive cells in preventing hypersensitivity reactions as a rationale for these observations (see section 3). In the HLA-B*57:01-Tg mice model, exposure to abacavir for 3 consecutive days in the local ear area resulted in enhanced proliferation of lymphocytes in the auricular lymph nodes, particularly in CD8⁺ T cells¹⁸⁾ (Fig. 1b). Moreover, these CD8⁺ T cells exhibited elevated expression of IL-2 and IFN-γ. Additionally, significant infiltration of lymphocytes into the dermal layer of the ear skin tissue was observed. These phenomena were not observed when abacavir was administered to the mice with HLA-B*57:03, which serves as a negative control molecule that abacavir cannot bind.²⁴⁾ However, oral administration of abacavir to the Tg mice did not induce significant skin rash symptoms, as reported in humans. Considering these findings, immune responses to abacavir were specifically elicited in the mouse model in an HLA polymorphism-dependent manner; however, other factors may mask the pathological conditions observed in humans.

HLA-B*15:02-Tg mice lacking murine MHC class I have been used to reproduce hypersensitivity reactions to carbamazepine, an antiepileptic drug.²⁵⁾ In this context, administering carbamazepine alone to Tg mice did not invoke toxic symptoms. T cells expressing specific α and β T cell receptors (TCRs) (referred as public TCRs) were clonally expanded at the lesion sites of SJS/TEN patients with HLA-B*15:02 and carbamazepine administration. Adaptive transfer of these T cells, to the Tg mice induced mild skin inflammation by oral administration of carbamazepine. Despite the various challenges of reproducing drug hypersensitivity reactions, HLA-Tg mouse models have provided a solid step toward understanding the mechanisms of HLA-mediated IDTs.

3. BREAKING IMMUNE TOLERANCE TO INDUCE HYPERSENSITIVITY REACTIONS BY ABACAVIR IN HLA-B*57:01-TG MICE

Administration of abacavir to HLA-B*57:01-Tg mice failed to induce clinical hypersensitivity symptoms despite HLA-mediated immune activation.^18,19) In patients, IDT does not occur in all subjects with specific HLAs. The rate of positive results in the epicutaneous patch test for abacavir is 47.9,⁵⁾ implying that environmental and patient-specific factors, other than the HLA-B*57:01 allele, are critical in determining the onset of hypersensitivity.

Abacavir is prescribed for human immunodeficiency virus (HIV) patients who have relatively low levels of CD4⁺ T cells, including immunosuppressive T regulatory cells (Tregs), since the virus directly infects CD4⁺ T cells.^26,27) Tregs down-regulate the expression of CD80/CD86 on dendritic cells and in a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)–dependent manner, suppressing the maturation of dendritic cells and weakening T cell co-stimulation through CD28.²⁸⁾ For T cell activation, two signals are required: signal 1 (antigen-specific TCR engagement), and signal 2 (co-stimulatory signal from antigen-presenting cells).^29,30) In the absence of the co-stimulatory signal 2, T cells enter an anergic state, where antigen-specific T cells are unable to respond to an immunogenic stimulus.³¹⁾ Removal of CD4⁺ T cells, including Tregs, upregulated CD86 expression on dendritic cells in HLA-B*57:01-Tg mice by exposure to abacavir.³²⁾ Cardone et al. observed skin scarring and immune cell infiltration associated with exposure to abacavir by depleting CD4⁺ T cells in HLA-B*57:01-Tg mice through intraperitoneal administration and skin sensitization.¹⁹⁾ In contrast, abacavir was orally administered to HLA-B*57:01-Tg mice with depleted CD4⁺ T cells for three weeks in our research group.³³⁾ Here, slight dermal infiltration of lymphocytes and CD8⁺ T cells in the ear skin, along with a significant increase in the percentage of effector memory (CD44^highCD62L^low) CD8⁺ T cells in the auricular lymph nodes, was observed. However, no significant rash symptoms were observed, in contrast to observations in clinical cases.

Depletion of CD4⁺ T cells activates the immune system but concurrently induces immune exhaustion. An increase in the expression of programmed cell death protein (PD-1) on activated CD8⁺ T cells has been observed in HLA-B*57:01-Tg mice with depleted CD4⁺ T cells following abacavir administration.³³⁾ By both CD4⁺ T cell depletion and PD-1 knockout, a significant increase in the proportion of cytokine- and cytolytic granule-secreting effector memory CD8⁺ T cells was observed in HLA-B*57:01-Tg mice with oral administration of abacavir³³⁾ (Fig. 2a). In these mice, pronounced redness and infiltration of CD8⁺ T cells in the ear skin were observed (Fig. 2b), suggesting a closer mimicry of the abacavir hypersensitivity observed in humans. Skin inflammation was confirmed by the serum thymus and activation-regulated chemokine (TARC),³⁴⁾ which is involved in many skin diseases and is a marker of atopic dermatitis.³⁵⁾ While TARC is a T-helper 2 (Th2) chemokine and its upregulation in the abacavir hypersensitivity model involving CD8⁺ T cells remains unclear, it might prove to be a valuable biomarker for assessing the pathophysiology of patients.

