Targeting Lopinavir to the Mesenteric Lymphatic System by Lipophilic Ester Prodrug Approach for Improved Treatment of HIV/AIDS

Human immunodeficiency virus (HIV) is a lifelong disease that can destroy human immune system, thereby leading to life-threatening acquired immune deficiency syndrome (AIDS). Combined antiretroviral therapy (cART) can efficiently suppress HIV replication, but the cessation of cART usually results in viral rebound, mostly due to the presence of viral reservoirs. The mesenteric lymphatic system, including mesenteric lymph nodes (MLNs), is an important viral reservoir. In this work, lopinavir (LPV), a protease inhibitor, was selected as a drug candidate for mesenteric lymphatic targeting. In this work we focused on two main aims as follows: (1) development and validation of a sensitive HPLC-UV bioanalytical method for determination of LPV for pharmacokinetics and biodistribution studies in rats; (2) discovery of a novel lipophilic ester prodrug approach, combined with oral lipid-based drug delivery, to efficiently deliver lopinavir (LPV) to the mesenteric lymph and MLNs. A simple and sensitive HPLC-UV bioanalytical method for determination of LPV in rat plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid-liquid extraction with n-hexane–ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini (C18, 150 mm × 2.0 mm, 5 μm) column at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10–10,000 ng/mL, with a lower limit of quantification (LLOQ) of 10 ng/mL using 100 μL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration (FDA). This method was successfully applied to a preliminary pharmacokinetic study in rats following intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma allowing its use in therapeutic drug monitoring. In conclusion, this novel and simple bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as therapeutic drug monitoring in human patients. The combined antiretroviral therapy (cART) can efficiently suppress HIV replication, but the cessation of cART usually results in viral rebound, mostly due to the presence of viral reservoirs. The mesenteric lymphatic system, including mesenteric lymph nodes (MLNs), is an important viral reservoir into which antiretroviral drugs poorly penetrate. In this work, we proposed a novel lipophilic ester prodrug approach, combined with oral lipid-based formulation, to efficiently deliver lopinavir (LPV) to the mesenteric lymph and MLNs. A series of prodrugs was designed using an in-silico model for prediction of affinity to chylomicrons (CMs), and then synthesised. The potential for mesenteric lymphatic targeting and bioconversion to LPV in physiologically relevant media was assessed in vitro and ex vivo. Subsequently, LPV and selected prodrug candidates were evaluated for their in vivo pharmacokinetics and biodistribution in rats. Oral co-administration of lipids alone could not facilitate the delivery of unmodified LPV to the mesenteric lymphatic system and resulted in undetectable levels of LPV in these tissues. However, a combination of the lipophilic prodrug approach with lipid-based formulation resulted in efficient targeting of LPV to HIV reservoirs in mesenteric lymph and MLNs. The maximum levels of LPV in mesenteric lymph were 1.6- and 16.9-fold higher than protein binding-adjusted IC90 (PA-IC90) of LPV for HIV-1 (140 ng/mL) following oral administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Moreover, the concentrations of LPV in MLNs were 1.1- and 7.2-fold higher than PA-IC90 following administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Furthermore, the bioavailability of LPV was also substantially increased following oral administration of activated ester prodrug compared to unmodified LPV. This approach, especially if can be translated to other antiretroviral drugs, has potential for reducing the size of HIV reservoirs within the mesenteric lymphatic system. Therefore, oral co-administration of lipophilic ester prodrugs and dietary lipids can improve the concentration of prodrugs and the release of the parent drug LPV in the mesenteric lymphatic system. The concentrations of LPV recovered in the mesenteric lymph and lymph nodes were significantly high and exceeded the therapeutic levels of LPV following administration of prodrug 7. In addition, the increase in systemic exposure to LPV following administration of prodrug 7 is of potentially high clinical importance as it could avoid drug-drug interaction issues caused by co-administration of ritonavir. Therefore, prodrug 7 could be potentially selected for future development for the treatment of HIV. Following the investigation of lipophilic ester prodrug approach, five self-immolative codrugs were also designed using in-silico model for prediction of affinity to chylomicrons (CMs), and then synthesised. The lead compound codrug 4 showed high affinity to artificial CMs (around 60%), good stability in FaSSIF (degradation half-life: 30.5 min). The half-life of codrug 4 was nearly 5-fold shorter and 24-fold longer compared to prodrugs 1 (shortest alkyl ester) and 7 (activated ester), respectively, in rat plasma. Therefore, codrug 4 was selected for the subsequent in vivo studies. Unfortunately, the levels of codrug or released LPV could not be detected in plasma at any sampled time points during pharmacokinetic study. Although the reason for these in vivo results is unclear, it is likely that first-pass intestinal or hepatic metabolic loss is the main cause of lack of measurable levels of codrug 4 or LPV in plasma. Further investigation will be needed to assess the feasibility of self-immolative codrug approach. In conclusion, in this PhD project, we have focused on the optimisation of drug delivery for maximising exposure of mesenteric lymphatic system, an important difficult-to-penetrate HIV reservoir, to the antiretroviral drug LPV. A sensitive bioanalytical method for the determination of LPV in rat plasma was a crucial part of this whole project as the measurement of drug concentrations in samples were achieved by means of HPLC-UV. Following the assessment of a series of stability, CM association, and in vivo studies, it was shown that the activated ester prodrug exhibited significantly efficient delivery of the active drug to the mesenteric lymph and lymph nodes. The results of the prodrug approach indicated that the delivery of high levels of antiretroviral drugs to viral reservoirs can be beneficial to improve the treatment of HIV/AIDS

