Intestinal Lymphatic Biology, Drug Delivery, and Therapeutics: Current Status and Future Directions.

Historically, the intestinal lymphatics were considered passive conduits for fluids, immune cells, dietary lipids, lipid soluble vitamins, and lipophilic drugs. Studies of intestinal lymphatic drug delivery in the late 20th century focused primarily on the drugs' physicochemical properties, especially high lipophilicity, that resulted in intestinal lymphatic transport. More recent discoveries have changed our traditional view by demonstrating that the lymphatics are active, plastic, and tissue-specific players in a range of biological and pathological processes, including within the intestine. These findings have, in turn, inspired exploration of lymph-specific therapies for a range of diseases, as well as the development of more sophisticated strategies to actively deliver drugs or vaccines to the intestinal lymph, including a range of nanotechnologies, lipid prodrugs, and lipid-conjugated materials that "hitchhike" onto lymphatic transport pathways. With the increasing development of novel therapeutics such as biologics, there has been interest in whether these therapeutics are absorbed and transported through intestinal lymph after oral administration. Here we review the current state of understanding of the anatomy and physiology of the gastrointestinal lymphatic system in health and disease, with a focus on aspects relevant to drug delivery. We summarize the current state-of-the-art approaches to deliver drugs and quantify their uptake into the intestinal lymphatic system. Finally, and excitingly, we discuss recent examples of significant pharmacokinetic and therapeutic benefits achieved via intestinal lymphatic drug delivery. We also propose approaches to advance the development and clinical application of intestinal lymphatic delivery strategies in the future. SIGNIFICANCE STATEMENT: This comprehensive review details the understanding of the anatomy and physiology of the intestinal lymphatic system in health and disease, with a focus on aspects relevant to drug delivery. It highlights current state-of-the-art approaches to deliver drugs to the intestinal lymphatics and the shift toward the use of these strategies to achieve pharmacokinetic and therapeutic benefits for patients.

Sex-specific adipose tissue imprinting of regulatory T cells.

Adipose tissue is an energy store and a dynamic endocrine organ1,2. In particular, visceral adipose tissue (VAT) is critical for the regulation of systemic metabolism3,4. Impaired VAT function-for example, in obesity-is associated with insulin resistance and type 2 diabetes5,6. Regulatory T (Treg) cells that express the transcription factor FOXP3 are critical for limiting immune responses and suppressing tissue inflammation, including in the VAT7-9. Here we uncover pronounced sexual dimorphism in Treg cells in the VAT. Male VAT was enriched for Treg cells compared with female VAT, and Treg cells from male VAT were markedly different from their female counterparts in phenotype, transcriptional landscape and chromatin accessibility. Heightened inflammation in the male VAT facilitated the recruitment of Treg cells via the CCL2-CCR2 axis. Androgen regulated the differentiation of a unique IL-33-producing stromal cell population specific to the male VAT, which paralleled the local expansion of Treg cells. Sex hormones also regulated VAT inflammation, which shaped the transcriptional landscape of VAT-resident Treg cells in a BLIMP1 transcription factor-dependent manner. Overall, we find that sex-specific differences in Treg cells from VAT are determined by the tissue niche in a sex-hormone-dependent manner to limit adipose tissue inflammation.

Acute Neuroinflammation Alters the Transport of a Model Therapeutic Protein from the Brain into Lymph and Blood.

