Various Approaches Employed to Enhance the Bioavailability of Antagonists Interfering with the HMGB1/RAGE Axis

Abstract

High-mobility group box 1 (HMGB1) is a nuclear protein that can interact with a transmembrane cell surface receptor for advanced glycation end products (RAGEs) and mediates the inflammatory pathways that lead to various pathological conditions like cancer, diabetes, cardiovascular diseases, and neurodegenerative disorders. Blocking the HMGB1/RAGE axis using various small synthetic or natural molecules has been proven to be an effective therapeutic approach to treating these inflammatory conditions. However, the low water solubility of these pharmacoactive molecules limits their clinical use. Pharmaceutically active molecules with low solubility and bioavailability in vivo convey a higher risk of failure for drug development and drug innovation. The pharmacokinetic and pharmacodynamics parameters of these compounds are majorly affected by their solubility. Enhancement of the bioavailability and solubility of drugs is a significant challenge in the area of pharmaceutical formulations. This review mainly describes various technologies utilized to improve the bioavailability of synthetic or natural molecules which have been particularly used in various inflammatory conditions acting specifically through the HMGB1/RAGE pathway.

1. Introduction

High-mobility group box 1 (HMGB1) is a protein that regulates several transcription factors including NF-κB and glucocorticoid receptors, by binding to DNA [1,2]. In extracellular space an excessive amount of it can cause internal injury and organ dysfunction that could lead to various pathological issues [3,4,5,6]. HMGB1 is widely expressed in nearly all eukaryotic cells and plays a crucial role in maintaining cellular structure, as well as regulating gene transcription and transcription factors [7,8]. Extracellular HMGB1 exerts its biological effects by interacting with receptors such as the receptor for advanced glycation end products (RAGEs) and Toll-like receptors (TLRs), thereby contributing to the regulation of inflammatory processes [9,10]. A RAGE is a transmembrane receptor with a molecular weight of approximately 45–50 kDa, composed of three immunoglobulin-like domains: the extracellular domain, hydrophobic transmembrane domain, and intracellular cytoplasmic domain [11]. The extracellular region is further subdivided into three subdomains: A variable domain (V-domain), which is connected to two constant domains C1 and C2 [12]. The VC1 domain of a RAGE plays a key role in binding a wide range of ligands, including HMGB1, which can activate multiple pathological inflammatory pathways by promoting the release of pro-inflammatory cytokines (Figure 1). The detailed functional description, expression along with the structural information of HMGB1 and individual domains of a RAGE are described in our previous manuscripts [13,14].

Targeting the HMGB1/RAGE signaling pathway has emerged as a promising therapeutic strategy for managing inflammatory conditions, as demonstrated by several recent studies. Several research groups published synthetic as well as natural molecules that can inhibit the interactions between HMGB1 and RAGE to treat various disease conditions. However, the low water solubility and low bioavailability of these therapeutic molecules limit their pharmacological potential and clinical use. In this review article various approaches towards the enhancement of bioavailability in terms of solubility of various natural or synthetic molecules reported to affect the HMGB1/RAGE pathway have been discussed. The article is a complete compilation and could be helpful for the research community striving hard to fight to enhance these physical parameters to meet desired bioavailability.

2. Methodology

For this compilation, the relevant literature published in the past 20 years (2005–2025) was collected from reputable online databases, including the National Center for Biotechnology Information (NCBI, https://pubmed.ncbi.nlm.nih.gov/) ScienceDirect (https://www.sciencedirect.com) accessed between 23 February 2023 and 12 April 2023 and as needed thereafter. A comprehensive search was conducted using various keywords and keyword combinations related to the discussed chemical entities. These keywords included: HMGB1/RAGE, HMGB1/RAGE antagonist, HMGB1/RAGE interaction inhibitors, solubility improvement of synthetic/natural molecules, bioavailability improvement of synthetic/natural molecules, and solubility/bioavailability issues in crocin, berberine, curcumin, etc. Exclusion Criteria: Articles were excluded if they were published before 2005 (unless showing some important information), were not in English, lacked peer review, or did not directly address the objectives of this review (e.g., studies not focused on HMGB1/RAGE or solubility/bioavailability enhancement strategies). Duplicate records and retracted studies were also omitted. Data Analysis Strategy: After initial screening by title and abstract, the full texts of eligible studies were reviewed to extract relevant information regarding HMGB1/RAGE interactions, molecular inhibitors, and solubility/bioavailability improvement approaches. The data were thematically grouped and qualitatively synthesized into three core focus areas: (1) HMGB1/RAGE-targeted inhibition mechanisms; (2) challenges associated with the solubility/bioavailability of natural products; and (3) emerging formulation technologies aimed at improving pharmacokinetic properties.

3. Various Approaches to Enhance the Solubility/Bioavailability of Synthetic or Natural Molecules

Undoubtedly, the oral route is the most preferable route of administration considering patient compliance and safety; still, it has many hurdles to overcome in order to achieve the highest possible bioavailability without affecting the chemical intactness of the drug. The poor solubility and bioavailability are among the greatest concerns as most of the molecules are hydrophobic in nature and further their insufficient gastrointestinal permeability also cannot be overlooked [15]. The poor aqueous solubility of the drug molecules could result in their inadequate concentration at the desired site of action with compromised drug distribution, protein binding, and absorption pattern. However, more than 70% of novel drug molecules are experiencing poor water solubility and related issues [16]. The biopharmaceutics classification system categorizes the drug compounds according to their solubility and permeability as described in Figure 2.

