In Vitro Predictive Model for Intestinal Lymphatic Uptake: Exploration of Additional Enhancers and Inhibitors

Introduction

Intestinal lymphatic drug transport has recently garnered attention owing to the many potential benefits it presents for drug delivery [1,2]. Following absorption, some drugs pass across the intestinal enterocytes, and during this transit, these drugs associate with the excretory enterocyte lipoproteins chylomicrons [3]. This process underscores the potential and significance of exploiting intestinal lymphatic transport for drug delivery purposes.

Chylomicrons are spherical particles that are composed mainly of triglycerides (85–90%) in addition to phospholipids (7–9%), cholesterol and cholesteryl esters (3–5 and 1–3%, respectively), and apolipoproteins (1–2%) [4]. They principally play a role in absorbing and facilitating the systemic distribution of dietary fats and lipophilic vitamins [5]. Following digestion, when dietary triglycerides transform into free fatty acids and monoglycerides, a subsequent process of re-esterification occurs inside enterocytes. During this phase, the resulting triglycerides are encapsulated within chylomicrons, which serve as transportation carriers in the bloodstream through the lymphatic network [2,6].
In the context of pharmaceutical applications, specifically lymphatic-targeting—or lymphotropic—drugs, these enterocyte-formed chylomicrons offer a unique avenue. By ‘hitchhiking’ on these carriers, candidate molecules gain entry into circulation.

Using chylomicrons as an approach holds the promise of evading the initial hepatic metabolism, commonly known as the first-pass effect, thereby elevating their bioavailability [5,7,8]. Alternatively, these drugs could accumulate within the lymphatic system, reaching increased concentrations at lymph node target sites. This concentration enhancement may translate into a more potent therapeutic impact with reduced off-target toxicity. This aspect is particularly important for compounds with immunomodulatory or anticancer properties, where maximizing their effect within the lymphatic system proves crucial [5,7].

In a previous study, we presented an in vitro model crafted to predict, inhibit, and enhance lymphatic uptake. Its foundation lies in the interaction of drugs with chylomicrons, a process documented for its ability to predict intestinal lymphatic uptake [9,10]. This model consists of two compartments: a donor compartment containing the drug solution under investigation and a receiver compartment filled with an artificial chylomicron medium (Intralipid®) [9]. These artificial chylomicrons serve as carriers for the drug molecules and mimic the behavior of naturally occurring chylomicrons in the body. To simulate the in vivo chylomicron-blocking effect and suppress drug release in an in vitro setting, pluronic L-81 (PL-81) was utilized. This chylomicron-blocking agent, which has been proven effective in both in vivo and Caco-2 cell culture models, demonstrated an inhibitory effect within the in vitro model [11,12,13]. Moreover, to enhance drug release into the receiver compartment and mimic lymphatic enhancement, peanut oil was used. This choice stemmed from peanut oil being contributes to the formation of chylomicrons and was guided by its potential to function as a carrier, facilitating increased drug entry into the receiver compartment [9,14].

In this study, the aim was to investigate other agents that would enhance or inhibit intestinal lymphatic uptake through the chylomicron pathway. Rifampicin served as the model drug in this study, consistent with the earlier investigation. Additionally, quercetin was used as a second xenobiotic to provide further confirmation in some experiments. Additional oils were explored to investigate their impact on enhancing intestinal lymphatic uptake. Olive, sesame, and coconut oils were chosen due to their varying percentages and chain lengths of different fatty acids, which are recognized for their impact on in vivo lymphatic uptake [4,15].

In order to deliver drugs through intestinal lymphatics, various formulation excipients and drug delivery systems have been and are being developed [1,7]. One example of the excipients used is Labrafil®. It consists of mono-, di-, and triglycerides and PEG6 (MW 300) mono- and diesters of linoleic (C18:2) acid. It is a non-ionic water-dispersible surfactant for lipid-based formulations to solubilize and increase the oral bioavailability of poorly water-soluble APIs [16]. A novel formulation of cannflavins was examined in this model system, with Labrafil® M 2125 CS acting as the enhancer. Moreover, this study delved into the impact of the zeta potential on either enhancing or inhibiting intestinal lymphatic uptake. For this purpose, racemic chloroquine (C18H26ClN3) and sodium lauryl sulfate (C12H25NaO4S) were utilized. Chloroquine is an antimalarial drug that has been shown to reduce plasma levels of triglycerides and cholesterol [17]. At a physiological pH, chloroquine carries a positive charge. The purpose was to investigate whether this positive charge could influence its interaction with chylomicrons, consequently reducing triglyceride levels and potentially drug transportation through chylomicrons. To further confirm the impact of this charge interaction, sodium lauryl sulfate, an anionic surfactant widely used in pharmaceutical formulations [18], was employed due to its negative charge, which contrasts with that of chloroquine. Both of these substances showed their capability to influence the zeta potential of artificial chylomicron particles in preliminary experiments. Using the previously developed in vitro model, this study investigated how the addition of these agents to the artificial chylomicron compartment could affect the uptake of model drugs into this compartment.

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Materials

Rifampicin (≤100%, CAS: 557303) was procured from EMD Millipore Corp, Burlington, MA, USA, while quercetin (≥95%, CAS: 117-39-5), 1-octanol (99%, CAS: 111-87-5), and chloroquine as diphosphate salt (98.5–101.0%, CAS:5 0-63-5) were sourced from Sigma–Aldrich Co. (Saint Louis, MO, USA). Intralipid^® (20%) was obtained from Fresenius Kabi (Toronto, ON, Canada). Peanut, olive and sesame oil products were acquired from a local Edmonton grocery, while coconut oil (CAS: 8001-31-8) was obtained from Medisca (Saint-Laurent, QC, Canada), and sodium lauryl sulphate (≤100%, CAS: 151-21-3) was obtained from Caledon Laboratories (Toronto, ON, Canada). Labrafil^® M 2125 CS was obtained from Gattefossé (Toronto, ON, Canada) while cannflavin (≥98%, CAS: 76735-57-4) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Additionally, synthetic hydrophobic polyvinylidene fluoride (PVDF) membranes were acquired from Millipore affiliated with Merck KGaA, Darmstadt, Germany. For HPLC analysis, methanol (99.9%, CAS: 67-56-1) and acetic acid (≥99.7%, CAS: 64-19-7) of HPLC grade were obtained from Fisher Scientific (Ottawa, ON, Canada); all other reagents were of analytical grade.

Yousef, M.; O’Croinin, C.; Le, T.S.; Park, C.; Zuo, J.; Bou Chacra, N.; Davies, N.M.; Löbenberg, R. In Vitro Predictive Model for Intestinal Lymphatic Uptake: Exploration of Additional Enhancers and Inhibitors. Pharmaceutics 2024, 16, 768. https://doi.org/10.3390/pharmaceutics16060768