The drainage of fluid and solutes along lymphatic pathways from the brain has been found to be impaired in mouse models of multiple sclerosis, Alzheimer's disease, and Parkinson's disease where neuroinflammation is present. We recently demonstrated that 3H-albumin, a model therapeutic protein (∼65 kDa), undergoes preferential lymphatic transport from the brain using a cervical lymph cannulation model in healthy rats. We thus hypothesized that neuroinflammation would impede the lymphatic transport of 3H-albumin from the brain. Our aim was to quantify the impact of acute neuroinflammation on drainage of the model therapeutic protein (3H-albumin) from the rat brain into blood and deep cervical lymph. To establish the required neuroinflammation model, male Sprague-Dawley rats were administered an intraperitoneal (IP) dose of 0.5-2 mg/kg lipopolysaccharide (LPS, Escherichia coli) or a saline control. After 12 or 24 h, brain samples were collected and analyzed for concentrations of interferon gamma (IFN-γ) using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The impact of neuroinflammation on the drainage of 3H-albumin from the brain was determined via IP administration of 2 mg/kg LPS or saline followed by cannulation of the carotid artery for blood collection 24 h later with/without cannulation or ligation at the efferent deep cervical lymph trunk. Rats were then administered 3H-albumin via direct injection into the brain striatum or via intravenous (IV) injection (lymph-intact group only). Blood ± lymph samples were collected for up to 8 h following dosing. At the end of the study, brain and lymph node samples were harvested for biodistribution analysis, with samples analyzed for radioactivity levels via scintillation counting. Brain concentrations of the pro-inflammatory cytokine IFN-γ were only significantly elevated 24 h after IP administration of 2 mg/kg LPS compared to saline control. Therefore, this induction regimen was utilized for subsequent studies. The plasma concentrations of 3H-albumin over time were elevated in LPS-induced rats compared to saline-injected rats in the lymph-intact and lymph-ligated groups but not in the lymph-cannulated group. In the deep cervical lymph-cannulated animals, the lymph transport of 3H-albumin was not increased and appeared to be slower in the LPS-administered rats. Acute LPS-induced neuroinflammation therefore led to an enhanced overall transport of 3H-albumin from the brain into the systemic circulation. This appeared to be primarily due to increased transport of 3H-albumin from the brain directly into the blood circulation as 3H-albumin transport from the brain via the lymphatics was not increased in the LPS-induced neuroinflammation model. Such changes in the clearance of therapeutic proteins from the brain in the setting of neuroinflammation may impact the therapeutic efficacy and safety.

Quantifying the Lymphatic Transport of Model Therapeutics from the Brain in Rats.

In recent years, the drainage of fluids, immune cells, antigens, fluorescent tracers, and other solutes from the brain has been demonstrated to occur along lymphatic outflow pathways to the deep cervical lymph nodes in the neck. To the best of our knowledge, no studies have evaluated the lymphatic transport of therapeutics from the brain. The objective of this study was to determine the lymphatic transport of model therapeutics of different molecular weights and lipophilicity from the brain using cervical lymph cannulation and ligation models in rats. To do this, anesthetized Sprague-Dawley rats were cannulated at the carotid artery and cannulated, ligated, or left intact at the cervical lymph duct. Rats were administered 14C-ibuprofen (206.29 g/mol, logP 3.84), 3H-halofantrine HCl (536.89 g/mol, logP 8.06), or 3H-albumin (∼65,000 g/mol) via direct injection into the brain striatum at a rate of 0.5 µL/min over 16 min. Plasma or cervical lymph samples were collected for up to 6-8 h following dosing, and brain and lymph nodes were collected at 6 or 8 h. Samples were subsequently analyzed for radioactivity levels via scintillation counting. For 14C-ibuprofen, plasma concentrations over time (plasma AUC0-6h) were >2 fold higher in lymph-ligated rats than in lymph-intact rats, suggesting that ibuprofen is cleared from the brain primarily via nonlymphatic routes (e.g., across the blood-brain barrier) but that this clearance is influenced by changes in lymphatic flow. For 3H-halofantrine, >73% of the dose was retained at the brain dosing site in lymph-intact and lymph-ligated groups, and plasma AUC0-8h values were low in both groups (<0.3% dose.h/mL), consistent with the high retention in the brain. It was therefore not possible to determine whether halofantrine undergoes lymphatic transport from the brain within the duration of the study. For 3H-albumin, plasma AUC0-8h values were not significantly different between lymph-intact, lymph-ligated, and lymph-cannulated rats. However, >4% of the dose was recovered in cervical lymph over 8 h. Lymph/plasma concentration ratios of 3H-albumin were also very high (up to 53:1). Together, these results indicate that 3H-albumin is transported from the brain not only via lymphatic routes but also via the blood. Similar to other tissues, the lymphatics may thus play a significant role in the transport of macromolecules, including therapeutic proteins, from the brain but are unlikely to be a major transport pathway from the brain for small molecule drugs that are not lipophilic. Our rat cervical lymph cannulation model can be used to quantify the lymphatic drainage of different molecules and factors from the brain.

