Qin, Chaolong
(2021)
Targeting Lopinavir to the Mesenteric Lymphatic System by Lipophilic Ester Prodrug Approach for Improved Treatment of HIV/AIDS.
PhD thesis, University of Nottingham.
Abstract
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.
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