Development of novel liver-targeting glucocorticoid prodrugs

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Medicine in Drug Discovery 21 (2024) 100172

Available online 21 November 2023

Development of novel liver-targeting glucocorticoid prodrugs

Yazheng Wang , Dandan Guo , Rebecca Winkler , Xiaohong Lei , Xiaojing Wang , Jennifer Messina , Juntao Luo , Hong Lu

^*

Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, United States

A R T I C L E I N F O Keywords:

Glucocorticoid receptor Alcoholic hepatitis Sepsis

Liver-specific Prodrug Zwitterionic linker

A B S T R A C T

Background: Glucocorticoids (GCs) are widely used in the treatment of inflammatory liver diseases and sepsis, but GC’s various side effects on extrahepatic tissues limit their clinical benefits. Liver-targeting GC therapy may have multiple advantages over systemic GC therapy. The purpose of this study was to develop novel liver-targeting GC prodrugs as improved treatment for inflammatory liver diseases and sepsis.

Methods: A hydrophilic linker or an ultra-hydrophilic zwitterionic linker carboxylic betaine (CB) was used to bridge cholic acid (CA) and dexamethasone (DEX) to generate transporter-dependent liver-targeting GC prodrugs CA-DEX and the highly hydrophilic CA-CB-DEX. The efficacy of liver-targeting DEX prodrugs and DEX were determined in primary human hepatocytes (PHH), macrophages, human whole blood, and/or mice with sepsis induced by cecal ligation and puncture.

Results: CA-DEX was moderately water soluble, whereas CA-CB-DEX was highly water soluble. CA-CB-DEX and CA-DEX displayed highly transporter-dependent activities in reporter assays. Data mining found marked dysregulation of many GR-target genes important for lipid catabolism, cytoprotection, and inflammation in patients with severe alcoholic hepatitis. These key GR-target genes were similarly and rapidly (within 6 h) induced or down-regulated by CA-CB-DEX and DEX in PHH. CA-CB-DEX had much weaker inhibitory effects than DEX on endotoxin-induced cytokines in mouse macrophages and human whole blood. In contrast, CA-CB-DEX exerted more potent anti-inflammatory effects than DEX in livers of septic mice.

Conclusions: CA-CB-DEX demonstrated good hepatocyte-selectivity in vitro and better anti-inflammatory effects in vivo. Further test of CA-CB-DEX as a novel liver-targeting GC prodrug for inflammatory liver diseases and sepsis is warranted.

1. Introduction

Alcoholic liver disease (ALD) is a major cause of death worldwide [1]. Patients with alcoholic hepatitis (AH) have severe inflammation and cholestatic liver injury [2], and severe AH (sAH) has a high mortality rate [3]. Glucocorticoids (GCs) are currently the only available drug therapy for sAH [4], however, prednisolone, the current standard of care for sAH, only marginally reduces 28-day mortality without long-term improvement [5]. Additionally, sepsis is a leading cause of death of sAH [6,7], and hepatic GR is essential to protect against liver failure and mortality in sepsis [8]. GCs are also frequently used in the treatment of sepsis to control the hyperinflammation. GC treatment improves liver failure in patients with sepsis [9]. However, there is no clear survival benefit for GC treatment of sepsis patients. There is an urgent unmet need to improve the current GC therapy of sAH and sepsis.

The markedly impaired urea synthesis in AH is associated with disease severity and hepatic encephalopathy [10,11]. Glucocorticoid receptor (GR) controls hepatic urea cycle [12], and prednisolone restores urea synthesis in survivors of sAH [13]. Additionally, cholestasis contributes to malnutrition and AH severity [14–17]. Activation of GR in hepatocytes protects against apoptosis and inflammation [18,19].

Activation of GR in hepatocytes induces the key BA transporters Na (+)-taurocholate transport protein (NTCP) and bile salt export pump (BSEP) [20,21], and GCs protect against cholestatic liver injury and steatohepatitis in patients [22] and mice [23]. These hepatoprotective and anti-inflammatory effects of GR activation on hepatocytes likely underly the benefits of current GC therapy in sAH and sepsis.

Long-term GC treatment can cause various adverse effects that limit AH therapy of sAH, the severity of which is proportional to the dose and duration of GC treatment [24]. Activation of GR in extrahepatic tissues can promote alcohol consumption and psychiatric problems [25,26],

* Corresponding author.

E-mail address: [email protected] (H. Lu).

Contents lists available at ScienceDirect

Medicine in Drug Discovery

journal homepage: www.elsevier.com/locate/medid

https://doi.org/10.1016/j.medidd.2023.100172

Received 14 September 2023; Received in revised form 7 November 2023; Accepted 19 November 2023

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adipose lipolysis [27], intestinal reabsorption of bile acid (BA) and cholestasis, gastrointestinal bleeding [28,29], and wasting of skeletal muscle [30]. A recent study shows that activation of GR in intestinal epithelia cells aggravates alcoholic steatohepatitis and liver injury due to increased intestinal epithelial permeability and gut dysbiosis [31].

Moreover, high doses of GC can aggravate alcoholic liver injury and impair liver regeneration by inhibiting macrophage-mediated phago- cytic and hepatic regenerative functions [32–35]. In contrast, GR deficiency in hepatocytes delays liver regeneration [36]. Therefore, extrahepatic and hepatic non-parenchymal side effects of GCs are likely the major factors that limit the therapeutic efficacy of GC therapy during high-dose and long-term GC treatment of sAH.