Increasing Linker Chain Length and Intestinal Stability Enhances Lymphatic Transport and Lymph Node Exposure of Triglyceride Mimetic Prodrugs of a Model Immunomodulator Mycophenolic Acid.

Targeted delivery of immunomodulators to the lymphatic system has the potential to enhance therapeutic efficacy by increasing colocalization of drugs with immune targets such as lymphocytes. A triglyceride (TG)-mimetic prodrug strategy has been recently shown to enhance the lymphatic delivery of a model immunomodulator, mycophenolic acid (MPA), via incorporation into the intestinal TG deacylation-reacylation and lymph lipoprotein transport pathways. In the current study, a series of structurally related TG prodrugs of MPA were examined to optimize structure-lymphatic transport relationships for lymph-directing lipid-mimetic prodrugs. MPA was conjugated to the sn-2 position of the glyceride backbone of the prodrugs using linkers of different chain length (5-21 carbons) and the effect of methyl substitutions at the alpha and/or beta carbons to the glyceride end of the linker was examined. Lymphatic transport was assessed in mesenteric lymph duct cannulated rats, and drug exposure in lymph nodes was examined following oral administration to mice. Prodrug stability in simulated intestinal digestive fluid was also evaluated. Prodrugs with straight chain linkers were relatively unstable in simulated intestinal fluid; however, co-administration of lipase inhibitors (JZL184 and orlistat) was able to reduce instability and increase lymphatic transport (2-fold for a prodrug with a 6-carbon spacer, i.e., MPA-C6-TG). Methyl substitutions to the chain resulted in similar trends in improving intestinal stability and lymphatic transport. Medium- to long-chain spacers (C12, C15) between MPA and the glyceride backbone were most effective in promoting lymphatic transport, consistent with increases in lipophilicity. In contrast, short-chain (C6-C10) linkers appeared to be too unstable in the intestine and insufficiently lipophilic to associate with lymph lipid transport pathways, while very long-chain (C18, C21) linkers were also not preferred, likely as a result of increases in molecular weight reducing solubility or permeability. In addition to more effectively promoting drug transport into mesenteric lymph, TG-mimetic prodrugs based on a C12 linker resulted in marked increases (>40 fold) in the exposure of MPA in the mesenteric lymph nodes in mice when compared to administration of MPA alone, suggesting that optimizing prodrug design has the potential to provide benefit in targeting and modulating immune cells.

Intra-articular Injection of a B Cell Depletion Antibody Enhances Local Exposure to the Joint-Draining Lymph Node in Mice with Collagen-Induced Arthritis.

Changes to the number, type, and function of immune cells within the joint-draining lymphatics is a major contributor to the progression of inflammatory arthritis. In particular, there is a significant expansion in pathogenic B cells in the joint-draining lymph node (jdLN). These B cells appear to clog the lymphatic sinuses in the lymph node, inhibit lymph flow, and therefore, reduce the clearance of inflammatory fluid and cells from the joint. Taken together, there is potential to treat inflammatory arthritis more effectively, as well as reduce off-target side effects, with localized delivery of B-cell depleting therapies to the jdLNs. We recently reported that joint-draining lymphatic exposure of biologic disease-modifying anti-rheumatic drugs (DMARDs), including the B cell depletion antibody rituximab, is increased in healthy rats following intra-articular (IA) compared to subcutaneous (SC) or intravenous (IV) administration. This suggests that IA administration of B cell depleting antibodies may increase delivery to target cells in the jdLN and increase the effectiveness of B cell depletion compared to standard SC or IV administration. However, whether enhanced local delivery of DMARDs to the jdLN is also achieved after IA injection in the setting of inflammatory arthritis, where there is inflammation in the joint and jdLN B cell expansion is unknown. We, therefore, assessed the lymph node distribution, absorption and plasma pharmacokinetics, and B cell depletion at different sites after IA, SC, or IV administration of a fluorescently labeled mouse anti-CD20 B cell depleting antibody (Cy5-αCD20) in healthy mice compared to mice with collagen-induced arthritis (CIA). The absorption and plasma pharmacokinetics of Cy5-αCD20 appeared unaltered in mice with CIA whereas distribution of Cy5-αCD20 to the jdLNs was generally increased in mice with CIA, regardless of the route of administration. However, IA administration led to greater and more specific exposure to the jdLNs. Consistent with increased Cy5-αCD20 in the jdLNs of CIA compared to healthy mice, there was a greater reduction in jdLN weight and a trend toward greater jdLN B cell depletion at 24 h compared to 4 h after IA compared to SC and IV administration. Taken together, this data supports the potential to improve local efficacy of B cell depletion therapies through a jdLN-directed approach which will enable a reduction in dose and systemic toxicities.