Fig. 2. Abacavir-Induced CD8⁺ T Cell Activation in HLA-B*57:01-Tg Mice without PD-1 Gene and CD4⁺ T Cells

(a) Percentage of effector memory (CD44^highCD62L^low) CD8⁺ T cells in the auricular lymph nodes of HLA-B*57:01-Tg mice. Mice were fed with 1% abacavir over 7 d. Each plot represents an individual mouse with the mean ± S.E. of three independent experiments using a total of 3 to 16 mice. *** p < 0.001, compared with other mice groups. (b) Representative image of a photo of the ear and immunostaining image with CD8 (Green) and nuclei (Blue) of an abacavir-treated HLA-B*57:01-Tg mouse without the PD-1 gene and CD4⁺ T cells. The scale bar represents 100 µm. (c) Proposed scheme of abacavir-induced skin hypersensitivity with HLA-B*57:01. Data modified from Susukida et al.³³⁾ (a), (b), and Song et al.³²⁾ (c).

In conclusion, combined with the depletion of PD-1 and CD4⁺ T cells in the HLA-B*57:01-Tg mouse model, CD8⁺ T cells were activated and further proliferated by abacavir stimulation, which was supported by dendritic cells (Fig. 2c). In patients prescribed abacavir, a reduction in the number of CD4⁺ T cells and Tregs was observed, yielding reasonable outcomes in these mouse studies. In addition, PD-1 is genetically and non-genetically regulated. Single-nucleotide polymorphisms (SNPs) exist in the PD-1 gene; for example, a missense variant of PD-1 has been reported as an additional determinant in ankylosing spondylitis, an autoimmune disease.^36,37) Moreover, environmental factors are important in altering the expression of PD-1. Hepatitis B virus (HBV) infection leads to increased expression of PD-1 in HBV-specific CD8⁺ T cells, resulting in the development of acute hepatitis due to their inhibitory immunity.³⁸⁾ In HLA-dependent immune-mediated IDTs, it is plausible that such inhibitory immunity may be involved. Whether the immune tolerance system truly plays a role in HLA-dependent IDTs is not yet clear, but it cannot be overlooked as a factor that may determine individual differences in adverse effects.

4. VARIOUS ATTEMPTS TOWARD REPRODUCING HLA-MEDIATED DRUG-INDUCED LIVER INJURY (DILI)

Hypersensitivity reactions to abacavir in patients with HLA-B*57:01, despite oral administration of abacavir, predominantly manifest on the skin. In our constructed HLA-B*57:01-Tg mouse model, HLAs were broadly expressed throughout the body, including in the liver and kidney,¹⁸⁾ yet the toxic manifestation was confined. The liver is an immunologically tolerogenic organ because it is exposed to various chemical compounds and antigens from the intestine, and reactive metabolites are produced in the liver.³⁹⁾ Immunosuppressive signaling and proteins provide immunological protection against external stimuli in the liver.⁴⁰⁾ A retrospective analysis of adverse drug event occurrences in Japan revealed that the infection status of patients influences the development of immune-mediated DILI and SJS/TEN.⁴¹⁾ Innate immune inflammation induced by pathogen infection may hinder immune tolerance in the liver. Co-administration of CpG-oligodeoxynucleotides (CpG-ODN), a toll-like receptor 9 (TLR9) agonist, and abacavir-induced immune-mediated liver injury in HLA-B*57:01-Tg mice.⁴²⁾ In this context, elevated alanine aminotransferase (ALT) and pathological changes (white spots) in the liver, an increase in the number of activated CD8⁺ T cells, and their infiltration into the liver tissue were observed. However, the elevation of ALT was transient, occurring approximately seven days after abacavir administration, and the liver injury was quite mild, implying that other factors might have mitigated the exacerbation of DILI. Agonists for TLR4 and TLR9 are employed in immune-mediated liver injury mouse models.⁴³⁾ In the case of HLA-B*57:01-Tg mice, TLR4 stimulation by lipopolysaccharide did not induce liver injury caused by abacavir (in-house data). The synergistic effects of a TLR9 agonist and abacavir-induced liver injury in HLA-B*57:01 remain unclear.

