Use of cannabidiol in oligopeptide transporter inhibitors

Abstract

The application provides application of cannabidiol in oligopeptide transporter inhibitors and belongs to the technical field of membrane transporter inhibitors. The application provides application of cannabidiol in preparation of oligopeptide transporter inhibitors, in particular, non-substrate oligopeptide transporter inhibitors. It is found that cannabidiol is a non-substrate oligopeptide transporter inhibitor and can specifically inhibit the uptake capacity of oligopeptide transporter to substrates.

Description

Technical Field

[0001] This invention belongs to the field of membrane transporter inhibitor technology, specifically relating to the application of cannabidiol in oligopeptide transporter inhibitors. Background Technology

[0002] Peptide transporters (PEPTs) are membrane protein carriers responsible for mediating the transmembrane transport of oligopeptides. They recognize and bind extracellular oligopeptides, and through conformational changes, transport the oligopeptides into the cell, thereby facilitating their absorption, distribution, and metabolism within the body. PEPTs are expressed in various tissues and organs, with higher expression levels in small intestinal epithelial cells and proximal tubular epithelial cells of the kidney. In the small intestine, PEPTs are responsible for absorbing oligopeptides produced after food digestion into the bloodstream, providing the body with essential amino acids and nutrients. In the kidneys, PEPTs participate in the reabsorption of oligopeptides in the renal tubular filtrate, reducing oligopeptide excretion and maintaining peptide homeostasis.

[0003] PEPTs play a crucial role in many physiological processes in the human body, particularly in the intestinal absorption of nutrients (such as dipeptides and tripeptides) and drug transport. In recent years, with the deepening research into drug transport mechanisms, PEPTs have received widespread attention as drug targets. The absorption and distribution of many drugs are closely related to the activity of PEPTs; therefore, developing inhibitors or modulators targeting PEPTs is of great significance for optimizing drug efficacy and reducing adverse drug reactions.

[0004] Current PEPTs inhibitors have limitations. Known PEPTs inhibitors are primarily competitive inhibitors, such as dipeptide analogs: some compounds structurally similar to natural dipeptides can competitively bind to PEPTs, thereby inhibiting the transport of natural substrates. Certain cephalosporins, such as cephalexin, can bind to PEPTs, competitively inhibiting the transport of other substrates, thus affecting drug absorption and bioavailability to some extent. Currently, there is a lack of discovery and use of non-substrate inhibitory agents with good PEPTs inhibitory effects. Summary of the Invention

[0005] The purpose of this invention is to provide the application of cannabidiol in oligopeptide transporter inhibitors. The cannabidiol of this invention is a non-substrate-based oligopeptide transporter inhibitor, capable of inhibiting the uptake of substrates by oligopeptide transporters.

[0006] This invention provides the application of cannabidiol in the preparation of oligopeptide transporter inhibitors.

[0007] This invention also provides the application of cannabidiol in the preparation of non-substrate oligopeptide transporter inhibitors.

[0008] Preferably, the oligopeptide transporter includes PEPT1 and / or PEPT2.

[0009] This invention also provides the application of cannabidiol as a substrate for screening and evaluating hPEPTs oligopeptide transporters as a non-substrate inhibitor in the MDCK / hPEPTs cell model.

[0010] Preferably, the MDCK / hPEPTs cell model uses genetic engineering methods to stably express human oligopeptide transporters in Madin-Darby canine renal epithelial cells.

[0011] Preferably, the human oligopeptide transporter includes hPEPT1 and / or hPEPT2.

[0012] The present invention also provides the application of an oligopeptide transporter inhibitor in screening tool drugs for optimizing drug efficacy and / or reducing adverse drug reactions, wherein the oligopeptide transporter inhibitor is cannabidiol.

[0013] The present invention also provides an application of a non-substrate oligopeptide transporter inhibitor in high-throughput drug screening and / or target validation based on the MDCK / hPEPTs cell model, wherein the non-substrate oligopeptide transporter inhibitor is cannabidiol.

[0014] The present invention also provides an oligopeptide transporter inhibitor, wherein the oligopeptide transporter inhibitor comprises cannabidiol and excipients.

[0015] Preferably, the excipients include MES buffer solution.