TPGS Decorated Liposomes as Multifunctional Nano-Delivery Systems.

Liposomes are sphere-shaped vesicles that can capture therapeutics either in the outer phospholipid bilayer or inner aqueous core. Liposomes, especially when surface-modified with functional materials, have been used to achieve many benefits in drug delivery, including improving drug solubility, oral bioavailability, pharmacokinetics, and delivery to disease target sites such as cancers. Among the functional materials used to modify the surface of liposomes, the FDA-approved non-ionic surfactant D-alpha-tocopheryl polyethylene glycol succinate (TPGS) is increasingly being applied due to its biocompatibility, lack of toxicity, applicability to various administration routes and ability to enhance solubilization, stability, penetration and overall pharmacokinetics. TPGS decorated liposomes are emerging as a promising drug delivery system for various diseases and are expected to enter the market in the coming years. In this review article, we focus on the multifunctional properties of TPGS-coated liposomes and their beneficial therapeutic applications, including for oral drug delivery, vaccine delivery, ocular administration, and the treatment of various cancers. We also suggest future directions to optimise the manufacture and performance of TPGS liposomes and, thus, the delivery and effect of encapsulated diagnostics and therapeutics.

Vmeasur: A software package for experimental and clinical measurement of mesenteric lymphatic contractile function over an extended vessel length.

OBJECTIVE: Conventionally, in vivo mesenteric lymphatic contractile function is measured using a high magnification transmission microscope (field of view 0.3-1.5 mm), which precludes visualization of extended lengths of vessels embedded in mesenteric fat. Existing software is not optimized for imaging at a low magnification using a contrast agent. We aimed to develop a simple and clinically transferable method for in situ visualization, image analysis, and quantitative assessment of mesenteric lymphatic contractile function over an extended area. METHODS: Subserosal injection of various blue dyes was taken up by mesenteric lymphatics and visualized under a stereomicroscope (25×), allowing for video recording of 1.4 × 1.1 cm of intact mesentery. A new R package ("vmeasur") that combines multiple high-performance image analyses into a single workflow was developed. The edges of each vessel were determined by applying an automated threshold to each frame (with real-time manual verification). The vessel width at every point in each frame was plotted to provide contractile parameters over time and along the lymphatic vessel length. RESULTS: Contractile parameters and their differences along the length of the vessel were accurately calculated in a rodent model. In a human mesenteric lymphatic, the algorithm was also able to measure changes in diameter over length. CONCLUSION: This software offers a low cost, rapid, and accessible method to measure lymphatic contractile function over a wide area, showing differences in contractility along the length of a vessel. Because the presence of mesenteric fat has less of an impact on imaging, due to the use of an exogenous contrast agent, there is potential for clinical application.

Systematic Down-Selection of Repurposed Drug Candidates for COVID-19.

