Application of lipoic acid trisulfide

Abstract

The invention discloses application of lipoic acid trisulfide, and belongs to the technical field of medicines. The invention discloses application of lipoic acid trisulfide or salt, isomer and derivative thereof as a drug penetration enhancer or / and an efflux inhibitor. The invention also discloses a medicinal preparation which comprises a medicine penetration enhancer or / and an efflux inhibitor, and the medicine penetration enhancer or / and the efflux inhibitor is lipoic acid trisulfide or salt and isomer thereof. The lipoic acid trisulfide is scientific in design and ingenious in conception, it is found for the first time that the lipoic acid trisulfide or the salt, isomer and derivative thereof not only can improve the drug permeability, but also shows an excellent drug excretion inhibition effect, and has a dual regulation function. As a drug penetration enhancer or / and an excretion inhibitor, the compound can significantly improve the bioavailability of drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of thioctic acid trisulfide or its salts, stereoisomers, and derivatives. Background Technology

[0002] Oral administration remains the most commonly used method of drug delivery due to its convenience and high patient compliance. However, the bioavailability of many drugs is significantly low after oral administration, becoming a key bottleneck restricting their clinical efficacy. The main reasons include poor drug permeability in the gastrointestinal tract and the ease with which drugs are recognized and pumped back into the intestinal lumen by efflux transport proteins (such as P-glycoprotein) expressed on the surface of intestinal epithelial cells, preventing them from effectively entering the systemic circulation and thus severely limiting their therapeutic effects.

[0003] To improve the bioavailability of low-permeability drugs, permeation enhancers (PEs), such as sodium caprylate (C10) and N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC), have been gradually introduced into the pharmaceutical field and have become one of the mainstream technologies for solving the problem of low permeability. Their mechanism of action mainly involves reversibly altering the structure of the intestinal epithelial cell membrane (transcellular pathway) and / or regulating the permeability of tight junctions between cells (paracellular pathway), thereby promoting drug absorption in the intestine. The application of PEs is particularly common in oral delivery systems for peptide and protein drugs.

[0004] However, existing technologies still have significant limitations: while efflux agents (PEs) can improve drug permeability in the intestinal mucosa, they cannot address the problem of drug efflux mediated by efflux transporters. As mentioned earlier, highly expressed efflux transporters on the surface of gastrointestinal epithelial cells are another key factor affecting drug absorption. Even if a drug successfully penetrates into the intestinal epithelial cells under the action of PEs, a large number of efflux transporters will actively expel it into the intestinal lumen, further reducing the amount of drug entering the bloodstream and resulting in a very limited improvement in bioavailability. Currently, even oral peptide / protein formulations using PEs generally have a bioavailability of less than 2%, far from meeting the needs of effective clinical treatment. Furthermore, none of the PEs reported in existing technologies have shown significant efflux inhibition, failing to simultaneously address both the low permeability and efflux pumping barriers.

[0005] Therefore, for drugs with low permeability, especially those easily recognized by efflux transport proteins, developing a novel absorption-enhancing excipient that can both enhance cell permeability and inhibit efflux has become a key research direction for improving their oral bioavailability. Summary of the Invention

[0006] The purpose of this invention is to provide the application of thioctic acid trisulfide or its salts, isomers, and derivatives as drug permeation enhancers and / or efflux inhibitors; it has the dual function of enhancing cell permeability and inhibiting efflux, and is applicable not only to low-permeability chemical small molecules and biological macromolecules, but also to nanomedicines, which can significantly improve the bioavailability of low-permeability drugs and have good biological safety.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses the use of lipoic acid trisulfide or its salts, isomers, and derivatives in the preparation of drug penetration enhancers and / or efflux inhibitors.

[0008] In some embodiments of the present invention, lipoic acid trisulfide and its derivatives include at least one of the compounds with the following structures: .

[0009] The compound shown in Formula I can be freely converted into the compound shown in Formula II or Formula III in vivo.

[0010] Dihydrolipoic acid trisulfide or its salts can be interconverted with lipoic acid trisulfide or its salts in the body.

