Microglial activation decreases retention of the protease inhibitor saquinavir: implications for HIV treatment

Background Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide. Methods To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS). Results [3H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters. Conclusions The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-κβ and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site

Additional file 1 of Novel localization of folate transport systems in the murine central nervous system

Additional file 1. Cellular localization of vimentin in immortalized cell cultures of mouse AB. Cells were stained with the following: DAPI nuclear marker, or anti-vimentin (1:50) (Panel 1) No primary antibody was used as a negative control (Panel 2). Sections were visualized using confocal microscopy (LSM 700; Carl Zeiss) operated with ZEN software using an oil-immersion 63x lens

Tumour and tissue distribution of E3S/[³H]-E3S, following a blocking dose, in mice bearing xenografts.

<p>Statistically significant (<i>p</i><0.05) differences in uptake were observed in the tumour and kidney in both MCF-7 and MDA-MB-231 xenograft models *<i>p</i><0.05. (Li (231): Liver from MDA-MB-231 tumour bearing mice, K (231): Kidneys from MDA-MB-231 tumour bearing mice, T (231): MDA-MB-231 xenograft, Li (MCF-7): Liver from MCF-7 tumour bearing mice, K (MCF-7): Kidney from MCF-7 tumour bearing mice, T (MCF-7): MCF-7 xenograft).</p

Estrone-3-Sulphate, a Potential Novel Ligand for Targeting Breast Cancers

<div><p>The current study investigates the potential of estrone-3-sulphate (E3S) as a ligand for targeting Organic Anion Transporting Polypeptides (OATP), a family of membrane associated uptake transporters, for detection and diagnosis of hormone dependent breast cancers. E3S, an OATP substrate, is a predominant source of tumour estradiol in post-menopausal patients. To assess the potential of E3S as a ligand, distribution of exogenous E3S was determined at the whole body, tumour and cellular levels in murine models of hormone-dependent (MCF-7) and independent (MDA-MB-231) breast cancers. The highest levels of tumour uptake were observed at 6 h post injection (p.i) with significant difference (p = 0.04) between the level in MCF-7 (13.9±3.1%ID/g) and MDA-MB-231 (10.4±1.1%ID/g) (%ID/g: percentage of the total injected dose per gram tissue). The highest tumour-to-blood ratios (MCF-7∶7.4±1.2; MDA-MB-231∶9.1±2.1) were observed at 48 p.i., and highest tumour-to-muscle ratios (MCF-7∶10.7±1.5; MDA-MB-231∶3.8±0.7) were observed at 6 h p.i. Analogous to total tumour uptake, <i>ex vivo</i> tumour cell uptake at 2 h p.i. was 6 fold higher in MCF-7 in comparison to MDA-MB-231 tumour cells. Blocking studies, conducted by pre-administration of 100-fold excess E3S, resulted in significantly lower (MCF-7: p = 0.01; MDA-MB-231: p = 0.02) tumour uptake in both xenograft models, suggesting the involvement of an active carrier-mediated process. The expression of OATP1A2 was detected in tumour sections from both xenografts, with significantly higher expression (p = 0.002) in the MCF-7 xenografts. Overall, the higher tumour uptake and tumour-to-muscle ratio, alongside the higher expression of OATP1A2, in the MCF-7 xenograft model suggests the potential of E3S to serve as a novel ligand for targeting hormone dependent breast cancers.</p></div

Pharmacokinetic profile of E3S/[³H]-E3S administered in healthy mice.

<p>Mathematical modeling was performed using the Scientist software (Micromath). Data were fit using a two-compartment model with an i.v. bolus injection, assuming clearance from the central compartment. (%ID/mL, % Injected Dose/mL).</p

Tumour uptake (A), tumour-to-blood ratios (B), and tumour-to-surrounding muscle ratios (C) of E3S.

<p>Tumour uptake (A), tumour-to-blood ratios (B), and tumour-to-surrounding muscle ratios (C) of E3S in mice bearing MCF-7 (hormone dependent) and MDA-MB-231 (non-hormone dependent) xenografts expressed as % injected dose per gram (%ID/g) at 2 h, 6 h and 48 h p.i. Uptake of E3S/[³H]-E3S was significantly higher in hormone dependent MCF-7 xenografts at all time points. Statistically significant (<i>p</i><0.05) difference is also observed in the ratio of the tumour uptake and the surrounding muscle uptake between the two xenograft models. *<i>p</i><0.05, **<i>p</i><0.01.</p

OATP1A2 expression in MCF-7 (A) and MDA-MB-231 (B) xenograft sections.

<p>A statistically significant (p<0.05) difference was observed in the OATP1A2 expression between the MCF-7 and MDA-MB-231 tumour xenografts expressed in % area (**p<0.005).</p

<i>Ex vivo</i> cellular uptake of E3S in MCF-7 and MDA-MB-231 xenografts.

<p>Significant differences in cellular uptake of ³H-E3S between the MCF-7 and MDA-MB-231 xenografts were observed at 2 h (***<i>p</i><0.001) and 6 h (*<i>p</i><0.05) p.i. but not at 48 h p.i.</p

Biodistribution of E3S/[³H]-E3S.

<p>Biodistribution of E3S/[³H]-E3S at 2 h, 6 h and 48 h p.i. in mice bearing MCF-7 (A) and MDA-MB-231 (B) xenograft expressed as % injected dose per gram (%ID/g). (Lu: Lung, Li: Liver, S+P: Spleen and Pancreas, St: Stomach, K: Kidneys, Ut: Uterus, In: Intestine, H: Heart, Bl: Blood, T: Tumour, Ms: Muscle).</p

Table1_SARS-CoV-2 infection dysregulates the expression of clinically relevant drug metabolizing enzymes in Vero E6 cells and membrane transporters in human lung tissues.docx

SARS-CoV-2-mediated interactions with drug metabolizing enzymes and membrane transporters (DMETs) in different tissues, especially lung, the main affected organ may limit the clinical efficacy and safety profile of promising COVID-19 drugs. Herein, we investigated whether SARS-CoV-2 infection could dysregulate the expression of 25 clinically relevant DMETs in Vero E6 cells and postmortem lung tissues from COVID-19 patients. Also, we assessed the role of 2 inflammatory and 4 regulatory proteins in modulating the dysregulation of DMETs in human lung tissues. We showed for the first time that SARS-CoV-2 infection dysregulates CYP3A4 and UGT1A1 at the mRNA level, as well as P-gp and MRP1 at the protein level, in Vero E6 cells and postmortem human lung tissues, respectively. We observed that at the cellular level, DMETs could potentially be dysregulated by SARS-CoV-2-associated inflammatory response and lung injury. We uncovered the pulmonary cellular localization of CYP1A2, CYP2C8, CYP2C9, and CYP2D6, as well as ENT1 and ENT2 in human lung tissues, and observed that the presence of inflammatory cells is the major driving force for the discrepancy in the localization of DMETs between COVID-19 and control human lung tissues. Because alveolar epithelial cells and lymphocytes are both sites of SARS-CoV-2 infection and localization of DMETs, we recommend further investigation of the pulmonary pharmacokinetic profile of current COVID-19 drug dosing regimen to improve clinical outcomes.</p