[0016] This invention provides the application of cannabidiol in the preparation of oligopeptide transporter inhibitors. Membrane transporter inhibitors have proven to be indispensable pharmaceutical and tool compounds. Experimental results show that the cannabidiol of this invention is a non-substrate oligopeptide transporter inhibitor, capable of inhibiting the uptake of substrates by oligopeptide transporters.

[0017] Compared to competitive inhibitors (which possess both substrate and inhibitor functions) as tool drugs, they have the following key advantages:

[0018] 1. Avoid interference from its own transport and simplify experimental design: Since cannabidiol is not actively transported by the target uptake transporter, its concentration remains stable in transmembrane experiments in cell models without the need for additional correction of its own transport rate; while substrate inhibitors are substrates themselves (such as cephalexin), and their experimental systems require simultaneous monitoring of their uptake and efflux processes, which increases experimental complexity and may cause changes in the inhibitor concentration gradient due to their own transport, affecting the stability of the inhibitory effect.

[0019] 2. Enhanced specificity of inhibition: Cannabidiol, as a non-substrate oligopeptide transporter inhibitor, can target the non-substrate binding sites of transporters, reducing the risk of non-specific binding with other transporters or enzymes.

[0020] 3. Eliminate confounding factors in competitive inhibition: Cannabidiol, as a non-substrate inhibitor, does not compete with the target substrate for binding sites and can directly reflect the baseline activity of the transporter and its degree of inhibition in experiments, making the data easier to quantify; while competitive inhibitors, as substrates, may inhibit the transporter through competitive mechanisms, resulting in the assessment of inhibition efficiency being highly correlated with substrate concentration, requiring the use of complex kinetic models (such as Dixon plots) to distinguish the type of inhibition.

[0021] 4. Reproducibility and linear response of experimental data: As a non-substrate inhibitor, cannabidiol exhibits a more linear dose-dependent inhibitory effect, making it suitable for constructing standard curves or calculating IC50. 50 Value; while competitive inhibitors that also function as substrates may exhibit competitive inhibition at low concentrations, but show nonlinear effects at high concentrations due to transporter saturation, increasing the difficulty of data interpretation.

[0022] 5. Avoid competitive interference

[0023] Cannabidiol (CBD), as a non-substrate inhibitor, typically does not enter cells via transporters to exert its effects. Therefore, in control cells lacking transporter expression, its background activity is lower, allowing for clearer validation of target specificity. In contrast, substrate-inhibitor drugs may experience unstable inhibitory effects due to active uptake by transporters, resulting in fluctuating intracellular and extracellular concentrations over time. For example, initially high concentrations may inhibit transport, but as the drug is transported into the cell, the extracellular concentration decreases, weakening the inhibitory effect and affecting experimental reproducibility.

[0024] 6. Suitable for high-throughput screening (HTS): As a non-substrate inhibitor, cannabidiol's stable concentration-response characteristics make it suitable for large-scale screening, reducing false positive or false negative results; while competitive inhibitors are themselves taken up by transporters, and may mask the true inhibitory activity at high concentrations due to the saturation effect, increasing noise in HTS data.

[0025] The non-substrate oligopeptide transporter inhibitors described in this invention offer significant advantages over substrate inhibitors in in vitro applications by avoiding self-transportation interference, improving target specificity, simplifying experimental design, and enhancing data reliability. Their advantages are particularly evident in high-throughput screening and target validation in cell models, making them an ideal tool for optimizing in vitro pharmacological studies. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Figure 1 shows the inhibitory effect of CBD on the uptake of CEPH, UBEN, and Gly in the MDCK / hPEPT1 / 2 and MDCK-MOCK cell models provided by this invention; Mean ± SD, n = 3;

[0028] Figure 2 Figure showing the results of CBD concentration-dependent inhibition of UBEN active uptake in the MDCK / hPEPT1 / 2 cell model provided by this invention; Mean±SD, n=3. Detailed Implementation

[0029] This invention provides the application of cannabidiol in the preparation of oligopeptide transporter inhibitors. The results of the examples show that cannabidiol can inhibit oligopeptide transporters, specifically by inhibiting the uptake of the substrates cephalexin, ubenimex, and glycylsarcosine of PEPT1 and / or PEPT2.