[0011] The compounds shown in Formulas IV, V, and VI are all homologues of the compound in Formula I.

[0012] In some embodiments of the present invention, the salt of thioctic trisulfide includes at least one of sodium salt, potassium salt, calcium salt, and magnesium salt.

[0013] The isomers described in this invention include racemic, levorotatory, and dextrorotatory isomers.

[0014] In some embodiments of the present invention, the drug includes a hypotonic drug.

[0015] In some embodiments of the present invention, the drug includes at least one of low-permeability small molecule chemical drugs, peptide drugs, protein drugs, and nanomedicines.

[0016] A second aspect of the present invention discloses a pharmaceutical formulation comprising a drug penetration enhancer and / or an efflux inhibitor, wherein the drug penetration enhancer and / or the efflux inhibitor is thioctic acid trisulfide or its salt or isomer.

[0017] In some embodiments of the present invention, the mass ratio of thioctic acid trisulfide or its salt, isomer to drug is 1:1 to 1000:1; preferably 5:1 to 500:1.

[0018] In some embodiments of the present invention, the formulation includes an oral formulation and an enteric formulation.

[0019] In some embodiments of the present invention, the orally administered formulation includes gastric-coated formulations, enteric-coated formulations, and oral formulations; Preferably, the gastric-soluble formulation includes gastric-soluble solid dosage forms and gastric-soluble liquid dosage forms; Preferably, the enteric-coated formulation includes enteric-coated solid dosage forms and enteric-coated liquid dosage forms; Preferably, the oral preparations include orally disintegrating tablets, lozenges, sublingual tablets, oral adhesive tablets, oral sprays, and mouthwashes.

[0020] In some embodiments of the present invention, the enteric administration preparations include suppositories and enemas.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention is scientifically designed and ingeniously conceived. It is the first discovery that lipoic acid trisulfide or its salts, isomers, and derivatives can not only enhance drug permeability but also exhibit excellent drug efflux inhibition, thus possessing a dual regulatory function. As a drug permeability enhancer and / or efflux inhibitor, it can significantly improve drug bioavailability.

[0022] Lipoic acid trisulfide effectively enhances cell membrane permeability and promotes intracellular absorption of drug molecules. Its mechanism of action includes altering epithelial cell membrane structure and reversibly opening tight junctions between cells, thereby significantly promoting bypass transport of drugs. Furthermore, lipoic acid trisulfide downregulates the expression of tight junction-related proteins, further enhancing the permeation-enhancing effect on low-permeability molecules; this effect is concentration-dependent, meaning it increases with increasing concentration.

[0023] Simultaneously, lipoic acid trisulfide can significantly reduce the expression level of extracellular proteins, effectively reducing drug efflux and increasing intracellular drug retention. This inhibitory effect also becomes more pronounced with increasing concentration. Therefore, lipoic acid trisulfide possesses the dual advantages of promoting drug uptake and inhibiting efflux at the cellular level.

[0024] Animal experiments have shown that lipoic acid trisulfide also exhibits excellent penetration-enhancing performance in vivo, further validating its application potential as a highly efficient penetration enhancer. Attached Figure Description

[0025] Appendix Figure 1 The figures show the safety results of sodium thiooctanoate trisulfide, sodium octanoate, and N-[8-(2-hydroxybenzoyl)amino]octanoate on intestinal epithelial cells at different time points; the top left figure shows the results after 2 hours, the top right figure shows the results after 4 hours, the bottom left figure shows the results after 12 hours, and the bottom right figure shows the results after 24 hours.

[0026] Appendix Figure 2The figure shows the experimental results of the effect of sodium thioctic acid trisulfide on the permeability of intestinal epithelial cell membranes.

[0027] Appendix Figure 3 The graph shows the effect and recovery results of sodium thioctic trisulfide on cell resistance; the vertical axis “%initial TEER” represents the percentage of the initial TEER value, and the horizontal axis “Time (min)” represents the time (minutes).

[0028] Appendix Figure 4 The figure shows the results of the investigation on the degree of enhancement of sodium luciferin uptake by cells by sodium lipoic acid trisulfide.

