Enhancing Oral Bioavailability of Poorly Water-Soluble Natural Products via Lipid–Drug Conjugates

Abstract

Background: The therapeutic potential of many natural products, including curcumin (CUR), betulinic acid (BA), and oleanolic acid (OA), is limited by poor oral exposure caused by low aqueous solubility, metabolic instability, and/or first-pass metabolism. Lipid–drug conjugate (LDC) strategies that mimic endogenous dietary lipid processing may provide a useful approach for improving oral absorption and lymphatic transport.

Methods: A 1,3-diolein-based lipidic promoiety (IN-4) was synthesized and conjugated to curcumin, betulinic acid, and oleanolic acid to generate three representative LDCs: CUR-PRO, BA-PRO, and OA-PRO. Their oral pharmacokinetic behavior was evaluated in rats. For CUR-PRO, matched-vehicle comparisons across three oral vehicles were performed, together with mesenteric lymph duct cannulation and in vitro stability/conversion studies in simulated gastrointestinal media, rat liver microsomes, and rat plasma.

Results: All three prodrugs were successfully synthesized and showed improved systemic exposure to the corresponding parent-drug-related analytes under the tested conditions. For CUR-PRO, dose-normalized AUC_0-last of released curcumin was markedly higher than direct curcumin administration across all three vehicles (increases of 15.0-, 70.9-, and 54.3-fold), and intact CUR-PRO was also detected in plasma. Mesenteric lymph sampling showed that CUR-PRO dosing, but not free-curcumin dosing, generated detectable curcumin-related signals under the present analytical conditions. In vitro, no free curcumin was detected during CUR-PRO incubation in enzyme-free simulated gastrointestinal media; CUR-PRO underwent rapid depletion in pancreatic-lipase-supplemented medium, showed greater microsomal stability than curcumin, and displayed plasma conversion that was markedly accelerated by exogenous LPL. BA-PRO and OA-PRO also increased systemic exposure of their released parent drugs, with 16.0- and 38.4-fold dose-normalized AUC_0-last increases, respectively.

Conclusions: These findings provide proof-of-concept evidence that 1,3-diolein-based lipidation can improve the oral exposure of selected poorly water-soluble natural products. The lymphatic transport data provide qualitative evidence supporting lymphatic access of CUR-PRO, although the quantitative contribution of this pathway to the overall exposure increase remains to be established.

Introduction

Natural products remain an important source of therapeutic leads and drug candidates because of their structural diversity and broad pharmacological activities [1,2,3,4,5]. However, the clinical translation of many natural products is frequently limited by poor oral bioavailability, which is often associated with low aqueous solubility, limited intestinal absorption, chemical or metabolic instability, and extensive first-pass metabolism [6,7,8]. Curcumin, betulinic acid, and oleanolic acid are representative examples of bioactive natural products with promising pharmacological activities but suboptimal oral pharmacokinetic profiles [9,10,11]. Strategies capable of improving their systemic exposure are therefore important for advancing their translational potential.

A variety of formulation and drug-delivery approaches have been investigated to overcome these limitations, including nanocarriers, lipid-based systems, surfactant-based solubilization, solid dispersions, particle engineering, salt or polymorph screening, and chemical modification [12,13,14,15,16]. Among these approaches, intestinal lymphatic drug delivery has attracted increasing interest because it can promote the absorption of highly lipophilic compounds through lipid-processing pathways and may reduce the impact of hepatic first-pass metabolism [17,18].

The intestinal lymphatic pathway is closely linked to dietary lipid digestion, absorption, and chylomicron assembly. Following intestinal uptake, long-chain lipids are re-esterified within enterocytes and incorporated into chylomicrons, which are subsequently transported through the mesenteric lymph before entering the systemic circulation [19,20,21,22,23,24]. Drug-delivery systems that interact with this endogenous pathway may therefore enhance oral exposure and alter tissue distribution profiles for appropriately designed lipophilic molecules.

Lipid-based formulations can facilitate lymphatic uptake by increasing solubilization in the intestinal lumen and promoting association with lipid digestion products. In parallel, covalent lipidation has emerged as a prodrug-based strategy in which drug molecules are chemically conjugated to fatty acid, glyceride, or other lipid motifs [25,26,27]. Compared with simple physical solubilization, lipid–drug conjugates can be designed to mimic endogenous lipid substrates and thereby engage specific enzymatic and trafficking processes involved in intestinal lipid absorption.