[0030] This invention also provides the application of cannabidiol in the preparation of non-substrate oligopeptide transporter inhibitors. In specific embodiments, the oligopeptide transporters include PEPT1 and / or PEPT2. Existing MDCK / hPEPTs models all use competitive inhibitors that simultaneously possess substrate properties, lacking the discovery and use of non-substrate inhibitory tools that effectively inhibit PEPTs. The results of these examples indicate that cannabidiol is not a substrate of PEPT1 / 2. This invention discovers that cannabidiol is a non-substrate oligopeptide transporter inhibitor, capable of inhibiting the transport capacity of oligopeptide transporters PEPT1 and / or PEPT2 for oligopeptides and other substrates. The SLC15 family of proton-dependent oligopeptide transporters PEPT1 and PEPT2 play important roles in human and mammalian physiology and drug intestinal absorption and renal excretion. The discovery of PEPT1 and / or PEPT2 inhibitors in this invention promotes the development of related drugs.

[0031] This invention also provides the application of cannabidiol (CBD) as a non-substrate inhibitor in the MDCK / hPEPTs cell model for screening and evaluating substrates for hPEPTs oligopeptide transporters. In a specific embodiment, the MDCK / hPEPTs cell model uses genetic engineering methods to stably express human oligopeptide transporters in Madin-Darby canine kidney epithelial cells. In a specific embodiment, the human oligopeptide transporters include hPEPT1 and / or hPEPT2. Cannabidiol can act as a non-substrate inhibitor to inhibit oligopeptide transporters in the MDCK / hPEPTs cell model, and can be used to screen and evaluate substrates for hPEPTs oligopeptide transporters.

[0032] This invention also provides the application of an oligopeptide transporter inhibitor in screening tool drugs for optimizing drug efficacy and / or reducing adverse drug reactions, wherein the oligopeptide transporter inhibitor is cannabidiol. Cannabidiol can be used to prepare products that can screen tool drugs for optimizing drug efficacy and / or reducing adverse drug reactions.

[0033] This invention also provides the application of a non-substrate oligopeptide transporter inhibitor in high-throughput drug screening and / or target validation based on the MDCK / hPEPTs cell model, wherein the non-substrate oligopeptide transporter inhibitor is cannabidiol. Cannabidiol can be used as a non-substrate oligopeptide transporter inhibitor to prepare products for high-throughput drug screening and / or target validation based on the MDCK / hPEPTs cell model.

[0034] The present invention also provides an oligopeptide transporter inhibitor, which comprises cannabidiol and an excipient. In a specific embodiment, the excipient comprises MES buffer.

[0035] Terminology Explanation:

[0036] Cellular uptake experiment:

[0037] Cultured cells are co-incubated with a culture medium containing a specific substance, and cells are collected at different time points. The content of the substance in the cells is determined by techniques such as radioactive counting, fluorescence detection, and high-performance liquid chromatography (HPLC) to assess the cells' ability to take up the substance and the uptake kinetics.

[0038] MDCK cells, or Madin-Darby Canine Kidney cells, were isolated from the kidney tissue of adult female Cork dogs in 1958. It is an adherent epithelial cell line with typical epithelial cell morphology and polarity. In vitro, it forms a tight monolayer and exhibits excellent transmembrane transport and barrier properties. Due to its well-known origin, relatively simple culture conditions, and ease of gene transfection, MDCK cells are widely used in various biological research fields, particularly in drug development, where they are frequently used to evaluate drug transmembrane transport properties, study drug absorption mechanisms, and screen potential drug transporter substrates.

[0039] hPEPTs (human peptide transporters) are a class of proteins located on human cell membranes, primarily responsible for mediating the transmembrane transport of oligopeptides (usually composed of 2-6 amino acid residues). hPEPTs play a crucial role in the absorption of oligopeptide nutrients from food and the in vivo transport of certain peptide drugs. Different types of hPEPTs vary in tissue distribution and substrate specificity, recognizing and binding to specific oligopeptide substrates and utilizing a proton gradient-driven approach to transport oligopeptides into cells.

[0040] MDCK / hPEPTs cells: This cell model uses genetic engineering to introduce the gene encoding human oligopeptide transporters (hPEPTs) into MDCK cells, enabling them to stably express these transporters. These cells retain the favorable growth characteristics and epithelial cell polarity of MDCK cells while also possessing the function of human oligopeptide transporters. Therefore, MDCK / hPEPTs cells can be used to study the functional properties and substrate specificity of human oligopeptide transporters, assess the transmembrane transport of peptide drugs or other related compounds, and provide a more physiologically accurate in vitro experimental model for drug development and research on intestinal absorption mechanisms.