The solubility issues can be overcome by employing numerous approaches including salt formation, co-crystallization, pH adjustment, conjugation, electrolytes, prodrug, etc. It is very important to focus here that the selection of method highly depends upon the type of target site, the nature of the drug molecule, and the dosage form [17]. For instance, the presence of the double bond in flavonoids resulted in a very tight and compact structural configuration which interrupts the penetration of solvent molecules, hence makes them poorly aqueous soluble moieties. In this regard, introducing more hydroxyl groups could make the molecules more inclined toward degradation under harsh physiological environments and this biotransformation will limit their efficacy. Therefore, chemical and physical alteration is not only the solution to develop an effective drug delivery; the drug transporters are also the backbone of drug therapy [18]. The design of conventional dosage form to deliver low-solubility drugs is very challenging as it not only fails to control the drug release precisely and suffers from a retarded onset of action and an inability to attain a steady-state plasma concentration (dose escalation is needed), but also the high dose frequency with poor patient compliance ultimately leads to toxicity at the physiological level [19,20].

3.1. Nano-Sized Carriers

Nano-sized carriers have also captured the attention of many researchers worldwide, in addition to the methodologies listed above. Nano-scale drug carriers offer various advantages including a high surface area due to their small diameter, protection of cargo from enzymatic degradation/extreme pH environments by enclosing them into polymeric, lipid, and polymer–lipid hybrid membranes. In addition, the possibility of surface modification enables them to achieve site-specific delivery [21].

3.2. Biodegradable Polymers

The nanoparticles made of biodegradable polymers, namely poly(ε-caprolactone), have the ability to cross the intestinal mucosa very efficiently even without affecting their intactness. They undergo endocytosis by mucosa-associated M-cells (microfold cells located in the follicle-associated epithelium in intestinal mucosa), the hydrogen bonding and Van der Waal forces of attraction are responsible for establishing interactions between nanoparticles and cells. These systems transfer drug molecules to the blood circulation via the lymphatic system. Adopting this approach, a 3.6-fold higher bioavailability of ellagic acid in New Zealand white rabbits has been quantified as compared to ellagic acid in the free solution form. Moreover, these polymeric nanoparticles demonstrated a 6.9-fold higher accumulation in colon adenocarcinoma, making them more clinically relevant [22].

PLGA nanoparticles have been fabricated for the delivery of doxorubicin and it has been found that their penetration and diffusion across the thick mucosa has been enhanced by several fold by surface modification with PEG. This concept has been proven by recording the bioavailability across the Caco-2 cell line in rat ileum [22].

3.3. Lipid-Based Nanosystems

Similarly to polymeric nanosystems, lipid-based nanosystems are also capable of delivering drug molecules to the systemic circulation by avoiding first-pass metabolism by the liver. The unique physiological structural configuration of these lipophilic macromolecules can improve permeability across the lymphatic vessels. Fascinatingly, the extent of lymphatic absorption can be modified using fatty acid chains of different lengths. Additionally, these lipids can interrupt P-gp (P-glycoprotein) efflux system and increase the absorption of the drugs [23,24,25,26].

However, the stability of lipid nanoparticles has always been a huge concern. In this regard, the fate of lipid nanoparticles (LPs) carrying siRNA has been followed across the GI track, wherein LPs were found to be stable and potent in terms of exposure to different pH conditions, bile salt, and pepsin. Further confocal microscopy confirmed the presence of unchanged LPs within endothelial cells of the intestine and colon, which reveals that LPs have the capacity to withstand the stomach and upper-intestine environment [27].

3.4. Hybrid Approach Combining Polymers and Lipids

Considering the fact that polymers and lipids are both very promising strategies to enhance oral bioavailability of natural and synthetic drugs, various attempts have been made to analyze the combined effect of both polymers and lipids as a single-unit model. More precisely, polymeric core and lipid shell can be employed together which can improve the bio-profile of molecules. A similar kind of model has been employed to deliver low-molecular-weight heparin, where drug–chitosan complex formation, followed by their encapsulation into the lipid shell, was carried out. The chitosan provides the muco-adhesiveness in the stomach while the lipid enhances the overall stability and loading efficiency of the drug [28,29]. One more attempt has been reported, wherein curcumin was conjugated with a water-soluble detachable polymer incorporated into a degradable lipid shell. Due to the improved mucus permeation and intracellular absorption; ultimately more curcumin was bioavailable in the rat model. Therefore, the synergic effect of lipids and polymers has been established [30].

3.5. Non-Ionic Surfactants

Bicelles composed of different chain lengths (long-chain phospholipid and short-chain non-ionic surfactants) can be designed for oral-route delivery. The main objective of using small chains is to protect the lipophilic tails of long-chain phospholipids from water. Therefore, this compact packing of chains results in the higher stability of the carriers. Bicelles derived from non-ionic surfactants—namely tween, labrasol, and pluronic F127—can be exploited along with a combination of phospholipids. These aforementioned systems have been selected to enhance the dissolution of curcumin as compared to liposomes and curcumin alone in anti-viral therapy for COVID-19 patients [31].