Triglyceride- and glyceride-mimetic prodrugs are particularly attractive because they can be processed in a manner analogous to dietary triglycerides. Previous studies have shown that appropriately designed glyceride-mimetic prodrugs can promote intestinal lymphatic transport, reduce direct portal exposure, and increase systemic oral exposure of selected model drugs. For example, triglyceride-based prodrugs of buprenorphine incorporating self-immolative linkers achieved up to 45% lymphatic delivery after intestinal lipolysis, compared with less than 0.1% for free buprenorphine, and resulted in a 22-fold increase in oral bioavailability [28]. Similarly, 1,3-diacylglycerol-based testosterone prodrugs containing self-immolative spacers redirected drug transport from the portal vein to the intestinal lymphatic pathway, leading to up to a 90-fold increase in oral plasma exposure relative to a conventional prodrug [29]. These findings provide a mechanistic basis for applying triglyceride-mimetic lipidation to poorly absorbed natural products.

The clinical translation of lymph-directed prodrug strategies has also shown encouraging progress. GlyphAllo™ (SPT-300, formerly LYT-300), an oral allopregnanolone prodrug developed using lymphatic-targeted prodrug technology, has demonstrated oral bioavailability, tolerability, and GABA_A receptor target engagement in early clinical studies and has advanced into a Phase 2b clinical trial for major depressive disorder with or without anxious distress [30,31]. More recently, preclinical and first-in-human data for GlyphAllo were reported, further supporting the clinical feasibility of triglyceride-mimetic prodrug design for achieving therapeutically relevant systemic exposure after oral dosing [32]. These advances provide a translational framework for the development of lymph-directed lipidized prodrugs and support further exploration of this strategy for improving the oral delivery of poorly absorbed natural products.

Despite these advances, the applicability of triglyceride-mimetic prodrug strategies to selected poorly water-soluble natural products remains insufficiently explored. In particular, it remains unclear whether representative natural products such as curcumin, betulinic acid, and oleanolic acid can be rationally converted into triglyceride-mimetic lipid–drug conjugate (LDC) prodrugs and subsequently generate measurable systemic exposure to their released parent drugs in vivo. This gap is especially relevant because natural products differ substantially in functional groups, lipophilicity, and metabolic liabilities, which may influence prodrug conversion, intestinal lipid processing, lymphatic transport, and systemic parent-drug release.

In this study, we developed a 1,3-diolein-based LDC strategy to improve the oral exposure of selected poorly water-soluble natural products. A common lipidic promoiety (IN-4) was synthesized and conjugated to curcumin, betulinic acid, and oleanolic acid to generate three representative prodrugs: CUR-PRO, BA-PRO, and OA-PRO. Figure 1 summarizes the study in a four-step experimental and conceptual sequence: (1) lipid conjugation of the selected natural products to the common 1,3-diolein-based promoiety; (2) proposed pancreatic lipase-mediated intestinal processing, followed by enterocyte uptake, reassembly into triglyceride-like species, and chylomicron assembly; (3) potential chylomicron-associated access to mesenteric lymph, evaluated by lymph collection in rats; and (4) increased systemic exposure to the released parent drugs after oral administration. This scheme is presented as a working hypothesis and does not imply that lymphatic transport is the sole determinant of the observed pharmacokinetic changes. The prodrugs were evaluated using exploratory rat pharmacokinetic studies, with detailed matched-vehicle analysis for CUR-PRO. Mesenteric lymph duct cannulation and complementary in vitro stability/conversion studies were further conducted to examine lymphatic access and enzymatic processing of CUR-PRO. Together, these studies provide an early-stage, multi-faceted proof-of-concept assessment of triglyceride-mimetic lipidation for selected poorly water-soluble natural products, while the relative contributions of dissolution or dispersion, permeability, metabolism, disposition, prodrug conversion, and lymphatic transport remain to be resolved. The principal novelty of this work is the integrated evaluation of a common 1,3-diolein-based promoiety across three selected natural products spanning two structural classes, combining cross-compound exposure assessment with matched-vehicle pharmacokinetics, direct mesenteric lymph sampling, and complementary enzymatic studies.