[0041] MDCK / hPEPTs cell model: The cell model constructed by transfecting MDCK cells with human PEPTs is a commonly used tool for studying PEPTs function and drug delivery mechanisms. This model can effectively simulate the activity and mode of action of PEPTs in vivo, providing a reliable in vitro experimental platform for screening and evaluating PEPTs inhibitors.

[0042] To further illustrate the present invention, the application of cannabidiol in oligopeptide transporter inhibitors provided by the present invention is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] Cannabidiol's inhibition of PEPT1 / 2 uptake:

[0045] Cell culture: MDCK cells expressing PEPT1 and PEPT2, as well as MDCK-MOCK, were cultured in DMEM medium containing 10% fetal bovine serum and penicillin-streptomycin (100 U / mL and 0.1 mg / mL)-purinemycin (500 ng / mL, used every other generation), and cultured in a cell culture incubator at 37°C containing 5% CO2.

[0046] Uptake experiment: MDCK-PEPT1, MDCK-PEPT2, and MDCK-MOCK cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded per well in a 24-well plate. When cell confluence reached 70%–80%, uptake assays were performed. Cells were washed twice with pre-warmed HBSS at 37°C, and then 0.5 mL of MES buffer (pH: 6.3) or MES buffer containing cannabidiol (CBD) (inhibitor group) was added to each well. The cells were pre-incubated at 37°C for 30 min, with three replicates per group. Aspirate the liquid from each well. Add 0.5 mL of MES buffer containing either the positive substrate (cephalosporin (CEPH) 200 μM / ubenimex (UBEN) 100 μM / glycylsarcosine (Gly) 100 μM) or the inhibitor group (CEPH 200 μM + CBD 100 μM / UBEN 100 μM + CBD 500 μM / Gly 100 μM + CBD 500 μM). Incubate at 37°C for 10 min. Aspirate the liquid from the wells, immediately add ice-cold HBSS to terminate the reaction, and wash three times. After air-drying, add 200 μL of pure water and lyse the cells by repeatedly freezing and thawing in liquid nitrogen three times. Collect the cell lysate, determine the protein content using the BCA method, and after protein precipitation, analyze the concentrations of CEPH, UBEN, and Gly by LC-MS / MS.

[0047] Protein concentration determination: according to the protein assay kit ( Protein concentration determination of cell samples was performed according to the BCA (Breast Cell Protein Acetate) instructions. Protein standards were prepared using purified water at concentrations ranging from 25 to 2000 μg / mL. 25 μL of standard or test sample was added to each well, followed by 200 μL of pre-prepared working solution. The mixture was thoroughly mixed by pipetting, and the microplate was sealed and incubated at 37°C on a shaker for 30 min. After incubation, the plate was cooled to room temperature, and absorbance was measured at 562 nm using a microplate reader. A standard curve was plotted, and the final protein concentration of the sample was calculated.

[0048] Data processing: Intracellular analyte concentrations were corrected by measuring the protein concentration in the cell lysate of each well.

[0049]

[0050] Calculate the analyte uptake rate (U) in cells using the following formula:

[0051]

[0052] In the formula:

[0053] C lysate —Drug concentration in cell lysate;

[0054] P—represents cellular protein content;

[0055] t — incubation time.

[0056] Calculate the uptake rate (UR) of the analyte in cells expressing the transporter using the following formula:

[0057]

[0058] Finally, the inhibition ratio (IR) is calculated according to the following formula to determine whether cellular uptake is inhibited:

[0059]

[0060] In the formula:

[0061] U withinhibitor —The average rate of drug uptake (in cells overexpressing transporters when selective inhibitors are available);

[0062] U mock with inhibitor —The average rate of drug uptake (MDCK-MOCK cells when selective inhibitors are available);

[0063] U withoutinhibitor —The average rate of drug uptake (in cells overexpressing the transporter when no selective inhibitor is available);

[0064] U mock without inhibitor —The average rate of drug uptake (MDCK-MOCK cells when there are no selective inhibitors).

[0065] According to the Technical Guidelines for Drug Interaction Studies issued by the National Medical Products Administration (NMPA) of China and the Food and Drug Administration (USFDA): when the uptake ratio (UR) of the test substance is ≥2 and the inhibition rate by the selective inhibitor is ≥50%, it is suggested that the test substance is the substrate of the transporter.