Vamorolone, a dissociative GC that only maintains GC’s transrepression activity, was developed to ameliorate GC’s side effects [37,38]; however, it has weaker anti-inflammatory effects and increases hepatic necrosis in mice with sick cell disease [39]. In contrast to GR, activation of mineralocorticoid receptor (MR) by GCs promotes steatohepatitis and fibrosis, vascular damage, and acute kidney injury (AKI) [40–44]. Prednisolone, the current standard of care for sAH [45], strongly activates MR at 100 nM [46]. Thus, sAH patients who are resistant to GR-mediated anti-inflammatory and cytoprotective effects will have elevated risk of the prednisolone-MR-mediated side effects such as AKI [47]. In contrast, DEX, a highly potent and selective GR agonist [48], prevents severe AKI in patients with kidney diseases [49].

Budesonide is a 2nd generation GC with extensive hepatic first-pass metabolism and limited systemic exposure. In a small clinical study, budesonide has similar efficacy and diminished side effects in sAH patients compared to prednisolone [50]. This provides a key proof of concept for our liver-targeting GC prodrug as improved sAH therapy.

However, oral GCs, even for short-term treatment, increase the risk of GI bleeding [28,51]. Moreover, GCs promote intestinal BA reabsorption, and activation of GR in intestinal epithelia disrupts intestinal permeability and gut microbiome and worsens steatohepatitis in ALD [29,31].

Therefore, liver-specific delivery of GCs to activate hepatic GR

should markedly improve GC therapy of sAH by ameliorating the adverse effects of GCs on extrahepatic tissues and immunosuppression.

BA-drug conjugates with a hydrophilic linker for liver-specific uptake by the liver-specific BA transporter NTCP have been synthesized as oral prodrugs for liver-specific drug delivery [52–56]. DEX is a lipophilic molecule, which compromises the liver-selectivity of prodrug. We further introduced ultra-hydrophilic zwitterionic moiety as a flexible linker to dramatically increase the water solubility of BA-drug conjugates to reduce the passive nonspecific diffusion into cells and maintain the high substrate specificity for NTCP. The purpose of this study was to characterize our novel liver-targeting GC prodrugs by comparing the differential effects of our liver-targeting GC prodrugs and the parent drug DEX on primary human hepatocytes (PHH), mouse macrophages, human whole blood, and mice with sepsis. Results from these studies showed that compared to the parent drug DEX, our liver-targeting GC prodrugs demonstrated high water solubility, transporter-dependent cellular activity, less suppressive effects on immune cells, and better anti-inflammatory effects on the livers of septic mice.

2. Materials and methods

2.1. Synthesis, purification, and validation of CA-DEX prodrugs 2.1.1. Materials

Dexamethasone (DEX) was obtained from Sigma. Rink Amide Resin was purchased from Hecheng Company (Tianjin, China). (Fmoc)-Lys (Dde)–OH, Fmoc-Glu-OtBu, and N-hydroxy benzotriazole (HOBt) were purchased from AnaSpec Inc. Cholic acid (CA), D-α-tocopherol succi- nate, diisopropyl carbodiimide (DIC), dichloromethane (DCM), methanol (MeOH), N,N-Dimethylformamide (DMF), Triisopropyl silane (TIPS), trifluoroacetic acid (TFA) and other chemical reagents were acquired from Sigma-Aldrich.

Abbreviations

AH alcoholic hepatitis AKI acute kidney injury ALD alcoholic liver disease BA bile acid

BSEP bile salt export pump CA cholic acid

CA-CB-DEX cholic acid-carboxylic betaine-dexamethasone conjugate

CA-DEX cholic acid-dexamethasone conjugate

CA-GPC-DEX cholic acid-glycerophosphorylcholine-dexamethasone conjugate

CB carboxylic betaine CLP cecal ligation puncture DCM dichloromethane DEX dexamethasone

DIC diisopropyl carbodiimide DMAP 4-dimethylaminopyridine DMF N,N-Dimethylformamide

DUSP1 dual specificity protein phosphatase 1 ERRFI1 ERBB receptor feedback inhibitor 1 G0S2 G0/G1 switch gene 2

G6PC glucose-6-phosphatase catalytic-subunit GADD45B growth arrest DNA damage-inducible gene 45β GC glucocorticoid

GILZ glucocorticoid-induced leucine zipper GNMT glycine N-methyltransferase

GPC glycerophosphorylcholine GR glucocorticoid receptor HAO2 hydroxy acid oxidase 2 HNF4A hepatocyte nuclear factor 4A HOBt N-hydroxybenzotriazole Il1b interleukin 1 beta IL1RN IL1 receptor antagonist IL6 interleukin 6

KLF15 Kruppel-like factor 15 LPS lipopolysaccharide

MALDI-TOF matrix-assisted laser desorption/ionization-time of flight

MeOH methanol

MR mineralocorticoid receptor MT1X metallothionein 1X

NAMPT nicotinamide phosphoribosyl-transferase NTCP Na(+)-taurocholate transport protein PDK4 pyruvate dehydrogenase kinase 4

PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 PGC1α peroxisome proliferator activated receptor gamma

coactivator 1α

PHH primary human hepatocytes

RORA retinoic acid receptor-related orphan receptor alpha sAH severe alcoholic hepatitis

TFA trifluoroacetic acid TIPS Triisopropyl silane TNF tumor necrosis factor

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2.1.2. Synthesis and characterization of DEX prodrugs

We synthesized three DEX prodrugs i.e. CA-DEX, CA-GPC-DEX, and CA-CB-DEX (Fig. 1), based on the rational design through a straight- forward method. DEX-COOH was prepared by conjugation of succinic anhydride on DEX by ester bond (Fig. 2A), followed by the coupling of targeting ligand cholic acid and hydrophilic zwitterionic groups i.e. GPC and CB via solid phase peptide chemistry. The final products were purified by column chromatography and further confirmed by matrix- assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (Fig. 3).