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Materials

Curcumin (purity ≥ 98%), betulinic acid (purity ≥ 98%), and oleanolic acid (purity ≥ 98%) were purchased from Macklin (Shanghai, China). Ethyl 4-oxopentanoate (IN-4a), tert-butyl (dimethoxyphosphoryl)acetate, and 1,3-dioleoylglycerol (1,3-diolein, IN-2b) were obtained from Chinasun Specialty Products Co., Ltd. (Changshu, China) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), 4-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIPEA), 1-chloroethyl chloroformate (CEOC), and pyridine were obtained from Shanghai Bepharm Science & Technology Co., Ltd. (Shanghai, China) Palladium on carbon (Pd/C, 10 wt%) and tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] were purchased from J&K Chemical Ltd. (Shanghai, China).

Sodium hydride (NaH, 60% dispersion in mineral oil), potassium carbonate (K₂CO₃), cesium carbonate (Cs₂CO₃), sodium hydroxide (NaOH), hydrochloric acid (HCl), trifluoroacetic acid (TFA), 1,3-dimethylbarbituric acid, and tetrabutylammonium iodide (TBAI), anhydrous sodium sulfate (Na₂SO₄), anhydrous tetrahydrofuran (THF), anhydrous dichloromethane (DCM), N,N-dimethylformamide (DMF), toluene, methanol, ethyl acetate, and petroleum ether (boiling range, 60–90 °C) were obtained from Chinasun Specialty Products Co., Ltd. (Changshu, China) Allyl bromide was purchased from Aladdin (Shanghai, China).

Polyethylene glycol 400 (PEG 400), diethylene glycol monoethyl ether (Transcutol^® HP), rice bran oil, Lipoid^® E 80, Tween 80, olive oil, oleic acid, ethanol, sodium carboxymethyl cellulose (CMC-Na), and propylene glycol were provided by Gattefossé (Lyon, France). Solutol^® HS 15 was purchased from Merck (Darmstadt, Germany), and sodium chloride for the preparation of 0.9% saline was obtained from Shaanxi Shengao Animal Pharmaceutical Co., Ltd. (Xi’an, Shaanxi, China) Polyethylene cannulation tubing (PE-50) was purchased from Instech Laboratories (Plymouth Meeting, PA, USA), and 5% glucose injection was obtained from Sichuan Kelun Pharmaceutical Co., Ltd. (Chengdu, China).

EDTA-K2 anticoagulant blood collection tubes were obtained from Nantong HaiRui Experimental Equipment Co., Ltd. (Nantong, China) Lipoprotein lipase (LPL) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and porcine pancreatic lipase was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) Fasted-state simulated intestinal fluid (FaSSIF), fed-state simulated intestinal fluid (FeSSIF), and fasted-state simulated gastric fluid (FaSSGF) powders were obtained from Biorelevant Limited (London, UK). Rat liver microsomes were purchased from IPHASE Bioscience Co., Ltd. (Kunshan, China) Sodium hydroxide, hydrochloric acid, sodium chloride, acetic acid, and sodium dihydrogen phosphate were purchased from Chinasun Specialty Products Co., Ltd. (Changshu, China) Tris-maleate and verapamil were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China), and calcium chloride monohydrate was obtained from Anhui Zesheng Science & Technology Co., Ltd. (Anqing, China) NADPH was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

For HPLC analysis, HPLC-grade acetonitrile and methanol were obtained from TEDIA (Fairfield, OH, USA). For LC–MS/MS bioanalysis, acetonitrile (LC–MS grade, TEDIA (Fairfield, OH, USA)), formic acid (LC–MS grade, Adamas-beta (Shanghai, China)), ammonium acetate (LC–MS grade, MREDA (Beijing, China)), and LC–MS-grade water were used. Deuterated solvents for NMR spectroscopy (CDCl3 and DMSO-d6) were obtained from TCI (Tokyo, Japan).

Unless otherwise stated, all reagents and solvents were of analytical or synthetic grade and were used as received without further purification.

Zou, X.; Zhang, B.; Mei, L.; Han, S.; Chen, K. Enhancing Oral Bioavailability of Poorly Water-Soluble Natural Products via Lipid–Drug Conjugates. Pharmaceutics 2026, 18, 899, https://doi.org/10.3390/pharmaceutics18070899

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