HLA-B*57:01 is a risk allele for the liver injury induced by flucloxacillin, a β-lactam antibiotic for narrow-spectrum Gram-positive bacterial infections.^13,14) But compared to abacavir hypersensitivity, its negative predictive value is low,¹³⁾ and there is suggested involvement of other HLA polymorphisms.⁴⁴⁾ Abacavir and HLA-B*57:01 interactions are explained by a model of altered peptide repertoire, whereas flucloxacillin is considered to be presented as a hapten (covalent conjugate of drug with peptide) on HLA-B*57:01, and its liver damage is metabolism-dependent.^4,45) Puig et al. reported that flucloxacillin forms conjugates with endogenous peptides in B-lymphoblastoid cells expressing HLA-B*57:01 using mass spectrometry.⁴⁶⁾ Furthermore, sensitizing the skin of HLA-B*57:01-Tg mice (also lacking the expression of the murine MHC-I molecules H-2K^b and H-2D^b) with the conjugate and subsequently stimulating isolated splenocytes with the same conjugate resulted in a robust release of IFN-γ, indicating the immunogenicity of the flucloxacillin-peptide conjugate.⁴⁶⁾

In the mechanism of immune-mediated liver injury by flucloxacillin, CD8⁺ T cells are strongly involved. Using human peripheral blood mononuclear cells (PBMCs), the proliferation of CD8⁺ T cell clones from patients with DILI and volunteers expressing HLA-B*57:01 was induced by flucloxacillin.¹⁴⁾ Similarly, CD8⁺ T cells derived from HLA-B*57:01-Tg mice were stimulated by flucloxacillin.⁴⁷⁾ However, similar to abacavir, simply treating HLA-B*57:01-Tg mice with flucloxacillin alone was insufficient to adequately activate T cells responsive to the drug, suggesting that immune tolerance systems might suppress T cell activation. Metushi et al. reported that the treatment of PD-1 knockout mice with amodiaquine and an anti-CTLA-4 antibody led to liver injury,⁴⁸⁾ as was the case in the abacavir-induced hypersensitivity model using HLA-B*57:01-Tg mice. However, in our study, even with the intragastric administration of flucloxacillin to HLA-B*57:01-Tg mice after the removal of PD-1 or CD4⁺ T cells, activation of the immune response or liver injury was not observed.⁴⁹⁾ Additionally, co-administration of CpG-ODN yielded similar negative results. In contrast, Ananthula et al. showed that flucloxacillin treatment with skin sensitization using retinoic acid resulted in signs of liver injury such as enlarged gallbladders and hepatocyte apoptosis in HLA-B*57:01-Tg mice without PD-1 or CD4⁺ T cells.⁴⁷⁾ However, no elevation in ALT was observed. Various investigations have been conducted, but it is evident that reproducing HLA-mediated DILI using animal models is extremely challenging. Hidden patient-specific factors beyond HLA and immunosuppressive factors need further consideration in animal models.

5. INTRACELLULAR BEHAVIOR OF HLA CLASS I MOLECULES ASSOCIATED WITH IDTS

HLA-B*57:01, well-known as a risk factor for adverse reactions to abacavir and flucloxacillin, is associated with the risk of carbamazepine-induced SJS/TEN in Europeans.⁵⁰⁾ Similarly, HLA-B*15:02 is a risk for carbamazepine, lamotrigine, and sulfamethoxazole,^51,52) and HLA-B*58:01 is implicated in the risk of adverse reactions to allopurinol, carbamazepine, and to nevirapine.^53,54) Given that a single HLA molecule can confer a risk for hypersensitivity reactions to multiple drugs, it is highly probable that these HLA molecules possess characteristics that may cause adverse drug effects.

HLA class I molecules form trimeric complexes with β₂m and antigen peptides in the endoplasmic reticulum and are transported to the cell surface.²⁰⁾ However, in our study, HLA-B*57:01-expressing cells exhibited it in a non-classical form that might consist of an HLA heavy chain alone, a dimer of HLA heavy chain and β₂m, or an HLA-peptide complex without β₂m, and exposure to abacavir increased the expression.⁵⁵⁾ Therefore, it is suggested that abacavir may influence HLA-B*57:01 molecules during the processes of complex formation in the endoplasmic reticulum and intracellular transport. Upon observing the intracellular behavior of HLA molecules, risk HLAs (HLA-B*57:01, HLA-B*58:01, HLA-B*15:02, and HLA-B*15:11) are predominantly localized within the intracellular endoplasmic reticulum, and their surface expression was relatively low compared to non-risk HLAs (HLA-B*57:03 and HLA-B*15:01)⁵⁶⁾ (Fig. 3a). This finding suggests that HLA molecules associated with the risk of adverse drug effects tend to have lower expression on the cell surface and are more prone to accumulate intracellularly. From in vitro and in silico studies, we have observed that these risk HLAs have a weak affinity for β₂m, likely due to weak hydrogen bonding formation between HLA and β₂m^56,57) (Fig. 3b). The weak binding of risk HLA molecules to β₂m may result in their accumulation in the endoplasmic reticulum. In the case of HLA-B*57:01, abacavir is speculated to bind to HLA in the endoplasmic reticulum, leading to alterations in intracellular trafficking.