SARS-CoV-2 is the cause of the COVID-19 pandemic which has claimed more than 6.5 million lives worldwide, devastating the economy and overwhelming healthcare systems globally. The development of new drug molecules and vaccines has played a critical role in managing the pandemic; however, new variants of concern still pose a significant threat as the current vaccines cannot prevent all infections. This situation calls for the collaboration of biomedical scientists and healthcare workers across the world. Repurposing approved drugs is an effective way of fast-tracking new treatments for recently emerged diseases. To this end, we have assembled and curated a database consisting of 7817 compounds from the Compounds Australia Open Drug collection. We developed a set of eight filters based on indicators of efficacy and safety that were applied sequentially to down-select drugs that showed promise for drug repurposing efforts against SARS-CoV-2. Considerable effort was made to evaluate approximately 14,000 assay data points for SARS-CoV-2 FDA/TGA-approved drugs and provide an average activity score for 3539 compounds. The filtering process identified 12 FDA-approved molecules with established safety profiles that have plausible mechanisms for treating COVID-19 disease. The methodology developed in our study provides a template for prioritising drug candidates that can be repurposed for the safe, efficacious, and cost-effective treatment of COVID-19, long COVID, or any other future disease. We present our database in an easy-to-use interactive interface (CoviRx that was also developed to enable the scientific community to access to the data of over 7000 potential drugs and to implement alternative prioritisation and down-selection strategies.

Intestinal lymphatic dysfunction: a new pathway mediating gut-kidney crosstalk in kidney disease.

The importance of kidney-gut crosstalk in driving kidney disease complications is increasingly being realized. However, little attention has been given to intestinal lymphatic changes in kidney disease. Zhong et al. report striking changes to intestinal lymphatic composition, structure, and function in proteinuric kidney injury models, including increased lymphangiogenesis, lymph flow, and transport of lipoproteins and proinflammatory mediators. These changes appear to be stimulated by isolevuglandin (IsoLG)-modified apolipoprotein AI (ApoAI). This intestinal lymphatic response may regulate systemic complications.

Lipophilic Conjugates of Drugs: A Tool to Improve Drug Pharmacokinetic and Therapeutic Profiles.

Lipophilic conjugates (LCs) of small molecule drugs have been used widely in clinical and pre-clinical studies to achieve a number of pharmacokinetic and therapeutic benefits. For example, lipophilic derivatives of drugs are employed in several long acting injectable products to provide sustained drug exposure for hormone replacement therapy and to treat conditions such as neuropsychiatric diseases. LCs can also be used to modulate drug metabolism, and to enhance drug permeation across membranes, either by increasing lipophilicity to enhance passive diffusion or by increasing protein-mediated active transport. Furthermore, such conjugation strategies have been employed to promote drug association with endogenous macromolecular carriers (e.g. albumin and lipoproteins), and this in turn results in altered drug distribution and pharmacokinetic profiles, where the changes can be 'general' (e.g. prolonged plasma half-life) or 'specific' (e.g. enhanced delivery to specific tissues in parallel with the macromolecular carriers). Another utility of LCs is to enhance the encapsulation of drugs within engineered nanoscale drug delivery systems, in order to best take advantage of the targeting and pharmacokinetic benefits of nanomedicines. The current review provides a summary of the mechanisms by which lipophilic conjugates, including in combination with delivery vehicles, can be used to control drug delivery, distribution and therapeutic profiles. The article is structured into sections which highlight a specific benefit of LCs and then demonstrate this benefit with case studies. The review attempts to provide a toolbox to assist researchers to design and optimise drug candidates, including consideration of drug-formulation compatibility.

Organ-specific lymphatics play distinct roles in regulating HDL trafficking and composition.

Recently, peripheral lymphatic vessels were found to transport high-density lipoprotein (HDL) from interstitial tissues to the blood circulation during reverse cholesterol transport. This function is thought to be critical to the clearance of cholesterol from atherosclerotic plaques. The role of organ-specific lymphatics in modulating HDL transport and composition is, however, incompletely understood. This study aimed to 1) determine the contribution of the lymphatics draining the intestine and liver (which are major sites of HDL synthesis) to total (thoracic) lymph HDL transport and 2) verify whether the HDLs in lymph are derived from specific organs and are modified during trafficking in lymph. The mesenteric, hepatic, or thoracic lymph duct was cannulated in nonfasted Sprague-Dawley rats, and lymph was collected over 5 h under anesthesia. Whole lymph and specific lymph lipoproteins (isolated by ultracentrifugation) were analyzed for protein and lipid composition. The majority of thoracic lymph fluid, protein, and lipid mass was sourced from the mesenteric, and to a lesser extent, hepatic lymph. Mesenteric and thoracic lymph were both rich in chylomicrons and very low-density lipoprotein, whereas hepatic lymph and plasma were HDL-rich. The protein and lipid mass in thoracic lymph HDL was mostly sourced from mesenteric lymph, whereas the cholesterol mass was equally sourced from mesenteric and hepatic lymph. HDLs were compositionally distinct across the lymph sources and plasma. The composition of HDL also appeared to be modified during passage from the mesenteric and hepatic to the thoracic lymph duct. Overall, this study demonstrates that the lipoproteins in lymph are organ specific in composition, and the intestine and liver appear to be the main source of HDL in the lymph.NEW & NOTEWORTHY High-density lipoprotein in lymph are organ-specific in composition and derive mostly from the intestine and liver. High-density lipoprotein also appears to be remodeled during transport through the lymphatics. These findings have implications to cardiometabolic diseases that involve perturbations in lipoprotein distribution and metabolism.