[0029] Appendix Figure 5 The figure shows the results of the investigation on the degree of enhancement of FD4 molecule uptake by sodium thioctic acid trisulfide.

[0030] Appendix Figure 6 The figure shows the results of the investigation on the degree of enhancement of FD10 molecule uptake by sodium thioctic acid trisulfide.

[0031] Appendix Figure 7 The figures show the effects of sodium thioctic acid trisulfide on the permeability and absorption enhancement of cefoxitin sodium in cell monolayers. The left figure shows the effect on permeability, and the right figure shows the absorption enhancement.

[0032] Appendix Figure 8 The figures show the effects of sodium thioctic acid trisulfide on the permeability of simulated protein molecules in a cell monolayer and the degree of absorption enhancement. The left figure shows the effect on permeability, and the right figure shows the degree of absorption enhancement.

[0033] Appendix Figure 9 The images show a comparison of the uptake results of FD4 and FD10 by sodium lipoic acid trisulfide and sodium lipoic acid. The top left image shows a comparison of the uptake results of FD4 by sodium lipoic acid trisulfide and sodium lipoic acid. The bottom left image shows a comparison of the quantitative results of the relative fluorescence intensity of FD4 uptake by sodium lipoic acid trisulfide and sodium lipoic acid. The top right image shows a comparison of the uptake results of FD10 by sodium lipoic acid trisulfide and sodium lipoic acid. The bottom right image shows a comparison of the quantitative results of the relative fluorescence intensity of FD10 uptake by sodium lipoic acid trisulfide and sodium lipoic acid.

[0034] Appendix Figure 10 The figure shows the results of the investigation on the enhanced uptake of negatively charged nanoparticles by sodium thioctic acid trisulfide.

[0035] Appendix Figure 11 The top figure shows the results of the investigation on the effects of sodium lipoic acid trisulfide on the expression of intercellular tight junction proteins and efflux proteins; the bottom figure shows the quantitative results of the relative expression levels of intercellular tight junction proteins and efflux proteins by sodium lipoic acid trisulfide.

[0036] Appendix Figure 12 The figure shows the results of the investigation on the inhibitory effect of sodium thioctic acid trisulfide and sodium thioctic acid on the efflux of doxorubicin hydrochloride. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0038] The present invention will now be described in detail.

[0039] This invention first discloses the application of lipoic acid trisulfides or their salts, isomers, and derivatives shown in Formulas I to VI in the preparation of drug penetration enhancers and / or efflux inhibitors: .

[0040] The compound shown in Formula I can be freely converted into the compound shown in Formula II or Formula III in vivo. The compounds shown in Formula IV, Formula V and Formula VI are all homologues of the compound shown in Formula I.

[0041] The present invention further discloses a pharmaceutical formulation comprising any one of the compounds shown in Formulas I to VI or their salts, isomers, or derivatives, wherein the compound is specifically used as a drug penetration enhancer and / or efflux inhibitor.

[0042] The drugs mentioned above all belong to the category of low-permeability drugs, which can specifically include low-permeability small molecule chemical drugs, peptide drugs, protein drugs, and nanomedicines.

[0043] In some preferred embodiments of the present invention, for low-permeability small molecule chemical drugs, cefoxitin, doxorubicin, methotrexate, acyclovir, metformin, cimetidine, and amphotericin B can be selected, and one of them can be used alone or in combination; for polypeptide drugs, at least one of low molecular weight heparin, calcitonin, and semaglutide can preferably be selected and included in the formulation system; for protein drugs, at least one of protamine, adrenocorticotropic hormone, and insulin is preferred; as for nanomedicines, any one or more of positively charged nanomedicines, electrically neutral nanomedicines, and negatively charged nanomedicines can preferably be selected.

[0044] In the pharmaceutical formulation of the present invention, the mass ratio of thioctic acid trisulfide or its salt, isomer, derivative and drug is set to 1:1 to 1000:1, wherein a more preferred ratio range is 5:1 to 500:1.

[0045] Meanwhile, the drug formulations of the present invention are mainly administered in two forms: oral administration and enteral administration.