Fig. 3. HLA Molecules Associated with Drug Hypersensitivity Accumulate in the Endoplasmic Reticulum and Have a Low Affinity with β₂m

(a) HLA-introduced HeLa cells were stained with HLA (introduced, Green), Calnexin (an endoplasmic reticulum protein, Red), and nuclei (Blue). Each scale bar represents 10 µm (upper) and 2.5 µm (lower, enlarged). (b) Lysates of HLA-introduced HeLa cells were subjected to immunoprecipitation (IP) for the introduced HLA. The immunoprecipitates and the whole cell lysate were subjected to Western blotting. Data modified from Shirayanagi et al.⁵⁶⁾

However, the immunological functions of intracellular HLA molecules are not clearly understood. Martin et al. observed that in human-derived PBMCs expressing HLA-B*57:01, exposure to abacavir increased the expression of heat shock protein 70 (HSP70), which was distributed largely to the endoplasmic reticulum and endosomes.⁵⁸⁾ Elevation occurred in PBMCs from individuals with or without abacavir sensitization within just 3 h of exposure. IFN-γ induction from the PBMCs was also observed,⁵⁸⁾ suggesting that interaction between abacavir and HLA-B*57:01 triggers innate immune responses. Furthermore, the intracellular assembly of HLA complexes is implicated in the occurrence of IDTs, since genetic variants of HSP70-Hom and endoplasmic reticulum aminopeptidase 1 (ERAP1) are associated with abacavir-induced hypersensitivity.^58,59) Therefore, elucidating the intracellular behavior of HLAs, such as protein synthesis, complex formation, drug binding within the endoplasmic reticulum, and subsequent intracellular distribution or trafficking of HLA, would be crucial for understanding individual differences in IDTs.

6. CONCLUSION AND FUTURE PERSPECTIVES

In conclusion, the use of HLA-Tg mice has significantly improved our understanding of the mechanisms underlying IDTs in humans. Although considerable progress has been made, the specific reasons for the pronounced manifestations of adverse effects on the skin or liver remain elusive. HLA-B*57:01-Tg mouse studies have revealed systemic immune reactions,⁶⁰⁾ consistent with the drug-induced immune stimulation observed in human PBMCs with high-risk HLAs. However, when translated into animal models, manifestations of marked toxicity are limited to specific tissues. Moreover, HLA molecules associated with adverse effects are potentially involved in innate immune-like reactions within cells. HLAs function as “immune sensors,” extending their role beyond interactions with T cells on the cell surface. Furthermore, although not extensively addressed in this manuscript, it is crucial to emphasize a noteworthy aspect: several antiepileptic drugs, such as carbamazepine, phenytoin, and lamotrigine, have been associated with severe hypersensitivity reactions linked to HLAs.²⁾ Although the pharmacological effects and unique structures of these drugs may contribute to IDTs, little information is available regarding their impact on immune activation.

In light of these findings, I advocate for a comprehensive approach that integrates insights from animal models (top-down) and molecular/cellular investigations (bottom-up) to enhance our understanding of HLA-mediated IDTs holistically. Exploring the factors that influence individual variations in drug reactions offers promising avenues for the prevention of severe adverse effects. Future research should prioritize addressing the knowledge gaps and delving into the intricate interplay between HLAs, drug pharmacology, and immune responses.

Acknowledgments

This work was supported by Grant-in-Aid for Young Scientists (B) (16K18932) and Grant-in-Aid for Scientific Research (B) (21H02640) from the Japan Society for the Promotion of Science. I would like to thank the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Takeda Science Foundation. I express my gratitude for the open innovation TaNeDS program at Daiichi Sankyo Co., Ltd. I wish to acknowledge Prof. Kousei Ito (Chiba University) and all the laboratory members at the Laboratory of Biopharmaceutics of the Graduate School of Pharmaceutical Sciences at Chiba University.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2023 Pharmaceutical Society of Japan Award for Young Scientists.

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