2.1.2a. Prodrug CA-DEX preparation

Dexamethasone prodrug CA-DEX was prepared by solid phase peptide synthesis as shown in Fig. 2B. Firstly, dexamethasone, succinic anhydride and 4-dimethylaminopyridine (DMAP) were dissolved in DMF and reacted at 50 ^◦C for 48 h to prepare DEX-COOH (Fig. 2A). Fmoc group of Rink Amide resin was deprotected via 20 % (v/v) 4-methylpi- peridine in DMF and the NH2 group was conjugated with Fmoc-Lys (Dde)–OH (three equiv) through coupling reagents HOBt and DIC con- firming by negative Kaiser test result. After deprotection of Fmoc group, Fmoc-Glu-OtBu and Cholic acid were coupled sequentially to achieve intermediate with cholic acid-targeting moiety. After removal of Dde group by 5 % (v/v) hydrazine in DMF, DEX-COOH was conjugated on NH2 group. The final product CA-DEX was acquired by deprotection of tert-butyl group and cleavage from rink resins by TFA: H2O: TIPs (18:1:1 v/v/v) solution for 3 times with 1 h each time. The final product was purified by column chromatography through silica gel (40–60 µm, 60A, Acros Organics) with mobile phase of DCM and MeOH (4:1 v/v). The molecular weight of intermediates (Fig. 3D-G) and final product CA-DEX were detected by MALDI-TOF mass (Bruker Microflex) [M +Na]⁺, m/z 1162.400 (calculated), m/z 1162.804 (found) (Fig. 3A). The solubility test showed that CA-DEX was slightly water soluble (up to 0.1 mg/ml).

2.1.2b. Preparation of prodrugs CA-GPC-DEX and CA-CB-DEX Dexamethasone prodrugs CA-GPC-DEX and CA-CB-DEX were prepared by solid phase peptide synthesis as shown in Fig. 2D and Fig. 2E.

Fmoc group of Rink Amide resin was deprotected via 20 % (v/v) 4-meth- ylpiperidine in DMF and the NH2 group was conjugated with Fmoc-Lys (Dde)–OH (three equiv) through coupling reagents HOBt and DIC con- firming by negative Kaiser test result. After deprotection of Fmoc group, Fmoc-Glu-OtBu and Cholic acid were coupled sequentially to achieve intermediate with cholic acid-targeting moiety. After removal of Dde group by 5 % (v/v) hydrazine in DMF, Fmoc-Lys(Dde)–OH was coupled.

Then glycerophosphorylcholine (GPC-COOH, Fig. 2C) or carboxylic betaine (CB) via both 4-(N,N-Dimethylamino)butanoic succinimide and tert-butyl bromoacetate at 60 ^◦C and DEX-COOH were conjugated after deprotection of Fmoc and Dde groups, respectively. The final product

CA-GPC-DEX and CA-CB-DEX were acquired by deprotection of tert- butyl group and cleavage from rink resins by TFA: H₂O: TIPs (18:1:1 v/

v/v) solution for 3 times with 1 h each time. The final products were purified by column chromatography through silica gel (40–60 µm, 60A, Acros Organics) with mobile phase of DCM and MeOH (2:1 v/v). The molecular weight of intermediates (Fig. 3D-J) final product CA-GPC- DEX and CA-CB-DEX were detected by MALDI-TOF mass (Bruker Microflex) CA-GPC-DEX [M + H]⁺, m/z 1607.874 (calculated), m/z 1607.035 (found) (Fig. 3B); CA-CB-DEX [M + H]⁺, m/z 1438.838 (calculated), m/z 1438.834 (found), [M +Na]⁺, m/z 1460.820 (calculated), m/z 1460.801 (found) (Fig. 3C). The solubility test showed that CA-CB-DEX was highly water soluble (up to 5 mg/ml). Thin-layer chromatography (TLC) revealed a single spot of CA-CB-DEX as visual- ized under UV 254 nm, indicating excellent purity (Fig. S1A).

Characterization of CA-CB-DEX in solution. CA-CB-DEX can be dissolved in phosphate buffered saline (PBS, pH 7.4) at the concentration of 5 mg/mL (3.5 mM) and micelle nanoparticles was detected by dynamic light scattering particle sizer at 10 nm (Fig. S1B). The critical micelle concentration (CMC) of CA-CB-DEX was measured by fluores- cence of Nile Red: 5 µL of 0.5 mg/mL methanol solution of Nile Red was added to the wells of 96-well plate. After methanol was evaporated under the vacuum, 100 µL of CA-CB-DEX solutions at different concentrations (1 to 1000 µg/mL) were added to each well and the 96-well plate was incubated with agitation overnight at room temperature covered with foil. Fluorescent intensity at 620 nm was measured using microplate reader (BioTek Synergy H1) with the excitation at 543 nm.

CMC of CA-CB-DEX was determined as 131.18 µg/mL (91 µM) at the intersection of two fitting lines (Fig. S1C).