[0046] Oral medications can be further divided into gastric-soluble, enteric-soluble, and oral medications: gastric-soluble medications specifically include gastric-soluble solid and liquid dosage forms; enteric-soluble medications include enteric-soluble solid and liquid dosage forms; and oral medications can take the form of orally disintegrating tablets, lozenges, sublingual tablets, oral adhesive tablets, oral sprays, mouthwashes, etc.

[0047] The specific types of enteral medications mainly include suppositories and enemas, which can be flexibly selected according to actual medication needs.

[0048] In the embodiments of this invention, %w / v refers to the mass-volume percentage concentration. For example, a 4% (w / v) paraformaldehyde solution means that every 100 mL of solution contains 4 g of paraformaldehyde.

[0049] Example 1 This embodiment discloses a systematic evaluation experiment on the biosafety of sodium lipoic acid trisulfide (LATNa) on intestinal epithelial cells Caco-2 at different concentrations and different treatment times. The specific steps are as follows: (1) Cell seeding: Caco-2 cells in good growth condition and in logarithmic growth phase were seeded into 96-well plates, DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) was added, and the cells were cultured in a cell culture incubator at 37°C, 5% CO2 and saturated humidity for 16 hours to allow the cells to adhere completely and recover to their normal morphology.

[0050] (2) Preparation of test samples and control samples: Using serum-free DMEM medium as solvent, LATNa solutions with concentrations of 1 mM, 2 mM, 5 mM, 8 mM, 10 mM and 20 mM were prepared respectively; at the same time, sodium decanoate (C10) solution and sodium N-[8-(2-hydroxybenzoyl)amino]octanoate (SNAC) solution with concentrations of 10 mM were prepared as positive control permeation enhancers.

[0051] (3) Drug treatment: Discard the original culture medium in the 96-well plate, wash gently once with PBS preheated to 37°C, and add 200 µL of LATNa solution, C10 solution and SNAC solution of different concentrations as mentioned above, with 6 replicates per group; at the same time, a blank control group (only serum-free DMEM is added) is set up. The 96-well plate is placed in a 37°C, 5% CO2 incubator for 2 hours, 4 hours, 12 hours and 24 hours respectively.

[0052] (4) Cell viability detection: After incubation to the preset time point, discard the drug solution, wash gently once with PBS, add 200 µL of serum-free DMEM medium containing 0.5 mg / mL MTT to each well, and continue incubation for 4 hours; then discard the MTT solution, add 150 µL of DMSO to each well, and shake on a shaker at low speed for 10 minutes to fully dissolve the formazan crystals. Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 490 nm, and calculate the relative cell viability using the formula "Cell viability (%) = (OD value of experimental group / OD value of blank control group) × 100%".

[0053] (5) Experimental results: such as Figure 1 As shown, under short-term treatment conditions of 2 hours and 4 hours, even with a LATNa concentration as high as 10 mM, the survival rate of Caco-2 cells remained above 80%, which was significantly better than that of the C10 and SNAC groups at the same concentration; even under long-term treatment conditions of 12 hours and 24 hours, LATNa was significantly less cytotoxic than commercial permeation enhancers.

[0054] (6) Conclusion: The above results show that LATNa has significantly lower toxicity to Caco-2 intestinal epithelial cells in the 10 mM concentration range and within a 2-24 hour action time than existing commercial permeation enhancers sodium decanoate and SNAC, demonstrating excellent biosafety and can be further used in the development of oral permeation enhancers.

[0055] Example 2 The amount of lactate dehydrogenase (LDH) released from the cytoplasm into the culture medium is considered a quantitative indicator of Caco-2 cell membrane integrity. This example uses "intracellular LDH release rate" as the endpoint to systematically evaluate the effects of different concentrations of LATNa on intestinal epithelial cell membrane permeability. The specific steps are as follows: (1) Cell seeding and pretreatment Caco-2 cells in good growth condition and in the logarithmic growth phase were seeded into 96-well plates, DMEM complete medium was added, and the cells were cultured at 37°C and 5% CO2 for 16 h. After the cells were completely attached, the original culture medium was discarded, and the cells were gently washed once with HBSS preheated to 37°C.

