Targeted bio-distribution of drugs to the lymphatic system

Jewell, Adelaide (2022) Targeted bio-distribution of drugs to the lymphatic system. PhD thesis, University of Nottingham.

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Targeted drug delivery to the intestinal lymphatic system has emerged as an important goal in drug development for the treatment of a number of clinical indications. These include, cancers, inflammatory disorders and infectious diseases. Following oral administration, small molecule drugs with specific physiochemical properties, have been shown to associate with lipoproteins called chylomicrons in the enterocytes of the small intestine. Chylomicron associated drugs then bypass hepatic uptake and enter the mesenteric lymph nodes (MLN). Oral coadministration of highly lipophilic small molecule drugs with lipids had subsequently been identified as an effective mechanism for delivering highly lipophilic small molecule drugs to the MLN in high concentrations. One example is Cannabidiol (CBD), which was shown to undergo lymphatic transport following oral administration with lipids and has received significant research interest for the treatment of inflammatory disorders.

The overarching aim of this thesis was to determine how small molecule drugs distribute in the lymphatic system following oral delivery. More specifically, work in this thesis was divided into two main research questions; 1) where within the cellular structures of lymph nodes drugs distribute and 2) which specific individual lymph nodes can be targeted. For the purposes of this thesis, CBD was selected as a model drug, with which drug distribution in the lymphatics was investigated.

Lymph nodes are comprised of distinct anatomical and function regions. The flow of lymph and lymph bourn molecules within lymph nodes is highly regulated. This aids appropriate immune surveillance. Based on this, it was hypothesised that small molecule drugs, such as CBD, may preferentially distribute in some regions of lymph nodes more than others.

Mass spectrometry imaging (MSI) has been adapted over recent years to enable detailed tissue analysis. However, so far, MSI had only been applied to image drug distribution in tissues at mg/g concentrations, following topical administration. A method using a hybrid instrument comprising a time-of-flight analyser with an Orbitrap mass spectrometer, termed OrbiSIMS, to image CBD in MLN tissues at in vivo relevant concentrations was described. The estimated limit of sensitivity for the [M-H]- ion of CBD was in the range of 5-10 µg/g, which correlates to concentrations observed in vivo. Subsequently, OrbiSIMS imaging of sectioned MLNs from rats dosed orally with CBD in sesame oil was performed. Critically, CBD could be visualised primarily in the paracortex of the lymph node, which is known to be dominated by T-cells. This work represents the first evidence of label- and matrix-free imaging of drug distribution at the time of peak absorption into intestinal lymph nodes. Although imaging reproducibility and drug-cell interaction would need to be confirmed, this work may therefore support the hypothesis that CBD exerts its immunoregulatory effects in vivo primarily through T-cells. This is likely aided through cross- talk with conduit resident dendritic cells of the paracortex. Assuming other lipophilic small molecule drugs are also distributed in the paracortex, this finding could have wider clinical implications for diseases where T-cells are primarily involved.

The MLNs are a large group of lymph nodes which drain lymph from the small intestine. Lipid uptake is understood to vary across the length of the small intestine. Based on this it was also hypothesised that individual nodes within the chain of MLNs may be exposed to differing concentrations of orally administered drugs entering the lymphatics via chylomicron association. In addition, following collection in the MLN, lymph is understood to drain into the retroperitoneal lymph nodes (RPLN) before entering the cisterna chyli and ultimately systemic circulation. Subsequently, it was also hypothesised that the RPLN may also be exposed to drugs undergoing lymphatic transport.

The second major research question of this project, relating to which specific lymph nodes can be targeted following oral delivery and to what extent, was addressed using two main approaches. Firstly, in a rat model, CBD concentrations in individual lymph nodes from animals dosed orally with CBD were determined using high-performance liquid chromatography (HPLC). It was shown that, at the time of peak absorption, drug concentrations were significantly higher in the upper middle nodes of the mesenteric chain and distribution was therefore non-uniform within the MLNs. Another key finding was that the RPLN could also be targeted. Moreover, at time points after 2 hours post administration, at which absorption into the MLN reaches a peak, concentrations were similar in RPLN and MLN. Concentrations in RPLN were also more than 20 times higher than previously reported in plasma, indicating lymphatic transport of drug rather than redistribution from systemic circulation. This widespread delivery of drugs to multiple groups of lymph nodes following oral administration had not previously been demonstrated and indicates the clinical potential of oral drug administration for diseases where lymphatic involvement is widespread. This includes inflammatory diseases and cancer metastases.

Finally, the question of which lymph nodes can be targeted using orally delivered lipid-based formulations was assessed in a human setting. It was hypothesised that MRI may be used to identify changes in lymph nodes as dietary lipids are absorbed. Subsequently, these changes could be used to map lipid uptake into individual lymph nodes and thus which nodes may be exposed to chylomicron associated drugs. An assessment of the feasibility of a label free, non-invasive MRI method for this indication was also performed. Repeated identification of individual lymph nodes at baseline and following a high fat meal was achieved in 3 healthy human volunteers. In all participants, the apparent diffusion coefficient (ADC) of lymph nodes was shown to increase following a high fat meal. The timings of these changes correlated with expected lymphatic lipid uptake. Based on this, increases in ADC may represent a novel measurable indicator for lipid tracking in the intestinal lymphatics in future work.

In conclusion, the work in this thesis has provided preliminary evidence that the paracortex is the predominant target following oral delivery of small molecule drugs. In addition, novel data support the notion that the MLN are differentially exposed to lymph associated drugs and that the RPLN may also be targeted.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Gershkovich, Pavel
Fischer, Peter
Stocks, Michael
Scurr, David
Keywords: Lymphatic system, Drugs, Bio-distribution
Subjects: R Medicine > RM Therapeutics. Pharmacology
Faculties/Schools: UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 71585
Depositing User: Jewell, Adelaide
Date Deposited: 14 Dec 2022 04:40
Last Modified: 14 Dec 2022 04:40

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