2.2. Cell culture and drug treatment

2.2.1. Treatment of PHH with DEX and DEX prodrugs

PHH (Liver Tissue Cell Distribution System, Pittsburg) was replaced with RPCD1 medium (without DEX) in the afternoon and cultured overnight. DEX, the prodrug CA-CB-DEX (1 μM in DMSO), and vehicle (0.1 % DMSO) were added in the next morning (final DMSO concentration 0.1 %). PHH were collected 6 h after drug treatment for total RNA isolation and real-time PCR quantification of mRNAs, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

2.2.2. Comparative study on effects of DEX and BA conjugates of DEX (DEX-BAs) on mouse macrophage activation by lipopolysaccharide (LPS, from escherichia coli O55:B5, L2880, Sigma-Aldrich)

Mouse macrophage RAW 264.7 cells were seeded in 12-well plates (5 ×10⁵cells/well) and incubated at 37 ^◦C for 18 h (overnight). Cells were co-treated with DEX or one of the three DEX-BA conjugates (100 nM and 1 uM) and LPS (200 ng/mL). After 6 h incubation periods, cells were collected for total RNA isolation and real-time PCR analysis of interleukin 1 beta (Il1b), normalized to beta-actin.

Fig. 1. Chemical structure of DEX prodrugs CA-DEX, CA-GPC-DEX, and CA-CB-DEX.

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Fig. 2. Route of synthesis of DEX-COOH (A), CA-DEX (B), GPC-COOH (C), CA-GPC-DEX (D), and CA-CB-DEX (E).

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2.2.3. Comparative study on effects of DEX and DEX-BAs on LPS- stimulated expression and release of cytokines in human whole blood

Fresh human whole blood samples were obtained from a healthy male volunteer (IRB#754811–13) in the morning via venipuncture into heparinized tube. Aliquots of 0.24 ml heparinized human whole blood

were transferred to Eppendorf tubes that contain 0.24 ml of RPMI-1640 medium as well as test drugs and 2 ng/mL LPS. There were 6 treatment groups (N =4 per group), namely DMSO control, LPS, LPS plus drugs (prednisolone 0.75 uM, DEX 0.1 uM, CA-CB-DEX 0.1 uM, and CA-GPC- DEX 0.1 uM). The final DMSO concentrations were 0.1 % in all the Fig. 3. MALDI-TOF mass spectrum of CA-DEX (A), CA-GPC-DEX(B), CA-CB-DEX(C) and intermediates (D-J).

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treatment groups. After 4 h incubation with LPS (at 37 ^◦C x 250 rpm), samples were centrifuged at 1500 g for 10 min at 25 ^◦C and supernatants stored at − 80 ^◦C for analysis of tumor necrosis factor (TNF) and IL6 by ELISA. Total RNAs were prepared from the lower layer of blood cells using the RiboPure RNA Purification kit, blood (AM1928, Invitrogen) for qPCR determination of mRNAs, normalized to akirin 1 (AKIRIN1) which has been validated as the most stable housekeeping gene in human blood cells during inflammation and LPS treatment [57].

2.3. Animals and treatments

The mouse model of polymicrobial sepsis induced by cecal ligation puncture (CLP) is a “gold standard” animal model of sepsis because it reproduces certain key features of secondary bacterial peritonitis in humans. Adult male C57/BL6 mice underwent CLP-induced sepsis following our previous protocol [58]. Briefly, after anesthesia with ke- tamine/xylazine and laparotomy, the cecum is ligated with a 5–0 silk suture at about 1.3 cm position from distal pole to the base of cecum to avoid the bowel obstruction. The cecum is punctured twice with a 22G- gauge needle at top and bottom, respectively, and gently squeezed to extrude a 1-mm³column of fecal material. The cecum is returned to the abdominal cavity. Control (sham) mice undergo laparotomy without CLP, and then the abdominal incision is closed with 5–0 silk suture. After CLP surgery, warm saline (1 ml sc) is given for resuscitation. Mice received 0.05 mg/kg buprenorphine every 12 h for pain control. CB-CA- DEX (0.25 μmole/kg), DEX (0.25 μmole/kg), or vehicle (0.5 % DMSO in saline 10 ml/kg) were ip administered at 3 h after CLP. Mice were sacrificed 24 h after CLP to collect blood for complete blood count using Auto Blood Analyzer and analyses of blood chemistry. Liver tissues were collected and snap-frozen in liquid nitrogen for storage at – 80C^◦. All procedures in animal experiments have been approved by Institutional Animal Care and Use Committee (IACUC) at SUNY Upstate Medical University (IACUC #459).

2.4. RNA isolation and real-time PCR quantification of mRNA

Total RNAs from liver tissues and/or cells were extracted using RNA STAT-60 (Tel-Test, Friendswood, TX, USA) according to the manufac- turer’s instructions. Equal amount of total RNAs from each sample in the given group was mixed to prepare the pooled RNA samples. cDNA was produced using a cDNA Synthesis kit (iScript™ cDNA Synthesis Kit, Bio- Rad, Hercules, CA, USA). The diluted cDNA was used for real-time PCR quantification of mRNA using iTaq SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) and CFX Real-Time PCR Detection System (Bio-Rad).

A list of primers used for qPCR quantification of mouse and human genes is provided in Supplemental Table 1. The data were analyzed by CFX Maestro qPCR Analysis Software (Bio-Rad), and the amounts of mRNA were calculated using the CQ value, normalized to an endogenous reference gene.