[0056] (2) Preparation of test sample and control solution Using HBSS as the solvent, LATNa solutions with concentrations of 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, and 20 mM were prepared. At the same time, 10 mM C10 solution and 10 mM SNAC solution were prepared as positive osmosis-promoting controls. A negative control (pure HBSS) and a positive membrane destruction control (0.1% w / v Triton-X-100, Tx) were also set up.

[0057] (3) Drug processing and centrifugation Add 200 μL of LATNa solution, C10 solution, SNAC solution, Tx solution or HBSS solution of the above concentrations to each well of a 96-well plate and incubate at 37°C and 5% CO2 for 2 h. After treatment, place the 96-well plate in a multi-well plate centrifuge and centrifuge at 25°C and 3000 rpm for 5 min to allow the detached cells to settle to the bottom. Then carefully aspirate 50 μL of supernatant from each well and transfer it to a new 96-well plate.

[0058] (4) LDH activity assay Add an equal volume of LDH detection working solution to a new 96-well plate containing the supernatant, following the instructions for the LDH detection kit (Beijing Regen Biotechnology Co., Ltd., TE0159). Incubate at 37°C in the dark for 30 min, then measure the absorbance (OD) at 440 nm using a microplate reader. 440 ); with the LDH release amount of group Tx as the 100% release baseline, calculate the relative LDH release rate (%) of each group.

[0059] (5) Experimental results like Figure 2 As shown, as the LATNa concentration increased from 1 mM to 20 mM, the LDH activity in the supernatant increased in a dose-dependent manner, indicating that the cell membrane permeability gradually increased. At the same 10 mM concentration, the LDH release in the LATNa group was similar to that in the commercial permeation enhancer C10 group, while the SNAC group basically overlapped with the negative control group, and no obvious LDH leakage was observed, indicating that it had almost no effect on membrane integrity.

[0060] (6) Conclusion The above results confirm that LATNa can dose-dependently increase the permeability of Caco-2 cell membranes and significantly promote the migration of intracellular LDH to the extracellular space, thus providing a structural basis for improving the transmembrane transport of macromolecules or poorly absorbed drugs and showing good prospects for permeation-enhancing applications.

[0061] Example 3 Cellular monolayer electrical resistance (TEER) is the gold standard for evaluating the integrity of the intestinal epithelial barrier and the opening and closing status of tight junctions between cells. This embodiment systematically examines the reversible regulatory ability of LATNa on tight junctions between Caco-2 cells by dynamically monitoring TEER values. The specific scheme is as follows: (1) Cell seeding and differentiation Healthy Caco-2 cells in logarithmic growth phase were seeded into 12-well Transwell chambers (0.4 μm pore size, polycarbonate PC membrane). 500 μL of DMEM complete medium was added to the upper chamber (AP side), and 1.5 mL of the same DMEM complete medium was added to the lower chamber (BL side). The chambers were incubated at 37°C, 5% CO2, and saturated humidity for 18–21 days. The medium was changed every other day for the first 7 days, and daily thereafter. TEER values ​​and apparent permeability coefficients (Papp) of sodium fluorescein were measured periodically. When TEER ≥ 400 Ω·cm... 2 And the Papp content of sodium fluorescein is ≤ 5×10 -7 When the flow rate reaches cm / s, the cells are considered to have formed a complete and compact monolayer, which can be used for subsequent experiments.

[0062] (2) Pre-experimental equilibrium One hour before the experiment, both sides of the Transwell medium were replaced with Ca-free medium. 2+ / Mg 2+ The HBSS was incubated at 37 °C for 30 min to remove exogenous ion interference; TEER was measured once before and after equilibration to confirm baseline stability and good monolayer integrity.

[0063] (3) LATNa treatment and TEER dynamic monitoring Discard the HBSS. Add 500 μL of HBSS solution containing LATNa (concentration gradient: 1, 2, 5, 8, 10, 20 mM, prepared according to the method in Example 2) to the AP side, and add 1.5 mL of blank HBSS to the BL side; a negative control (HBSS only) is also set up. During the period from 0 to 120 min, administer Millicell every 20 min. @ Use an ERS 3.0 resistance meter to measure TEER once and record the resistance change curve.