[0066] Results: CEPH, UBEN, and Gly were positive substrates for PEPT transporters. The inhibitory effects of CBD on CEPH, UBEN, and Gly are shown in Table 1 and 2. Figure 1 The uptake rates of the positive substrates CEPH, UBEN, and Gly in this model were 9–93%, demonstrating the effectiveness of the system. CBD inhibited the uptake of CEPH, UBEN, and Gly by PEPT1 / 2, suggesting that CBD is an inhibitor of PEPT1 / 2.

[0067] Table 1. Uptake of CEPH, UBEN, and Gly in MDCK / hPEPT1 / 2 and MDCK-MOCK cell models ( n=3)

[0068]

[0069]

[0070] -: Do not include CBD; +: Include CBD; / : Not applicable.

[0071] Tests to verify whether CBD is a substrate for hPEPT1 / 2.

[0072] After incubating CBD (100 μM) for 10 min in MDCK cell models expressing and not expressing hPEPT1 / 2, the intracellular concentrations of CEPH, UBEN, and Gly were measured. The results showed that the drug uptake rate in cells expressing the transporter was not significantly increased compared to MDCK-MOCK cells, suggesting that CBD is not a substrate for PEPT1 / 2. See Table 2 for detailed results.

[0073] Table 2. CBD uptake rates on MDCK cells expressing and not expressing hPEPT1 / 2.

[0074] Cell model Uptake rate (nmol / mg / min) UR MDCK-MOCK 1169.31±226.73 / MDCK-hPEPT1 987.88±224.50 0.8 MDCK-hPEPT2 1498.25±321.97 1.3

[0075] / indicates that it is not applicable.

[0076] Example 2

[0077] Concentration-dependent inhibition of UBEN uptake by PEPT1 / 2 by cannabidiol:

[0078] Cell culture: MDCK cells expressing PEPT1 and PEPT2, as well as MDCK-MOCK, were cultured in DMEM medium containing 10% fetal bovine serum and penicillin-streptomycin (100 U / mL and 0.1 mg / mL)-purinemycin (500 ng / mL, used every other generation), and cultured in a cell culture incubator at 37°C containing 5% CO2.

[0079] Uptake experiment: MDCK-PEPT1, MDCK-PEPT2, and MDCK-MOCK cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded per well in 24-well plates. When cell confluence reached 70%–80%, uptake assays were performed. Cells were washed twice with pre-warmed HBSS at 37°C, and then 0.5 mL of MES buffer (pH: 6.3) (without inhibitors) or MES buffer containing cannabidiol (CBD) (5, 10, 20, 50, 100, 200, 500, 1000, 2000 μM) was added to each well. Pre-incubation was performed at 37°C for 30 min, with three replicates per group. Aspirate the liquid from each well. Add 0.5 mL of MES buffer containing 100 μM of the positive substrate ubenimex (without inhibitor), 100 μM of ubenimex, and inhibitors (cannabidiol 5, 10, 20, 50, 100, 200, 500, 1000, 2000 μM) to each well. Incubate at 37°C for 10 min, then aspirate the liquid from the well. Immediately add ice-cold HBSS to terminate the reaction and wash three times. After air-drying, add 200 μL of pure water and lyse the cells by repeatedly freezing and thawing in liquid nitrogen three times. Collect the cell lysates, determine the protein content using the BCA method, and analyze the UBEN concentration by LC-MS / MS after protein precipitation.

[0080] The protein concentration determination and data processing methods are the same as in Example 1.

[0081] The results are as follows Figure 2 As shown, according to Figure 2 It is known that cannabidiol can inhibit the uptake of UBEN via PEPT1 and PEPT2 in a concentration-dependent manner.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of cannabidiol as a non-substrate inhibitor for screening the ability of hPEPTs oligopeptide transporter in substrate uptake in a MDCK / hPEPTs cell model.

2. Use according to claim 1, characterized in that, The MDCK / hPEPTs cell model utilizes a genetic engineering method to stably express a human oligopeptide transporter in Madin-Darby canine kidney epithelial cells.

3. Use according to claim 2, characterized in that, The human oligopeptide transporter includes hPEPT1 and / or hPEPT2.

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