2.5. Statistical analysis

Data are presented as mean ±standard error (SE). Statistical significance was set at * p <0.05. For comparison of two groups, Student’s t-test was used. For comparison of multiple groups, ANOVA tests and Dunnett T3 post hoc testing of selected pairings were utilized.

3. Theory

The introduction of novel ultra-hydrophilic zwitterionic linker into the design of liver-targeting GC prodrug will markedly increase the water solubility and decrease the non-specific passive diffusion of GC prodrug across cell membranes, leading to markedly improved liver- specific delivery of GCs and the resultant enhanced anti-inflammatory effects on the liver and decreased immunosuppression on blood cells and side effects on extrahepatic tissues.

4. Results and discussion

4.1. Development of novel DEX-BA conjugates as liver-targeting GC prodrugs

In contrast to GR, activation of MR by GCs worsens non-alcoholic steatohepatitis (NASH) and liver fibrosis [43]. Many natural GCs and synthetic corticosteroid hormones can activate both the GR and the MR, and we would like to avoid the deleterious side effects of activation of hepatic MR. Thus, we used DEX, a highly potent and selective agonist of GR over MR [59]. BA conjugates have been successfully synthesized as oral prodrugs for liver-specific drug delivery [52–56]. DEX is hydrophobic, so it is easily absorbed by essentially all types of cells. We used a hydrophilic flexible linker or a zwitterionic linker, namely carboxybetaine (CB) or glycerylphosphorylcholine (GPC) [60], to bridge CA and DEX to generate CA-DEX, CA-CB-DEX, and CA-GPC-DEX, via solid- phase peptide chemistry. We purified these GC prodrugs with chromatography, and confirmed the molecular weight and structure by mass spectrometry and nuclear magnetic resonance (NMR), respectively. We found that CA-DEX was slightly water-soluble, whereas the zwitterionic CA-CB-DEX and CA-GPC-DEX were highly water-soluble. The CA molecule allows the prodrug to be absorbed into hepatocytes by the liver-specific BA uptake transporter NTCP. This bridge of CA and DEX to generate CA-DEX, CA-CB-DEX and CA-GPC-DEX allows the prodrugs to be absorbed by the liver but minimizes the passive diffusion across the cell membrane.

4.2. Verification of transporter-dependent cellular activity of liver- targeting GC prodrugs

We first verified the transporter-dependent cellular activity of these prodrugs (Fig. 4). The liver-specific organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are essential for hepatic uptake of unconjugated BAs [61,62], whereas NTCP and apical sodium- dependent BA transporter (ASBT) mediate hepatic and intestinal uptake of conjugated BAs [63–65]. To verify the transporter-dependent cellular uptake, we co-transfected HEK293 cells with a luciferase reporter vector for bile salt export pump (BSEP) [66], a target gene for the BA receptor farnesoid X receptor (FXR), and expression vectors for FXR, OATP1B1, OATP1B3, ASBT, and/or NTCP. CA-DEX and CA-CB-DEX at 2 µM failed to activate BSEP promoter in the absence of transporters or the presence of OATP1B1/OATP1B3, but caused moderate and strong transactivation of BSEP promoter in the presence of ASBT and NTCP, respectively. We found that the DEX prodrugs CA-DEX and CA-CB-DEX had very poor activities in the activation of reporter for GR in HEK293 cells, even in the presence of NTCP (data not shown), which is consistent with the dramatic decreased GR agonist activity when the 21-hydroxyl of DEX is esterfied [67]. Thus, cellular uptake of CA-DEX and CA-CB- DEX are largely dependent on the liver-specific NTCP and intestine- predominant ASBT. CA-DEX and CA-CB-DEX will exhibit liver- selective uptake after iv/ip/sc injection and have the potential to be developed as oral drugs via the ASBT-NTCP pathway.

4.3. Differential effects of DEX prodrugs and DEX on LPS-stimulated macrophages

Macrophages have a high capacity of chemical uptake via endocytosis, which is independent of the BA transporters. To test whether the prodrugs have decreased uptake and activity in the macrophages, we conducted a comparative study using DEX and DEX-BAs (100 nM and 1 μM) to treat the macrophage RAW264.7 cells activated by LPS (200 ng/

mL). Compared to vehicle (VEH), LPS treatment for 6 h caused a dramatic induction of Il1b mRNA (Fig. 5A). DEX at 100 nM largely inhibited Il1b induction by LPS. CA-DEX had weaker, and CA-CB-DEX had much weaker inhibition compared to DEX at 100 nM. Even at 1 μM of CA-CB- DEX, its inhibitor effect still tended to be weaker than the parent drug

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DEX at 100 nM (Fig. 5A).