[0064] (4) Reversible recovery test After 120 min, the drug-containing HBSS was discarded, and both sides were gently washed twice with preheated DMEM complete medium at 37℃. Fresh DMEM was then added, and the culture was continued for 24 h. TEER was measured again to assess the tight junction reconstitution ability.

[0065] (5) Results and Judgments like Figure 3As shown, LATNa significantly reduced TEER within 20 min, and the reduction was concentration-dependent; in the 20 mM group, TEER decreased by approximately 60% at 120 min, indicating that it can rapidly open cellular bypass pathways. When LATNa ≤ 10 mM, TEER recovered to more than 85% of baseline within 24 h, suggesting that tight junction opening and closing is reversible; when LATNa > 10 mM, the recovery rate was still greater than 70% at 24 h, indicating that although barrier function repair is delayed at high concentrations, it can still be largely restored.

[0066] (6) Conclusion LATNa can reversibly reduce the TEER of the Caco-2 monolayer in a concentration-dependent manner within 2 hours, rapidly and transiently opening tight junctions between cells and providing a transient permeation window for bypass drug delivery. Within the concentration range of ≤10 mM, the barrier function can be basically fully restored within 24 hours, meeting the clinical demand for permeation enhancers that are "rapid onset and reversible".

[0067] Example 4 To visually verify the enhancing effect of LATNa on the uptake capacity of Caco-2 cells, this embodiment uses fluorescence imaging to systematically investigate the promoting effect of LATNa on the endocytosis of three fluorescently labeled model molecules—fluorescein (MW376.27), FD4 (FITC-Dextran-4 kDa), and FD10 (FITC-Dextran-10 kDa)—at different concentrations. Among them, sodium fluorescein is a classic mimic of low-permeability small molecule drugs (see Sam Maher et al., Advanced Drug Delivery Reviews, 2016, Vol. 106, Chapter B, pp. 277-319); FD4 and FD10 are used to mimic peptide drugs such as GLP-1 receptor agonists and protein drugs, respectively (see Huyen Tran et al., Molecular Pharmaceutics, 2023, Vol. 20, Issue 2, pp. 924-941 and Angelika et al., Translational Stroke Research, 2011, Vol. 2, pp. 106-111). The specific experimental procedure is as follows: (1) Cell seeding and pretreatment Caco-2 cells in good condition and in the logarithmic growth phase were seeded into 12-well plates, DMEM complete medium was added, and the plates were incubated at 37°C and 5% CO2 for 48 h. The old culture medium was discarded, and the cells were gently washed twice with PBS at 37°C to remove residual serum and metabolites.

[0068] (2) Preparation and dosing of test solution Using serum-free DMEM as solvent, mixed solutions containing 200 μg / mL of fluorescent model molecules (fluorescein sodium, FD4, or FD10) and LATNa concentrations of 0, 1, 2, 5, 10, and 20 mM were prepared. 1 mL of the above solution was added to each well. The blank control group contained no LATNa. The 12-well plate was incubated at 37°C in a 5% CO2 incubator for 1 h.

[0069] (3) Cell fixation and nuclear staining Discard the drug solution in the wells and wash three times with PBS; add 500 μL of 4% (w / v) paraformaldehyde solution to each well and fix at room temperature for 15 min; wash three more times with PBS, add 200 μL of DAPI staining solution to each well and stain at room temperature in the dark for 10 min; finally wash three times with PBS for 3 min each time, and finally cover the cells with an appropriate amount of PBS for fluorescence observation.

[0070] (4) Fluorescence microscopy Five fields of view images were randomly acquired using an inverted fluorescence microscope.

[0071] (5) Experimental results like Figure 4 , Figure 5 and Figure 6 As shown: only weak intracellular fluorescence signals were detected in the blank group, indicating that Caco-2 cells had extremely low background uptake of the three model molecules; when the LATNa concentration was ≥ 1 mM, the intracellular fluorescence signal was significantly enhanced, and showed a dose-dependent increasing trend with increasing concentration.