High-Density Lipoprotein Composition Influences Lymphatic Transport after Subcutaneous Administration.

Interstitial administration (e.g., subcutaneous (SC) administration) of immunotherapies and vaccines within nanoparticles can improve access to lymph-resident immune cells, leading to enhanced efficacy and reduced off-target effects. Recently, endogenous high-density lipoproteins (HDLs) were found to return from peripheral tissue back to the systemic circulation via the lymphatic vessels and nodes. This suggests the potential utility of HDLs as biocompatible lymphatic-targeted therapeutic carriers. However, we have a limited understanding of the mechanisms that drive HDL uptake into peripheral lymphatics from the interstitium. This study investigated the influence of HDL physicochemical properties on lymphatic transport and lymph node (LN) retention of HDL after SC administration. A range of HDL particles was prepared and characterized. Sphere-shaped endogenous HDLs were isolated from biological fluids (rat lymph, rat plasma, and human plasma) and separated into two subclasses based on the density. Discoidal-shaped synthetic (reconstituted) HDLs (rHDLs) of similar sizes were assembled from lipids and apolipoprotein A-I. All HDLs had similar sizes of 10-20 nm and a slightly negative surface charge. All HDLs were radiolabeled with 3H-cholesteryl ester (3H-CE) and/or 14C-free cholesterol (14C FC) and administered SC into the hind leg of thoracic lymph-cannulated rats. The recovery of radiolabels in lymph, plasma, LN, and tissues was determined. From the interstitial injection site, all HDLs were preferentially transported into the lymph and not blood vessels as indicated by high lymph-to-plasma concentration ratios of the radiolabels (up to 100:1 during the absorption phase) and greater radiolabel recovery in LNs draining the injection site compared to the contralateral side. Several HDLs with unique composition demonstrated significantly higher lymphatic transport compared to other HDLs despite possessing similar physical properties, suggesting that HDL lymphatic transport is less influenced by physical properties. The LN retention of HDL was positively correlated to increasing the negative charge of HDL, which was related to surface composition. Overall, this study informs the optimal design of HDL-based nanoparticles to promote lymphatic targeting of immunotherapies and vaccines.

Lymphatic Uptake of Liposomes after Intraperitoneal Administration Primarily Occurs via the Diaphragmatic Lymphatics and is Dependent on Liposome Surface Properties.

Drugs are commonly administered via the intraperitoneal (IP) route to treat localized infections and cancers in patients and to test drug efficacy and toxicity in preclinical studies. Despite this, there remain large gaps in our understanding of drug absorption routes (lymph vs blood) and pharmacokinetics following IP administration. This is particularly true when drugs are administered in complex delivery systems such as liposomes which are the main marketed formulation for several drugs that are administered intraperitoneally. This study investigated the impact of liposome surface properties (charge and PEGylation) on absorption into lymph and blood, and lymphatic disposition patterns, following IP administration. To achieve this, stable 3H-dipalmitoyl-phosphatidylcholine (DPPC) and 14C-sucrose-radiolabeled liposomes of 100-150 nm diameter with negative, neutral, or positive surface charge, or a PEGylated surface, were prepared and administered intraperitoneally to rats. Radiolabel concentrations were measured in lymph, blood, and lymph nodes (LNs). Lymph was collected from the thoracic lymph duct at either the abdomen (ABD) or the jugular-subclavian junction (JSJ). The lymphatic recovery of the radiolabels was substantially lower after administration in positively charged compared to the neutral, negative, or PEGylated liposomes. Radiolabel recovery was substantially greater (up to 18-fold) in the thoracic lymph collected at the JSJ when compared to that at the ABD, suggesting that liposomes entered the lymphatics at the diaphragm. Consistent with this, the concentration of the liposome labels was substantially higher (up to seven-fold) in mediastinal than in mesenteric LNs. Overall, this study shows how the peritoneal absorption and lymphatic disposition of drugs administered intraperitoneally can be manipulated through a careful selection of the drug delivery system and may thus be optimized to treat localized conditions such as cancers, infections, inflammatory diseases, and acute and critical illness.