4.4. Differential effects of DEX prodrugs and DEX on LPS-treated human whole blood

In the blood circulation, the ester bond of these GC prodrugs may be broken by esterases to release the parent drug DEX, and these prodrugs may be taken up by the blood cells via endocytosis and/or passive

diffusion. We further compared the effects of our GC prodrugs with DEX and prednisolone, the current standard care for sAH, on the LPS- stimulated induction and release of cytokines in human whole blood.

qPCR data showed that LPS (2 ng/ml) tended to moderately induce TNF mRNA (Fig. 5B), and LPS strongly induced mRNA expression of proin- flammatory IL6 (Fig. 5C) and IL1B (Fig. 5D) as well as the anti- inflammatory IL1 receptor antagonist (IL1RN, Fig. 5E) and IL10 (Fig. 5F). In contrast, LPS down-regulated the key GR-target anti- Fig. 4.Roles of transporters in mediating transactivation of BSEP promoter by bile acid-dexamethasone conjugates. HEK293 cells were co-transfected with BSEP promoter reporter vector, pRL-CMV reporter vector, and expression vectors for FXR, OATP1B1/1B3 (1B1/3), ASBT, and/or NTCP. Medium was replaced with chemical-containing medium 18 h after transfection and dual-luciferase assay was conducted 6 h after chemical treatment. N =4 per group, mean ±SE. * p <0.05 versus control.

Fig. 5.(A) Effects of dexamethasone (DEX) and DEX prodrugs on Il1b mRNA expression (qPCR) in mouse RAW264.7 cells stimulated with lipopolysaccharides (LPS) for 6 h; N =3, mean ±SE. * p <0.05 versus LPS. (B-H) Effects of Prednisolone (Pred, 0.75 μM), DEX (0.1 μM), CA-GPC-DEX (GPC, 0.1 μM), and CA-CB-DEX (CB, 0.1 μM) on mRNA expression (qPCR) of TNF (B), IL6 (C), IL1B (D), IL1RN (E), IL10 (F), GILZ (G) and release of TNF (H) and IL6 (I) proteins in human whole blood 4 h (at 37 ^◦C) after stimulation with LPS (2 ng/ml). N =4 per group, mean ±SE. * p <0.05 versus LPS; # p <0.05 versus DEX.

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inflammatory gene glucocorticoid-induced leucine zipper (GILZ) (Fig. 5G). The equipotent dose of DEX (0.1 μM) and prednisolone (0.75 μM) caused similarly potent inhibition of the LPS-induction of IL6, IL1B, and IL1RN. Additionally, DEX and prednisolone further enhanced the LPS-induction of IL10 (Fig. 5F) and completely blocked the down- regulation of GILZ by LPS (Fig. 5G). Compared to DEX group, the CA- GPC-DEX (GPC) group tended to have higher expression of TNF, IL6, and IL1B, whereas the CA-CB-DEX (CB) group had significantly higher expression of TNF (Fig. 5B), IL1B (Fig. 5D), IL1RN (Fig. 5E) but lower IL10 (Fig. 5F). In contrast, all these GCs similarly reversed the LPS- down-regulation of GILZ, a major mediator of GC’s anti-inflammatory effects (Fig. 5G). Overexpression of GILZ in monocytes/macrophages inhibits the inflammatory response, enhances the phagocytosis of bac- teria, and prolongs the survival of CLP mice [68]. ELISA results showed that DEX and prednisolone similarly attenuated the LPS-induced release of TNF proteins (Fig. 5H), whereas only DEX attenuated the release of IL6 proteins in the whole blood (Fig. 5I). These results clearly

demonstrate weaker immunosuppressive effects of GC prodrugs CA-CB- DEX and CA-GPC-DEX compared to DEX on the human blood cells.

Because of the trend of lower GC activities of CA-CB-DEX compared to CA-GPC-DEX in non-hepatocytes, CA-CB-DEX was used in the further in vitro and in vivo studies.

4.5. Down-regulation of known GR-target genes in severe human AH Currently, alterations of hepatic GR signaling in human AH remain poorly understood. Thus, we analyzed the microarray data of hepatic mRNA expression in patients with sAH (Maddrey’s discriminant function >32) (GSE28619) [69]. Compared to normal liver, liver from sAH had moderate down-regulation of GR (NR3C1) and the GR-target bile acid uptake transporter NTCP [70] (Fig. 6A), consistent with cholestasis in AH patients [71]. Moreover, sAH liver had marked down-regulation of known GR-target genes hepatocyte nuclear factor 4A (HNF4A) (↓69 %) [72], Kruppel-like factor 15 (KLF15,↓69 %) [73], GILZ (↓87 %)

Fig. 6.(A) Data mining of microarray analysis (GSE28619) of mRNAs in humans with severe AH (normalized to β-actin). Mean ±SE. N =7–15 per group. (B) qPCR analysis of mRNAs in primary human hepatocytes (PHH) treated with 1 µM DEX, 1 µM CA-CB-DEX, or vehicle (0.1 % DMSO) for 6 h, normalized to GAPDH. N =3 per group, mean ±SE. * p <0.05 versus normal livers (A) or control PHH (B).

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[74], dual specificity protein phosphatase 1 (DUSP1, ↓92 %) [75,76], growth arrest DNA damage-inducible gene 45β (GADD45B, ↓81 %) [77], peroxisome proliferator activated receptor gamma coactivator 1α (PGC1α, ↓65 %) [78], glucose-6-phosphatase catalytic-subunit (G6PC,

↓82 %), pyruvate dehydrogenase kinase 4 (PDK4, ↓85 %) [79], glycine N-methyltransferase (GNMT, ↓88 %) [80], and metallothionein 1X (MT1X, ↓60 %) [81] (Fig. 6A). Hepatic KLF15 enables rapid switch between lipogenesis and gluconeogenesis to ameliorate hyper- triglyceridemia [82]. DUSP1 is essential in protecting against TNF- induced inflammation in liver [76]. GADD45β is a key hepatoprotective gene by inhibiting the JNK signaling [83–85]. The PGC1α is a master regulator of mitochondria biogenesis [86]. Hepatic deficiency of the key gluconeogenic enzyme G6PC aggravates HFD-induced steatosis and liver injury [87]. Hepatic PDK4 is critical in FAO [88], and loss of PDK4 switches the hepatic NF-κB pathway from pro-survival to pro-apoptosis [89]. GNMT maintains DNA methylation and protects against steatohepatitis and cholestatic liver injury [90]. Metallothionein protects against non-alcoholic fatty liver disease and ALD by inhibiting oxidative stress [91,92]. Thus, marked down-regulation of these known GR-target genes likely play important roles in steatohepatitis and cholestatic liver injury in sAH patients.