3.6. Polymer—Surfactant Combinations

Moving along, a positive synergism can be established using a polymer along with a surfactant. This hypothesis can be further explained by considering an approach wherein the dissolution of efavirenz is modified with a combination of a polymer (hydroxypropyl methylcellulose phthalate) along with surfactants (either sucrose palmitate or polysorbate 80). It has been concluded that the surfactant can play a key role in tuning the dissolution pattern of various drugs. Thus, it can improve the overall stability and bioavailability of poorly soluble drugs [32].

4. Drug Molecules Affecting HMGB1/RAGE Axis and Methods to Enhance Their Bioavailability

4.1. Crocin

Crocin (Figure 3) exerts a suppressive effect on the HMGB1-RAGE signaling pathway in the context of cigarette smoke-induced cognitive impairment. Specifically, Crocin reduced HMGB1 protein expression, thereby downregulating the downstream RAGE/TLR4-NF-κB pathway. Although the exact effects on mRNA levels, nuclear translocation, or phosphorylation of HMGB1 are unknown, crocin inhibits the signaling cascade primarily at the protein level. This suppression is linked to an increase in GLP-1 expression, a reduction in pro-inflammatory cytokines (TNF-α and IL-1β), and the attenuation of oxidative stress and neuronal apoptosis, suggesting that crocin action involves inhibition of HMGB1/RAGE-mediated neuroinflammatory signaling [33]. Crocin is the active metabolite of saffron. However, only trace levels of crocin can be found in the systemic circulation after its oral administration due to the fact that they are glycosidic esters [34,35,36]. Crocins and crocetin (Figure 3) have low stability, poor absorption, and low bioavailability [37]. In vitro studies have shown that crocins of saffron are probably not bioavailable in the systemic compartment after oral application because they are rapidly hydrolyzed, mainly by esterase enzymes in the intestinal epithelium [36]. It is interesting to observe, given that the pharmacological effect is attributed to crocetin, that the oral administration of 60 mg/kg of crocins, once daily for 4 days, reaches a concentration 56–81 times greater than crocetin in rat serum and than the oral administration of crocetin. Interestingly, oral crocin administration leads to significantly higher serum levels of its active form, crocetin [38], compared to direct crocetin administration, suggesting a more effective delivery route. This insight opens promising avenues for leveraging crocin over crocetin in therapeutic applications.

4.2. Berberine

Shi et al. [39] found that treatment with berberine led to a significant reduction in inflammatory cytokines, including TNF-α and IL-1α, in mice, accompanied by neuroprotective effects. Their findings indicate that berberine’s anti-inflammatory action may be specifically mediated through inhibition of the HMGB1/RAGE signaling pathway. Specifically, berberine treatment reduced HMGB1-mediated activation of microglia and A1 astrocytes in the hippocampus, thereby alleviating neuroinflammation and neuronal damage. The data suggest that berberine acts primarily at the protein signaling level to suppress HMGB1-RAGE activity. This suppression led to decreased levels of inflammatory markers (TNF-α, IL-1α, and C1qA) and improved cognitive function in septic mice, highlighting berberine’s potential as a therapeutic agent targeting HMGB1-RAGE signaling [39]. It has been reported that Berberine is hydrophilic and undergoes extensive metabolism; thus, the bioavailability upon oral administration is extremely low. However, several nano strategies have been implemented by researchers to increase its bioavailability. Moreover, certain drugs, when co-administered, significantly increase its absorption. Talking about its physical properties, it is a yellow colored, isoquinoline alkaloid, crystalline, stable, quaternary amine (Figure 4).

It is readily soluble in hot ethanol, and slowly dissolves in water. Moreover, it is not very soluble in organic solvents like chloroform and benzene [40]. Berberine is a permanently charged compound and its water solubility depends primarily upon the buffer and temperature and is independent of pH due to the absence of any ionisable group. It has been observed by Battu et al. [41] in their study that, as temperature increases, the solubility of berberine chloride in water slightly increases. At a physiological pH, it has been found that berberine is a positively charged moiety because of the presence of an iminium cation (C = N +), which is polar in nature, and the absence of an acceptor or proton donor group [42].

It has been found that various parameters affect the oral bioavailability of berberine such as the rate of its dissolution and dispersion, its permeability, its stability in the environment of the gastrointestinal tract, its solubility, and physiological aspects like metabolism. Although a number of contradictory bioavailability values of berberine have been reported so far, its absolute bioavailability is less than 1% [43,44,45]. Although the exact mechanism of its low bioavailability is still unknown, some studies have proposed various possible reasons for this. Firstly, the hydrophilic nature of the compound, which may be the primary reason that limits its absorption. The strong hydrophilicity of berberine is contributed to by various factors such as the octanol–water partition coefficient with a Log p-value of −1.51, topological Polar Surface Area (tPSA) of 40.8 A°, a percentage of Hydrophilic Surface Area (HAS) of 25.97% [41,46]. Secondly, there was an extensive intestinal first-pass metabolism observed. Finally, ATP-binding cassette (ABC) transporters such as P-glycoproteins (P-gp) could interfere with absorption by directly effluxing the absorbed berberine back into the intestine [43,47]. Researchers have used several nanoparticulate approaches to enhance bioavailability and solubility, such as solid-lipid nanoparticles, polymeric nanoparticles, magnetic silica-based nanoparticles, gold nanoparticles, micelles, liposomes, and many more [48]. For example, Fei et al. developed novel self-assembled berberine-loaded PEG–lipid–PLGA nanoparticles using a Berberine–soybean phosphatidylcholine complex as a lipo-solubility enhancer via the solvent evaporation technique for enhancing oral bioavailability. They reported a significant increase in oral bioavailability by 343% compared to raw berberine [49]. In another study, Xiong et al. developed a Brij-S20-modified nanocrystal formulation with the aim of improving the intestinal absorption of berberine. Their findings revealed that the relative bioavailability of BBR-BS20-NCs compared to pure berberine was found to be 404.1% [50]. Several other recent nanoformulation studies of berberine published in recent patents are summarized in