Development and validation of a cost-effective and sensitive bioanalytical HPLC-UV method for determination of lopinavir in rat and human plasma

© 2020 The Authors. Biomedical Chromatography published by John Wiley & Sons Ltd A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid–liquid extraction with n-hexane–ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini column (C18, 150 mm × 2.0 mm, 5 μm) at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10–10,000 ng/mL, with a lower limit of quantification of 10 ng/mL using 100 μL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration. This method was successfully applied to a preliminary pharmacokinetic study in rats following an intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma, allowing its use in therapeutic drug monitoring (TDM). In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as TDM in human patients

Oral administration of tipranavir with long-chain triglyceride results in moderate intestinal lymph targeting but no efficient delivery to HIV-1 reservoir in mesenteric lymph nodes

The introduction of combination antiretroviral therapy (cART) led to substantial improvement in mortality and morbidity of HIV-1 infection. However, the poor penetration of antiretroviral agents to HIV-1 reservoirs limit the ability of the antiretroviral agents to eliminate the virus. Mesenteric lymph nodes (MLNs) are one of the main HIV-1 reservoirs in patients under suppressive cART. Intestinal lymphatic absorption pathway substantially increases the concentration of lipophilic drugs in mesenteric lymph and MLNs when they are co-administered with long-chain triglyceride (LCT). Chylomicrons (CM) play a crucial role in the intestinal lymphatic absorption as they transport drugs to the lymph lacteals rather than blood capillary by forming CM-drug complexes in the enterocytes. Thus, lipophilic antiretroviral drugs could potentially be delivered to HIV-1 reservoirs in MLNs by LCT-based formulation approach. In this study, protease inhibitors (PIs) were initially screened for their potential for intestinal lymphatic targeting using a computational model. The candidates were further assessed for their experimental affinity to CM. Tipranavir (TPV) was the only-candidate with substantial affinity to both artificial and natural CM in vitro and ex vivo. Pharmacokinetics and biodistribution studies were then performed to evaluate the oral bioavailability and intestinal lymphatic targeting of TPV in rats. The results showed similar oral bioavailability of TPV with and without co-administration of LCT vehicle. Although LCT-based formulation led to 3-fold higher concentrations of TPV in mesenteric lymph compared to plasma, the levels of the drug in MLNs were similar to plasma in both LCT-based and lipid-free formulation groups. Thus, LCT-based formulation approach alone was not sufficient for effective delivery of TPV to MLNs. Future efforts should be directed to a combined highly lipophilic prodrugs/lipid-based formulation approach to target TPV, other PIs and potentially other classes of antiretroviral agents to viral reservoirs within the mesenteric lymphatic system

Natural sesame oil is superior to pre-digested lipid formulations and purified triglycerides in promoting the intestinal lymphatic transport and systemic bioavailability of cannabidiol

© 2021 Elsevier B.V. Lipid-based formulations play a significant role in oral delivery of lipophilic drugs. Previous studies have shown that natural sesame oil promotes the intestinal lymphatic transport and oral bioavailability of the highly lipophilic drug cannabidiol (CBD). However, both lymphatic transport and systemic bioavailability were also associated with considerable variability. The aim of this study was to test the hypothesis that pre-digested lipid formulations (oleic acid, linoleic acid, oleic acid with 2-oleoylglycerol, oleic acid with 2-oleoylglycerol and oleic acid with glycerol) could reduce variability and increase the extent of the intestinal lymphatic transport and oral bioavailability of CBD. The in vivo studies in rats showed that pre-digested or purified triglyceride did not improve the lymphatic transport and bioavailability of CBD in comparison to sesame oil. Moreover, the results suggest that both the absorption of lipids and the absorption of co-administered CBD were more efficient following administration of natural sesame oil vehicle compared with pre-digested lipids or purified trioleate. Although multiple small molecule constituents and unique fatty acid compositions could potentially contribute to a better performance of sesame oil in oral absorption of lipids or CBD, further investigation will be needed to identify the mechanisms involved