4.6. Dysregulation of noncanonical GR-target genes in sAH

Our data mining found that sAH liver had marked dysregulation of a group of noncanonical GR-target genes (Fig. 6A), including the retinoic acid receptor-related orphan receptor alpha (RORA, ↓76 %), ERBB receptor feedback inhibitor 1 (ERRFI1, ↓80 %) [93], 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3, ↓74 %) [94], hydroxy acid oxidase 2 (HAO2, ↓83 %), nicotinamide phosphoribosyl-transferase (NAMPT, ↓93 %) [95], and G0/G1 switch gene 2 (G0S2, ↑105 %). The orphan receptor RORα protects against NASH by inhibiting lipogenesis and inflammation [96–98]. ERRFI1, a negative EGFR regulator, protects against fatty liver and insulin resistance [99,100]. Increased glycolysis can provide the energy and intermediate metabolites to permit the survival of hypoxic hepatocytes [101]. The peroxisomal enzyme HAO2 promotes lipid catabolism to eliminate lipid accumulation [102].

NAMPT is a rate-limiting enzyme for the biosynthesis of NAD+[103]

that is depleted in AH. NAMPT is down-regulated in human AH and ethanol-fed mice [104]. In contrast, NAMPT overexpression ameliorates alcoholic liver injury by restoring NAD +level and activity of Sirtuin 1 [104], a crucial mitochondrial biogenesis regulator that protects against AH [105]. Conversely, G0S2 is a potent inhibitor of lipolysis and lipid droplet degradation [106]. Thus, dysregulation of these noncanonical GR-target genes also plays an important role in steatohepatitis and cholestatic liver injury in AH patients.

4.7. Modulation of mRNA expression of GR-target genes in PHH by GC treatments

PHH (Liver Tissue Cell Distribution System, Pittsburg) were cultured overnight in the serum-free medium for PHH [107] without GC, and then treated with CA-CB-DEX and DEX at 1 μM for 6 h to determine the direct effect of GR activation on transcriptome by RNA-sequencing, followed by qPCR verification. DEX at 1 μM does not activate the human pregnane X receptor [108]. Excitingly, we found that treatment of PHH with 1 μM DEX and CA-CB-DEX for 6 h caused similarly rapid and marked down-regulation of G0S2 and strong induction of all those known and noncanonical GR-target genes down-regulated in sAH (Fig. 6B). Taken together, hepatic GR signaling is markedly impaired in sAH patients, and GR in hepatocytes protects against AH via anti- inflammatory, anti-apoptotic, lipid-catabolic, and anti-cholestatic effects. Our novel highly hydrophilic CA-CB-DEX displayed good selectivity toward hepatocytes over immune cells which strongly supports the further development of CA-CB-DEX as liver-targeting GC prodrug to improve the GC therapy of sAH.

4.8. Better anti-inflammatory efficacy of CA-CB-DEX than DEX in mice with sepsis

Systemic GC treatment may increase the risk of infection in AH patients [109]. Currently, GC treatment is avoided in sAH patients with infections [45]. In contrast, hepatic GR deficiency occurs in septic patients, and GR in hepatocytes is critical in protecting against liver failure and mortality in mice with sepsis induced by CLP [8]. Thus, liver- selective activation of GR may improve the efficacy and decrease the side effects in AH patients with infections. We conducted a comparative study on the effects of CA-CB-DEX and DEX on CLP-induced sepsis in WT mice. CLP mice were ip injected DEX (0.25 μmole/kg =0.1 mg/kg), CA- CB-DEX (0.25 μmole/kg), or vehicle 3 h after CLP surgery, and mice were sacrificed 24 h after surgery. CA-CB-DEX, but not DEX, attenuated weight loss in CLP mice (Fig. 7A). Both CA-CB-DEX and DEX attenuated hypoglycemia (Fig. 7B). White blood cells were markedly decreased in CLP mice (Fig. 7C); CA-CB-DEX group tended to have less lymphocytopenia than DEX group (p =0.06). Hepatocytes are a major site of production of IL6 in mice [110] which has a key role in hyperinflammation and weight loss in sepsis. CA-CB-DEX attenuated hepatic induction of IL6 (Fig. 7D) and IL1b (Fig. 7E). Importantly, CA-CB-DEX had much weaker inhibitory effect than DEX on IL1b induction in macrophages (Fig. 5A). Thus, the stronger inhibitory effects on hepatic IL1b and IL6 by CA-CB-DEX than DEX in CLP mice are most likely due to pharmacokinetics, namely higher NTCP-mediated liver delivery of CA- CB-DEX (it is hard to determine DEX in liver 21 h after a low dose).