Table 1. Various patented formulation forms of berberine to enhance solubility and bioavailability.

S No.	Type of Formulation	Year	Patent Number
1	Berberine hydrochloride self-microemulsion	2018	CN104825389B
2	Mangiferin-6-o-berberine salt	2019	US10285969B2
3	Extracted from phellodendron amurense	2019	CN1100551118B
4	Fenofibric acid salt with berberine or its analogs	2020	US10577379B10
5	Gelatin-loaded berberine hydrochloride nanoparticles encapsulated by erythrocyte membranes	2020	CN108113977B
6	Berberine succinate crystals	2022	CN115572292A and CN115477647A

.

Collectively, berberine exhibits strong anti-inflammatory effects, particularly by targeting the HMGB1/RAGE axis, but its clinical translation is hindered by its hydrophilic nature, low permeability, and extensive first-pass metabolism. Its oral bioavailability is reported to be less than 1%. Researchers have explored numerous nanoformulations (liposomes, solid-lipid nanoparticles, and nanocrystals) and co-administration strategies to significantly enhance its absorption and stability. This section highlights the promising outcomes of these technologies, some achieving over 400% bioavailability improvements, demonstrating the drug’s viable future via advanced delivery systems.

4.3. Curcumin

Curcumin is the major biologically active polyphenolic constituent in turmeric, also called diferuloylmethane (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione). (Figure 5). Curcumin is found primarily in the roots and rhizomes of the turmeric plant (Curcuma longa). Curcumin exhibits a wide range of beneficial effects including anti-inflammatory, antioxidant, chemoprotective, tissue protective, antibacterial, anti-fungal, antiviral, metabolism-regulating, immuno-modulating, antineoplastic, and anti-depressant properties [51,52,53,54,55]. Han et al. [56] demonstrated that curcumin (Figure 5) may help alleviate cognitive deficits in a transgenic mouse model, potentially by targeting the HMGB1/RAGE inflammatory pathway. Their findings indicated that curcumin did not alter amyloid plaque accumulation in the hippocampus, suggesting that its neuroprotective effects might be specifically linked to modulation of the HMGB1/RAGE axis rather than plaque reduction. More specifically, curcumin attenuates neuroinflammation in Alzheimer’s disease by inhibiting the HMGB1-RAGE/TLR4-NF-κB signaling pathway. In APP/PS1 transgenic mice, chronic oral administration of curcumin significantly reduced HMGB1 protein expression along with downstream molecules RAGE, TLR4, and NF-κB in the hippocampus. While curcumin did not affect amyloid plaque accumulation, it improved cognitive performance and decreased glial activation, as evidenced by the reduced number of GFAP-positive cells. Although the study does not elaborate on HMGB1 mRNA levels, nuclear translocation, or phosphorylation, the observed effects suggest curcumin’s anti-inflammatory action is mediated through downregulation of HMGB1 protein signaling, contributing to improved memory and reduced neuroinflammation [56]. Issues which greatly limit the effectiveness and usefulness of curcumin are its low bioavailability, attributed to water insolubility, and rapid metabolism to inactive metabolites. Curcumin is an oil-soluble compound, practically insoluble at room temperature in water at acidic and neutral pH. While it is soluble in alkali, it is very susceptible to auto-degradation. The water solubility of curcumin is estimated to be 11 ng/mL.

Curcumin is a highly reactive compound because of its unique molecular structure. It is sensitive to visible and UV light, breaking down into a series of compounds with vanillin and ferulic acid being the most prominent end products [57,58]. Uncontrolled and unknown light exposure has cast a shadow of unreliability on curcumin research to date. Therefore, when studying curcumin, great care must be exercised to avoid exposure to light. Curcumin is unstable in aqueous solutions at pH > 6.5 at 37 °C through an autoxidation reaction, resulting in the formation of cleavage products with the ultimate products becoming vanillin and ferulic acid, cyclopentadione internal cyclization structures, or dimerization compounds [57,58,59,60]. Curcumin can also be oxidized by free radicals and oxyradicals, with many decomposition products, accounting for some of the well-known antioxidant properties of curcumin. Curcumin has been found to be metabolized by various aldo-keto reductase enzymes inside the human body that can only capture and degrade its di-ketone group [61]. To overcome this limitation, mono carbonyl analogs of curcumin have been synthesized by deleting the reactive β-diketone moiety (which was considered to be responsible for the pharmacokinetic limitation of curcumin) (Figure 5) through several chemical modifications in the basic structure of curcumin in order to increase its potential, stability, and bioavailability. Nevertheless, some of the recently developed mono carbonyl curcumin analogs (1–5) (Figure 5) exhibit their improved biological activity [62,63,64,65,66].