Transient Supersaturation Supports Drug Absorption from Lipid-Based Formulations for Short Periods of Time, but Ongoing Solubilization Is Required for Longer Absorption Periods.

The current studies sought to explore the impact of drug supersaturation and precipitation during the dispersion and digestion of lipid-based formulations (LBFs), on in vivo absorption using a coupled in vitro digestion-in vivo perfusion absorption model. Fenofibrate absorption was evaluated from a number of LBFs with different solubilization and supersaturation capacities, and conditions at the absorptive membrane manipulated by changing perfusion conditions, intestine segment lengths, and by the conduct of experiments in the presence or absence of suspended/precipitated drug. LBF dispersion and digestion resulted in varying periods of supersaturation across the different formulations. Even fleeting (5-10 min) periods of supersaturation were able to drive flux across a perfused 10 cm intestinal segment for up to 60 min, although over longer infusion periods (60-80 min) flux dropped in the absence of ongoing drug solubilization and supersaturation. In contrast, the presence or absence of precipitated/suspended drug, had little impact on drug flux. When perfused intestinal segment lengths were extended, the role of initial supersaturation was attenuated and ongoing solubilization conditions became the primary driver of absorptive flux. The data suggest that for highly permeable drugs such as fenofibrate, a short period of supersaturation at the absorptive membrane may be sufficient to drive absorptive drug flux in spite of significant drug precipitation on formulation dispersion or digestion in vitro. In contrast, where longer periods of absorption are required, for example, at higher doses, the requirement for ongoing solubilization and supersaturation becomes more apparent.

Correlating in Vitro Solubilization and Supersaturation Profiles with in Vivo Exposure for Lipid Based Formulations of the CETP Inhibitor CP-532,623.

Lipid based formulations (LBFs) are a promising formulation strategy for many poorly water-soluble drugs and have been shown previously to enhance the oral exposure of CP-532,623, an oral cholesteryl ester transfer protein inhibitor. In the current study, an in vitro lipid digestion model was used to probe the relationship between drug solubilization and supersaturation on in vitro dispersion and digestion of LBF containing long chain (LC) lipids and drug absorption in vivo. After in vitro digestion of LBF based on LC lipids, the proportion of CP-532,623 maintained in the solubilized state in the aqueous phase of the digest was highest in formulations containing Kolliphor RH 40, and in most cases outperformed equivalent formulations based on MC lipids. Subsequent administration of the LC-LBFs to beagle dogs resulted in reasonable correlation between concentrations of CP-532,623 measured in the aqueous phase of the in vitro digest after 30 min digestion and in vivo exposure (AUC); however, the LC-LBFs required greater in vitro drug solubilization to elicit similar in vivo exposure when compared to previous studies with MC-LBF. Although post digestion solubilization was enhanced in LC-LBF compared to MC-LBF, equilibrium solubility studies of CP-532,623 in the aqueous phase isolated from blank lipid digestion experiments revealed that equilibrium solubility was also higher, and therefore supersaturation lower. A revised correlation based on supersaturation in the digest aqueous phase and drug absorption was therefore generated. A single, linear correlation was evident for both LC- and MC-LBF containing Kolliphor RH 40, but this did not extend to formulations based on other surfactants. The data suggest that solubilization and supersaturation are significant drivers of drug absorption in vivo, and that across formulations with similar formulation composition good correlation is evident between in vitro and in vivo measures. However, across dissimilar formulations, solubilization and supersaturation alone are not sufficient to explain drug exposure and other factors also likely play a role.