Genome-wide DNA methylation profiling in blood reveals epigenetic signature of incident acute coronary syndrome

DNA methylation (DNAm) has been implicated in acute coronary syndrome (ACS), but the causality remains unclear in cross-sectional studies. Here, we conduct a prospective epigenome-wide association study of incident ACS in two Chinese cohorts (discovery: 751 nested case-control pairs; replication: 476 nested case-control pairs). We identified and validated 26 differentially methylated positions (DMPs, false discovery rate [FDR] <0.05), including three mapped to known cardiovascular disease genes (PRKCZ, PRDM16, EHBP1L1) and four with causal evidence from Mendelian randomization (PRKCZ, TRIM27, EMC2, EHBP1L1). Two hypomethylated DMPs were negatively correlated with the expression in blood of their mapped genes (PIGG and EHBP1L1), which were further found to overexpress in leukocytes and/or atheroma plaques. Finally, our DMPs could substantially improve the prediction of ACS over traditional risk factors and polygenic scores. These findings demonstrate the importance of DNAm in the pathogenesis of ACS and highlight DNAm as potential predictive biomarkers and treatment targets

Vegetable oils composition affects the intestinal lymphatic transport and systemic bioavailability of co-administered lipophilic drug cannabidiol

Although natural sesame oil has been shown to facilitate the lymphatic delivery and oral bioavailability of the highly lipophilic drug cannabidiol (CBD), considerable variability remains an unresolved challenge. Vegetable oils differ substantially in composition, which could lead to differences in promotion of intestinal lymphatic transport of lipophilic drugs. Therefore, the differences in composition of sesame, sunflower, peanut, soybean, olive and coconut oils and their corresponding role as vehicles in promoting CBD lymphatic targeting and bioavailability were investigated in this study. The comparative analysis suggests that the fatty acids profile of vegetable oils is overall similar to the fatty acids profile in the corresponding chylomicrons in rat lymph. However, arachidonic acid (C20:4), was introduced to chylomicrons from endogenous nondietary sources. Overall, fatty acid composition of natural vegetable oils vehicles affected the intestinal lymphatic transport and bioavailability of CBD following oral administration in this work. Olive oil led to the highest concentration of CBD in the lymphatic system and in the systemic circulation in comparison to the other natural vegetable oils following oral administration in rats

A novel nucleoside rescue metabolic pathway may be responsible for therapeutic effect of orally administered cordycepin

Although adenosine and its analogues have been assessed in the past as potential drug candidates due to the important role of adenosine in physiology, only little is known about their absorption following oral administration. In this work, we have studied the oral absorption and disposition pathways of cordycepin, an adenosine analogue. In vitro biopharmaceutical properties and in vivo oral absorption and disposition of cordycepin were assessed in rats. Despite the fact that numerous studies showed efficacy following oral dosing of cordycepin, we found that intact cordycepin was not absorbed following oral administration to rats. However, 3′-deoxyinosine, a metabolite of cordycepin previously considered to be inactive, was absorbed into the systemic blood circulation. Further investigation was performed to study the conversion of 3′-deoxyinosine to cordycepin 5′-triphosphate in vitro using macrophage-like RAW264.7 cells. It demonstrated that cordycepin 5′-triphosphate, the active metabolite of cordycepin, can be formed not only from cordycepin, but also from 3′-deoxyinosine. The novel nucleoside rescue metabolic pathway proposed in this study could be responsible for therapeutic effects of adenosine and other analogues of adenosine following oral administration. These findings may have importance in understanding the physiology and pathophysiology associated with adenosine, as well as drug discovery and development utilising adenosine analogues