Besides chemical modifications, various formulations have been developed to enhance solubility or dispersibility with the goal of enhancing bioavailability and consequent bioefficacy [67,68,69,70]. The reported delivery systems for curcumin include micelles, liposomes, phospholipid complexes, microemulsions, nano-emulsions, emulsions, solid lipid nanoparticles, nanostructured lipid carriers, biopolymer nanoparticles, and microgels. They not only enhance efficacy, but also increase curcumin bioavailability by optimal permeation in the small intestine and preventing possible degradation in the gastrointestinal tract [71]. In addition to the above commercial formulations, a wide range of micellar and nanoparticle formulations of curcumin have been prepared involving the use of ingredients such as Tween 80, polysorbate 80, ceramic particles, polyethylene glycol (PEG), alginate, poly(lactic-co-glycolic acid) (PLGA), omega-3 fatty acids, chitosan, and other substances [72,73,74,75]. The cellular uptake of a substance depends on size and surface properties. A study compared the bioavailability of free curcumin powder and five curcumin formulations involving hydroxy propyl methyl cellulose, poly(lactic-co-glycolic acid) (PLGA), cyclodextrin, dendrimeric (globular, branched macromolecular structures), and magnetic nanoparticles [76]. The curcumin formulations had spherical particle sizes ranging from ~5 to 58 nm, while unformulated curcumin exhibited highly aggregative, larger clusters (>1.2 μm). All formulations showed significantly higher cellular uptake compared with free curcumin powder. The hydroxy propyl methyl cellulose–curcumin formulation, which had the smallest particle size of 5.2 nm and, compared to the other four formulations, displayed its highest bioavailability and efficacy in prostate cancer cells. Various polymer or surfactant surface stabilizers such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and the surfactants sodium dodecyl sulfate (SDS), carboxymethylcellulose sodium salt, Tween 80, polysorbate 20, and D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) have been utilized to achieve the stability of curcumin nanoparticles in physiological conditions. Their incorporation also increases the permeability of cellular membranes which allows higher absorption of nanoparticles [72,73].

So far, mono-carbonyl analogs have been developed and various delivery systems (nanoparticles, liposomes, and micelles) have been employed to overcome the limitations associated with raw curcumin. These strategies not only enhance its stability and absorption but also reinforce its therapeutic potential, suggesting curcumin could be more effective through optimized formulations.

4.4. Pentoxifylline

Pentoxifylline (PTX), chemically named 3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1H-purine-2,6-dione or 1-(5-oxohexyl)-3,7-dimethylxanthine, is a BCS (biopharmaceutics classification system) class II drug (Figure 6). It is a methyl-xanthine derivative, and is used as a hemorheologic agent for the therapy of peripheral artery disease. It has been found that this hemorheological agent shows its anti-epileptic effect by affecting the HMGB1/RAGE pathway [77]. Due to the slow dissolution rate of PTX, the oral administration of the drug has a bioavailability of only 20 to 30%—this needs to be enhanced.

It is well known that microsponges are designed to efficiently deliver a pharmaceutical active ingredient at a minimum dose while enhancing stability, reducing side effects, and improving drug release. Inspired by these findings, Mehetre and colleagues prepared polymer-based Pentoxifylline microsponges using a quasi-emulsion solvent diffusion method. These microsponges were associated with a better aqueous solubility and dissolution rate. In addition to their use for transdermal delivery, microsponges also play a role in oral administration. A standard oral dissolution study was further performed to determine the drug release profile. The data also suggested an improved oral bioavailability of Pentoxifylline from microsponges.

Varshosaz et al. [78] also undertook successful efforts to enhance the oral bioavailability of Pentoxifylline by preparing solid lipid nanoparticles (SLNs). The mean particle size and size distribution, drug entrapment efficiency (EE%), zeta potential, and drug release of the SLNs were investigated. A pharmacokinetic study was conducted in male Wistar rats after oral administration of 10 mg kg(⁻¹) of Pentoxifylline in the form of the free drug or SLNs. The z-average particle size, zeta potential, and EE% of the SLNs were at least 250 nm, −30.2 mV, and 70%, respectively. They reported that the lipid type, surfactant type, and percentage had a significant effect on the particle size. Zeta potential was claimed to be more affected by lipid type, the acetone–DCM ratio, and sonication time. The speed of the homogenizer and the acetone–DCM ratio had a significant effect on the EE%. The optimized SLN was prepared by combining 80 mg of cetyl alcohol and 10 mg of lecithin, with an acetone–DCM ratio (1:2), for 30 s of sonication, 3% Tween 20, and a mixing rate of 800 rpm. In vitro drug release lasted for about 5 h. It was found that the relative bioavailability of Pentoxifylline in SLNs was significantly increased, compared to that of the Pentoxifylline solution [78].

Novel delivery systems such as polymer-based microsponges and solid lipid nanoparticles (SLNs) have been explored to improve Pentoxifylline release profile and absorption. These advancements underline the potential for improved clinical efficacy of PTX with modified formulations aimed at enhancing solubility and bioavailability.