Single Intravenous Dose of Novel Flurbiprofen-Loaded Proniosome Formulations Provides Prolonged Systemic Exposure and Anti-inflammatory Effect.

Vesicular and colloidal delivery systems can be designed to control drug release spatially and temporally to improve drug efficacy and side effect profiles. Niosomes (vesicles prepared from nonionic surfactants in aqueous media) are gaining interest as an alternative vesicular delivery system as they offer advantages such as biocompatibility, chemical stability, low cost, high purity, and versatility. However, the physical stability of niosomes, like other vesicular systems, is limited by vesicle fusion, aggregation, and leakage. Proniosomes (dehydrated powder or gel formulations that spontaneously form niosomes on hydration with aqueous media) can overcome these physical stability problems and are more convenient for sterilization, storage, transport, distribution, and dosing. Proniosomes have mostly been explored for their potential to enhance transdermal and oral absorption. In this study we assess, for the first time, the potential for hydrated proniosomes to sustain systemic exposure and therapeutic effect after intravenous delivery. Proniosomes carrying the anti-inflammatory drug, flurbiprofen, were prepared by spraying different nonionic surfactants (span 20, span 40, and span 60 in varying ratios with span 80) and cholesterol onto a sorbitol carrier. The proniosome powders were characterized for surface morphology and flow properties. Niosome formation was assessed at three different hydration temperatures (25, 37, and 45 °C), and the niosomes were assessed for vesicle size, entrapment efficiency, and sterility. OLP proniosomes prepared with a high ratio of span 80 to span 20 were found to spontaneously form vesicles of small size and high drug loading on hydration with aqueous media. The OLP derived niosomes successfully sustained in vitro drug release, in vivo pharmacokinetics, and the anti-inflammatory effect of flurbiprofen in an acute (rat paw edema) model of inflammation when compared to a control solution formulation. The study demonstrates that hydrated proniosomes can prolong systemic drug exposure over 3 days and provide a sustained therapeutic effect. The developed proniosomes represent a novel approach to treat acute pain and inflammation with the potential to be administered as a single intravenous dose by a clinician at the time of injury or surgery that provides adequate relief for several days and reduces fluctuations in therapy. Similar systems loaded with different drugs have potential for broader application in anesthesia, anti-infective, antiemetic, and cancer therapy.

Lymphatic Transport and Lymphocyte Targeting of a Triglyceride Mimetic Prodrug Is Enhanced in a Large Animal Model: Studies in Greyhound Dogs.

In previous studies, a triglyceride (TG) mimetic prodrug of the model immunomodulator mycophenolic acid (MPA) was shown to significantly enhance lymphatic transport of MPA-related species in the rat. The rat gastrointestinal tract, however, is somewhat different from that in higher order species such as dogs and humans and may underestimate lymphatic transport. Here the effectiveness of the prodrug strategy has been examined in conscious greyhound dogs, the GI physiology of which is more representative of that in humans. The bioavailability and lymphatic transport of free MPA and total MPA related materials were examined following oral administration of the parent drug (MPA) and the prodrug (2-MPA-TG) to both thoracic lymph duct cannulated and intact (noncannulated) greyhound dogs. The enrichment of free MPA in lymph nodes and lymph-derived lymphocytes was also determined to examine the efficiency of drug targeting to potential sites of action within the lymph. Via biochemical integration into a series of site-specific metabolic processes, the prodrug markedly increased (288-fold) lymphatic transport of total MPA related material (present as re-esterified 2-MPA-TG) when compared to the parent MPA and the extent of lymphatic transport was significantly greater in the dog (36.4% of the dose recovered in lymph) when compared to the previous data in the rat (13.4% of the dose). Conversion from 2-MPA-TG derivatives to parent MPA occurred in vivo, resulting in a marked increase in MPA concentrations in lymph nodes (5-6-fold) and lymph lymphocytes (21-fold), when compared to animals administered the parent drug. In conclusion, the data demonstrate that the TG prodrug of MPA facilitates efficient delivery of MPA to the lymphatic system in dogs and suggest that the TG prodrug strategy may more effectively facilitate targeted delivery in large animals than in rats.