Sepsis causes translational inhibition and induction of Eif4ebp3, a repressor of Eif4e assembly to the cap complex [111]. Both CA-CB-DEX and DEX treatment abolished hepatic induction of Eif4ebp3 in CLP mice (Fig. 7F), suggesting that GC may ameliorate sepsis-induced translational repression in the liver. Overall, these data strongly suggest that our novel liver-targeting GC prodrug will have better efficacy in sAH and may be safer in AH patients with infections, in view of the better anti- inflammatory effects of CA-CB-DEX in septic livers and the trend of less lymphocytopenia in CA-CB-DEX group than DEX group (Fig. 7C).

5. Discussion

In the present study, we developed the first-in-class liver-targeting GC prodrugs and verified their transporter-dependent and liver-selective actions. CA-CB-DEX caused weaker immunosuppressive effects than DEX and prednisolone on human whole blood, whereas CA-CB-DEX exerted stronger anti-inflammatory effects on livers in septic mice. Re- sults from the present study strongly support further testing of our novel liver-targeting GC prodrug CA-CB-DEX as an improved therapy for sAH.

Hydrophilic linkers have been used to synthesize BA-drug conjugates to improve NTCP-mediated liver-selective drug delivery [52–56]. Our study is the first to use a highly hydrophilic zwitterionic linker to synthesize novel second-generation BA-drug conjugates, CA-CB-DEX and CA-GPC-DEX, to further enhance liver-specific drug delivery via increasing the water solubility and decreasing the passive diffusion of BA-drug conjugate across the cell membrane. Compared to DEX and the first-generation CA-DEX, CA-CB-DEX and CA-GPC-DEX exert much- enhanced water solubility and decreased immunosuppressive activities in mouse macrophages and/or human whole blood. To develop CA-CB- DEX as a novel improved therapy for sAH and sepsis, further test of CA- CB-DEX in translational animal model(s) of sAH and sepsis is warranted.

An interesting observation in this study is the formation of uniform nanoparticles (10 nm) by CA-CB-DEX, which likely contributes to its high water solubility. Zwitterionic polymers are known to form self- assembled nanoparticles [112]. The CA-CB-DEX nanoparticles are likely formed with a shell of zwitterionic CB and an inner core of CA and hydrophobic DEX. The CMC value of 91 µM for CA-CB-DEX suggests that the iv injected CA-CB-DEX will be in the form of nanoparticles in the blood initially. After dissociation from nanoparticle, the conjugated bile acids will mediate NTCP-dependent liver-specific uptake of the prodrug.

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The filtration clearance of nanoparticles by kidney has the effective size cutoff of 10 nm [113]. The uptake of nanoparticles by macrophages decreases with decreasing particle size [113]. Drugs delivered by nanoparticles have prolonged half-life due to the retention of drug nanoparticles in the circulation [113,114]. Therefore, the formation of stable small (10–20 nm) nanoparticles is desirable for persistent liver- specific delivery of GC. Interestingly, a zwitterionic polymer micelle with superhydrophilic zwitterionic CB polymer and superhydrophobic lipid has an ultra-low CMC and ultra stability [115]. Thus, we will further fine tune the density of zwitterionic CB polymer and the hydrophobic linkers to optimize the pharmacokinetics of enhanced liver- targeting DEX prodrugs. It can also be applied to other drugs to syner- gize liver disease treatments.

6. Conclusions

The ultra-hydrophilic zwitterionic linker can be used in the design of next-generation tissue/cell-specific prodrugs to markedly improve the water solubility and decrease the non-specific passive diffusion across cell membranes. Compared to DEX, CA-CB-DEX demonstrated good transporter-dependent hepatocyte-selectivity in vitro and better anti- inflammatory effects on livers of septic mice. Further test of the novel liver-targeting GC prodrug CA-CB-DEX as an improved GC therapy for inflammatory liver diseases and sepsis is warranted.

CRediT authorship contribution statement

Yazheng Wang: Investigation, Data curation, Formal analysis, Fig. 7. Effects of CA-CB-DEX and DEX on (A) Weight loss, (B) Blood glucose, (C) Blood lymphocytes, (D-F) qPCR quantification of hepatic mRNA expression (normalized to PGK1) 24 h after surgery in adult male sham and/or CLP mice ip injected CA-CB-DEX (0.25 μmole/kg), DEX (0.25 μmole/kg), or vehicle (VEH, 0.5 % DMSO 10 ml/kg) 3 h after surgery. N =3 (sham groups) or 6 (CLP groups) per group, mean ±SE. * p <0.05 versus sham control. # p <0.05 versus VEH CLP group.

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Methodology, Visualization, Writing – original draft. Dandan Guo:

Investigation, Data curation, Formal analysis, Methodology, Visualiza- tion, Writing – original draft. Rebecca Winkler: Investigation, Data curation, Formal analysis, Methodology, Visualization. Xiaohong Lei:

Investigation, Data curation, Formal analysis, Methodology. Xiaojing Wang: Investigation, Methodology. Jennifer Messina: Investigation, Data curation, Formal analysis. Juntao Luo: Conceptualization, Su- pervision, Resources, Investigation, Writing – review & editing. Hong Lu: Conceptualization, Funding acquisition, Supervision, Resources, Investigation, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Funding was provided partly by grants to H.L. from the National Institute of Health (R21AA027349 and R03CA241781).

Ethical statement

All animal experiments comply with the ARRIVE guidelines and have been carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Funding source

Funding was provided partly by grants to H.L. from the National Institute of Health (R21AA027349 and R03CA241781).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.medidd.2023.100172.

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