4.5. Doxorubicin/Adriamycin and FPS-ZM1

Doxorubicin HCl (doxorubicin, Figure 7) also known as Adriamycin is a widely used chemotherapeutic agent. It is usually given in combination with other drugs. Lai et al. [79] reported that Doxorubicin in combination with FPS-ZM1 (a known inhibitor of RAGE, Figure 7), could inhibit the interactions between HMGB1 and RAGE to treat leukemia. Doxorubicin combined with FPS-ZM1 effectively disrupts the HMGB1/RAGE signaling pathway. This combination attenuates HMGB1-induced autophagy by downregulating ERK1/2 activation and restoring mTOR phosphorylation, while simultaneously promoting apoptosis through the p53/Bcl-2 axis. Blocking HMGB1/RAGE also suppresses NF-κB activation and reduces the expression of drug efflux proteins P-gp and MRP at both mRNA and protein levels, thereby mitigating chemoresistance. In vivo, this intervention improves survival and reduces pathological severity in T-ALL mice, highlighting its potential as a therapeutic strategy against relapsed or refractory leukemia. FPS-ZM1 is generally sparingly soluble in aqueous solution but to enhance its solubility in the aqueous buffer, it is recommended to dissolve it into highly polar organic solvents like DMSO or DMF, then it can be diluted to its desired concentration. Its reported solubility is approximately 0.12 mg/mL of 1:7 DMF:PBS solution at pH 7.2 but the aqueous solution is very unstable. Besides its solubility, it is a highly permeable compound that can readily cross the blood–brain barrier [80]. Meanwhile, Doxorubicin is a low-permeability, class III drug (according to biopharmaceutical classification system (BCS) (Figure 2) with a low oral bioavailability which is less than 5% [81]. Some efforts have been made to alleviate this problem [82,83]. However, researchers are continuously fighting with its oral delivery challenges. In 2018, Ahmad et al. reported a method to enhance the oral bioavailability using surface-modified biodegradable polymeric nanoparticles [84]. They developed a sphere-shaped smooth-surface nanoformulation (PEGylated-doxorubicin-loaded-poly-lactic-co-glycolic acid (PLGA)-nanoparticles (NPs)) with a particle size of 183.10 nm and a zeta potential of −13.10 mV, containing a drug content of 42.69 μg/mg. They found a marked improvement (6.8 fold) in the developed nanoparticles as compared to the standard Doxorubicin as a linear dynamic range with R² ≥ 0.9985 over a concentration range of 1.00–2500.0 ng/ml was observed [84]. Similarly, Daeihamed et al. found a four-fold improvement in the oral bioavailability of doxorubicin when administered as liposomes [85]. This improvement was observed with the use of non-PEGylated, 120 nm positively charged rigid liposomes. The extent of the drug’s first-pass metabolism as well as the endocytosis of nanoparticles were markedly affected by liposomal formulation [85]. Manickam et al. also published their reports in support of these results stating that Liposomal Doxorubicin–Hydrochloride shows significant advantages of using liposomes as a carrier vehicle, which reduced cardio toxicity and had an enhanced antitumor effect against various cancers.

Ultimately, when used in combination with the RAGE inhibitor FPS-ZM1, it shows promise in disrupting HMGB1/RAGE-mediated pathways in leukemia. However, Doxorubicin’s poor oral bioavailability and FPS-ZM1′s aqueous instability present formulation challenges. Researchers have addressed this by creating nanoparticle and liposome-based drug delivery systems, which significantly enhance bioavailability while also potentially reducing systemic toxicity. These innovations support the potential for oral formulations of Doxorubicin and improved solubility of FPS-ZM1 in targeted cancer therapies.

4.6. Dexmedetomidine

Dexmedetomidine is a highly selective α2 adrenergic agonist with anxiolytic, sedative, and mild analgesic properties. Recently, it has been identified with the potential to act through the HMGB1/RAGE axis for the treatment of acute lung injury (ALI) [86]. Dexmedetomidine (Figure 8) exerts protective effects against acute lung injury (ALI) by modulating the HMGB1/RAGE signaling pathway [86]. Specifically, it significantly downregulates HMGB1 and RAGE expression at both the mRNA and protein levels, inhibits NF-κB activation, and reduces pyroptosis-related protein expression. Importantly, dexmedetomidine suppresses the nuclear-to-cytoplasmic translocation of HMGB1, thereby limiting its extracellular pro-inflammatory activity. These effects are diminished by RAGE overexpression, indicating the centrality of HMGB1/RAGE signaling in Dexmedetomidine’s mechanism. Overall, Dexmedetomidine mitigates inflammation and pyroptosis in ALI through inhibition of the HMGB1/RAGE/NF-κB axis and blockade of HMGB1 nuclear export [86]. No significant reports have been published on the oral bioavailability of this compound except the improvement in the nasal spray that is generally used in very particular disease conditions where the HGMB1/RAGE pathway has not been found to be involved. The absence of significant oral delivery studies suggests an area for future exploration, particularly if HMGB1/RAGE modulation becomes a clinical target in related pathologies.