Development of cordycepin formulations for preclinical and clinical studies

There is extensive literature on in vivo studies with cordycepin but these studies were generally conducted without validation of the various formulations, especially in terms of the solubility of cordycepin in the dosing vehicles used. Cordycepin is a promising drug candidate in multiple therapeutic areas and there is a growing interest in studies aimed at assessing the pharmacological activity of this compound in relevant animal disease models. It is likely that many reported in vivo studies used formulations in which cordycepin was incompletely soluble. This can potentially confound the interpretation of pharmacokinetics and efficacy results. Furthermore, the presence of particles in intravenously administered suspension can cause adverse effects and should be avoided. Here we present the results from our development of simple and readily applicable formulations of cordycepin based on quantitative solubility assessment. Homogeneous solutions of cordycepin were prepared in phosphate-buffered saline (PBS) at different pH levels, suitable as formulations for both intravenously and oral administration. For the purpose of high-dose oral administration we also developed propylene glycol (PPG)-based vehicles in which cordycepin is completely soluble. The stability of the newly developed formulations was also assessed, as well the feasibility of their sterilisation by filtration. Additionally, an HPLC-UV method for the determination of cordycepin in the formulations, which may also be useful for other purposes, was developed and validated. Our study could provide useful information for improvement of future preclinical and clinical studies involving cordycepin

Targeted delivery of lopinavir to HIV reservoirs in the mesenteric lymphatic system by lipophilic ester prodrug approach

© 2020 Elsevier B.V. The combined antiretroviral therapy (cART) can efficiently suppress HIV replication, but the cessation of cART usually results in viral rebound, mostly due to the presence of viral reservoirs. The mesenteric lymphatic system, including mesenteric lymph nodes (MLNs), is an important viral reservoir into which antiretroviral drugs poorly penetrate. In this work, we proposed a novel lipophilic ester prodrug approach, combined with oral lipid-based formulation, to efficiently deliver lopinavir (LPV) to the mesenteric lymph and MLNs. A series of prodrugs was designed using an in-silico model for prediction of affinity to chylomicrons (CMs), and then synthesized. The potential for mesenteric lymphatic targeting and bioconversion to LPV in physiologically relevant media was assessed in vitro and ex vivo. Subsequently, LPV and selected prodrug candidates were evaluated for their in vivo pharmacokinetics and biodistribution in rats. Oral co-administration of lipids alone could not facilitate the delivery of unmodified LPV to the mesenteric lymphatic system and resulted in undetectable levels of LPV in these tissues. However, a combination of the lipophilic prodrug approach with lipid-based formulation resulted in efficient targeting of LPV to HIV reservoirs in mesenteric lymph and MLNs. The maximum levels of LPV in mesenteric lymph were 1.6- and 16.9-fold higher than protein binding-adjusted IC90 (PA-IC90) of LPV for HIV-1 (140 ng/mL) following oral administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Moreover, the concentrations of LPV in MLNs were 1.1- and 7.2-fold higher than PA-IC90 following administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Furthermore, the bioavailability of LPV was also substantially increased following oral administration of activated ester prodrug compared to unmodified LPV. This approach, especially if can be translated to other antiretroviral drugs, has potential for reducing the size of HIV reservoirs within the mesenteric lymphatic system

Lipophilic activated ester prodrug approach for drug delivery to the intestinal lymphatic system

The intestinal lymphatic system plays an important role in the pathophysiology of multiple diseases including lymphomas, cancer metastasis, autoimmune diseases, and human immunodeficiency virus (HIV) infection. It is thus an important compartment for delivery of drugs in order to treat diseases associated with the lymphatic system. Lipophilic prodrug approaches have been used in the past to take advantage of the intestinal lymphatic transport processes to deliver drugs to the intestinal lymphatics. Most of the approaches previously adopted were based on very bulky prodrug moieties such as those mimicking triglycerides (TG). We now report a study in which a lipophilic prodrug approach was used to efficiently deliver bexarotene (BEX) and retinoic acid (RA) to the intestinal lymphatic system using activated ester prodrugs. A range of carboxylic ester prodrugs of BEX were designed and synthesised and all of the esters showed improved association with chylomicrons, which indicated an improved potential for delivery to the intestinal lymphatic system. The conversion rate of the prodrugs to BEX was the main determinant in delivery of BEX to the intestinal lymphatics, and activated ester prodrugs were prepared to enhance the conversion rate. As a result, an 4-(hydroxymethyl)-1,3-dioxol-2-one ester prodrug of BEX was able to increase the exposure of the mesenteric lymph nodes (MLNs) to BEX 17-fold compared to when BEX itself was administered. The activated ester prodrug approach was also applied to another drug, RA, where the exposure of the MLNs was increased 2.4-fold through the application of a similar cyclic activated prodrug. Synergism between BEX and RA was also demonstrated in vitro by cell growth inhibition assays using lymphoma cell lines. In conclusion, the activated ester prodrug approach results in efficient delivery of drugs to the intestinal lymphatic system, which could benefit patients affected by a large number of pathological conditions