4.7. Epigallocatechin-3-Gallate

Green tea is rich in catechins composed of more than eight polyphenolic compounds. According to different sources, the most abundant catechin in tea is Epigallocatechin-3-gallate (EGCG) (Figure 9). [87,88]. Many in vitro studies have shown that tea catechins had various beneficial effects through various pharmacological mechanisms. It has also been reported that by targeting the HMGB1/RAGE axis it can treat the inflammatory condition. Nishioku et al. [89] claimed EGCG suppresses the extracellular release of HMGB1 and downregulates the expression of its receptor, RAGE, during osteoclast differentiation. This is accompanied by the upregulation of HO-1 and a reduction in key osteoclastogenic markers, including NFATc1, cathepsin K, c-Src, and ATP6V0d2. These effects suggest that EGCG interferes with HMGB1/RAGE signaling at both the ligand and receptor levels, likely preventing downstream inflammatory and differentiation signals, thereby reducing bone resorption. However, inconsistent results between in vitro and in vivo studies or between laboratory tests and epidemical studies are observed. The low solubility as well as bioavailability of tea catechins was an important factor leading to these inconsistencies. In vitro studies revealed that therapeutic doses of EGCG ranged from 1 to 100 μmol/L. However, the peak plasma levels of tea catechins were usually in the very low micromolar range in both human subjects and animals models after their oral administration [90,91], which was lower than the effective concentration of in vitro tests.

Therefore, the therapeutic potential is limited, due to its poor stability in the gastrointestinal tract and very limited membrane permeability via the intestine [92,93]. The inconsistency between the in vitro biological activity and in vivo studies can also be recognized to its low stability, which lead to the formation of pro-oxidant molecules and degradative products [94]. They are unstable under physiological conditions and they usually degrade or metabolize through interactions owing to their hydroxyl groups on the phenol rings [94]. EGCG generally performs its cellular functioning at relatively high concentrations to affect disease conditions [95,96]. It is believed that taking green tea polyphenol products in amounts equivalent to the EGCG content in 8–16 cups of green tea daily may mitigate the poor bioavailability of EGCG [97]. The systemic bioavailability of EGCG increased at higher doses, possibly due to the saturable pre-systemic elimination of oral green tea polyphenols (GTPs) [98]. However, it is not recommended to use excessive doses of catechins to improve effectiveness. The schematic diagram of metabolism of green tea catechins in EGCG is given in Figure 10 [99,100,101].

Therefore, the bioavailability of catechins can be improved by nanostructure-based drug delivery systems, molecular modification, and co-administration with some other bioactive ingredients. Encapsulation of tea catechins on protein-based, carbohydrate-based, and lipid-based nanoparticles improved stability, sustainable release, and cell membrane permeation of catechins, resulting in increased bioavailability. Molecular modification such as synthesizing peracetylated EGCG (AcEGCG) protects the hydroxyl groups on EGCG from oxidative degradation until it is deacetylated into its parent EGCG by the esterases in cells [102], which decreases the biotransformation and efflux of EGCG. Co-administrating or formulating catechins with appropriate other drugs or bioactives will produce synergism effects through interaction of catechins with the selected drugs, resulting in improvement of absorption and inhibition of the efflux transporter [103,104,105]. Strategies such as nanostructured delivery systems, peracetylation, and synergistic formulations with other bioactives are being explored to overcome these hurdles. These approaches hold promise for aligning in vitro potential with in vivo effectiveness.

4.8. Glycyrrhizin

Glycyrrhizin (GL, Figure 11), a glucuronide consisting of two molecules of glucuronic acid and one molecule of glycyrrhetinic acid (GA), is one of the main components extracted from Glycyrrhiza glabra L. which is the most commonly used herb in traditional Chinese medicine [106]. Glycyrrhizin has been identified as a compound that binds to HMGB1, potentially preventing its interaction with the RAGE receptor and thereby disrupting the associated inflammatory signaling pathway. To determine whether glycyrrhizin’s neuroprotective effects are mediated specifically through the HMGB1/RAGE axis, studies were conducted using RAGE^−/− knockout mice. Mice treated with 4 mg/kg of glycyrrhizin did not produce any inhibitory effects, suggesting that glycyrrhizin specifically affects the HMGB1/RAGE pathway to improve the neuro-inflammatory condition [107]. Glycyrrhizin mitigates traumatic brain injury (TBI) by directly binding to HMGB1, thereby preventing its interaction with the RAGE receptor. This inhibition blocks HMGB1-mediated inflammatory signaling without directly binding to RAGE. Glycyrrhizin suppresses HMGB1 translocation from the nucleus to the cytoplasm, reducing its extracellular release [108]. Consequently, GL treatment decreases blood–brain barrier permeability, inflammatory cytokine expression (TNF-α, IL-1β, IL-6), and improves motor and cognitive function post TBI. Notably, these protective effects are absent in RAGE-deficient mice, confirming that Glycyrrhizin’s efficacy depends on disrupting HMGB1/RAGE signaling [108]. Besides its potential for treating diseases, its poor absorption has seriously limited its clinical efficacy. Previous findings suggested that its low bioavailability after administration via the oral route is mainly attributed to the impermeability of GL across the gastrointestinal mucosa and/or hydrolysis to its metabolite GA by gastric fluid and β-glucuronidases in the gastrointestinal flora [109,110]. Considering such arising absorption problems, the development of a drug delivery system with a high solvent power and/or an ability to penetrate is a challenging research area in the pharmaceutical technology of GL. Many useful methods have been applied in the absorption of GL so far, such as incorporating it into the phospholipid complex via enteric-coated capsules and tablets. However, most of them have been based on making GL into a salt and few have produced a marked effect via the oral route. Therefore, in order to further improve the bioavailability of GL, other sites with a potential high permeability for GL need to be utilized and/or the membrane permeability needs to be increased by the use of an absorption enhancer [111]. Previous studies have shown that sodium deoxycholate has stronger compatibilization compared with other bile salts [112]. Therefore, sodium deoxycholate/phospholipid-mixed nanomicelles (SDC-PL-MMs) might be the ideal drug carrier for GL for oral administration. Inspired by these findings, Jin et al. prepared a formulation of GL as sodium deoxycholate/phospholipid-mixed nanomicelles (SDC/PL-MMs) to improve its oral bioavailability [112]. Pharmacokinetics- and pharmacodynamic-based studies on carbon tetrachloride (CCl4)-induced acute liver injury were performed to verify the theoretical hypothesis. They claimed that nanomicelles with a mean particle size of 82.99 nm and a zeta potential of −32.23 mV were obtained. In contrast to the control group, for pharmacokinetics, GL-SDC/PL-MMs show a significant superiority in AUC0–t, Cmax. In the pharmacodynamic studies, compared with the bifendate control group, GL-SDC/PL-MMs showed an equivalent effect in reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and improving the pathological changes in liver tissue. These results revealed that SDC/PL-MMs could enhance GL absorption in the gastrointestinal tract and the pharmacodynamic effect [112].

Recently, Li et al. [113] used Probiotic Lactobacillus rhamnosus R0011 supplementation to enhance the bioavailability of GL in rats, especially under a liver fibrosis state. They claimed that L. rhamnosus R0011 intervention directly improved the microbial glucuronidase activity. On the other hand, L. rhamnosus R0011 also helps to restore the gut microbial composition and repair intestinal homeostasis, thus promoting the bioabsorption of GL [113]. Overall, data associated with glycyrrhizin suggests novel formulations like sodium deoxycholate/phospholipid nanomicelles and probiotic interventions have shown success in terms of enhancing its absorption and systemic availability. These studies underscore the importance of innovative drug delivery and microbiome-targeted strategies for realizing glycyrrhizin’s full therapeutic potential in neuroinflammatory conditions.

Table 2. Overall summary of drugs targeting HMGB1/RAGE axis and bioavailability enhancement methods.

Drugs	Mechanism of Action	Bioavailability Issues	Enhancement Strategies
Crocin	Suppresses HMGB1/RAGE axis in neuroinflammation	Low bioavailability due to poor absorption and stability	Oral crocin yields higher serum crocetin levels than crocetin itself
Berberine	Inhibits inflammatory cytokines via HMGB1/RAGE	Very low (<1%) due to poor solubility, metabolism, P-gp efflux	Nanoparticles, self-emulsions, salts, nanocrystals; e.g., 343–404% improved formulations
Curcumin	Neuroprotection via HMGB1/RAGE; anti-inflammatory agent	Poor solubility, instability, rapid metabolism	Mono-carbonyl analogs, liposomes, micelles, PLGA nanoparticles, PEG, surfactants
Pentoxifylline	Anti-epileptic via HMGB1/RAGE axis	Low (20–30%) due to poor dissolution rate	Microsponges, solid lipid nanoparticles (SLNs)
Doxorubicin + FPS-ZM1	Combination inhibits HMGB1–RAGE in leukemia	Doxorubicin: <5% oral bioavailability; FPS-ZM1: unstable in water	Nanoparticles, liposomes (4–6.8× improvement); use of solvents for FPS-ZM1
Dexmedetomidine	Blocks HMGB1 translocation in acute lung injury	Limited data on oral bioavailability	Nasal spray; no significant oral formulation reported
EGCG	Decreases extracellular HMGB1 and RAGE expression	Low solubility, stability, and permeability; in vivo < in vitro	Nanoparticles, peracetylated EGCG, co-administration with bioactives
Glycyrrhizin	Inhibits HMGB1–RAGE interaction	Poor oral absorption due to hydrolysis and GI impermeability	Sodium deoxycholate/phospholipid nanomicelles (SDC-PL-MMs), probiotics (L. rhamnosus)

presents an overall summary of the various drugs that target the HMGB1/RAGE signaling axis described above, along with the methods employed to enhance their bioavailability.

5. Conclusions

The low bioavailability of synthetic as well as natural drug-like molecules is an important factor leading to the observed inconsistency between their in vitro and in vivo results. Stability, absorption rate, and efflux influence bioavailability. Extreme pH conditions in the stomach and intestinal tract as well as related digestive enzymes are confirmed to be factors inducing instability, including the degradation and conjugation of these molecules. Enhancement of the bioavailability and solubility of drugs is a significant challenge in the area of pharmaceutical formulations. This compilation describes various pharmaceutical techniques which have been utilized to improve the bioavailability of synthetic or natural molecules particularly used in various inflammatory conditions acting specifically through the HMGB1/RAGE pathway. This literature review comes to the conclusion that various molecules behave differently in various carrier systems inside in vivo models. Oral absorption is highly dependent on liposomal properties, and optimum formulations are effective for low-permeability drugs. It was also found that chemical or structural modification still needs a pharmaceutical carrier system to enhance the physiology of the compound to better deliver it inside the animal or human body. This article would be helpful for the research community to choose better carrier systems for their medicinal compounds, particularly in the field of inflammatory diseases.