[0072] (6) Conclusion The above results indicate that LATNa can significantly and dose-dependently enhance the uptake of small molecules, peptides, and protein model molecules by Caco-2 cells, providing direct cellular evidence for its use as an oral penetration enhancer.

[0073] Example 5 This embodiment systematically investigated the regulatory ability of LATNa on the permeability of Caco-2 cell monolayers. Cefoxitin Sodium (CS, molecular weight 449.4) was used as a low-permeability small molecule drug model, and its apparent permeability coefficient (Papp) was measured to evaluate the permeation-enhancing effect of LATNa. The specific experimental procedure is as follows: (1) Preparation of cell monolayers Caco-2 cells were seeded into 12-well Transwell chambers according to the method described in Example 3 and cultured continuously until a dense cell monolayer was formed before they could be used for osmosis experiments.

[0074] (2) Preparation of test solution With no Ca 2+ / Mg 2+ Using HBSS as the solvent, a series of mixed solutions containing 200 μg / mL CS and different concentrations (0, 1, 2, 5, 10, 20 mM) of LATNa were prepared. Simultaneously, CS solutions containing 10 mM C10, SNAC, or sodium lipoate (LANa) were prepared as positive osmosis-enhancing controls. All solutions were filtered through a 0.22 µm microporous membrane and preheated to 37°C before use.

[0075] (3) Two-way drug administration and sampling Discard the original Transwell medium and wash twice with HBSS at 37°C. Add 500 µL of drug-containing mixed solution to the AP side and 1.5 mL of blank HBSS to the BL side. Incubate at 37°C with 5% CO2. Accurately pipette 100 µL from the BL side at 120 min. Centrifuge the sample at 4°C and 10,000 rpm for 5 min, and collect the supernatant for analysis.

[0076] (4) Drug concentration determination and Papp calculation CS concentration was determined by high-performance liquid chromatography (HPLC): a C18 column (4.6 × 250 mm, 5 µm), a mobile phase of water-acetonitrile-glacial acetic acid (81:19:1, v / v / v), a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, and a column temperature of 30 °C. Papp was calculated using the formula Papp = (dQ / dt) / (A·C0), where dQ / dt is the transepithelial transport rate (mol·s⁻¹). -1 A is the surface area of ​​a cell monolayer, calculated at 1.12 cm². 2 C0 is the initial CS concentration (mol / mL) on the AP side.

[0077] (5) Experimental results like Figure 7 As shown: the Papp value of the blank group (0 mM LATNa) CS was (0.62 ± 0.07) × 10⁻⁶. -7 The velocity (cm / s) is consistent with the characteristics of low-permeability drugs; LATNa increases CS permeability in a concentration-dependent manner, with Papp increasing to (54.83 ± 3.51) × 10 at 10 mM. -7 cm / s, with a permeability enhancement ratio >90 times; significantly better than the C10, SNAC and LANa groups at the same concentration (approximately 58, 37 and 45 times, respectively, p < 0.001).

[0078] (6) Conclusion LATNa can significantly increase the transmembrane transport rate of low-permeability small molecule cefoxitin sodium within 120 min in a concentration-dependent manner. A 10 mM solution can achieve a permeation enhancement of more than 90 times, and a 20 mM solution can enhance the permeation by more than 120 times, demonstrating its excellent efficacy as a permeation enhancer for oral small molecule chemical drugs.

[0079] Example 6 This embodiment systematically evaluated the permeation-enhancing effect of LATNa on the transepithelial transport of protein drugs. FD10 was selected as a protein drug model, and the apparent permeability coefficient (Papp) of FD10 in the presence of different concentrations of LATNa was measured to quantitatively evaluate the permeation-enhancing effect of LATNa. The specific procedure is as follows: (1) Preparation of cell monolayers Caco-2 cells were seeded into 12-well Transwell chambers according to the method described in Example 3 and cultured until a dense cell monolayer was formed before subsequent permeation experiments could be performed.

[0080] (2) Preparation of test solution With no Ca 2+ / Mg 2+ Using HBSS as the solvent, a series of mixed solutions containing 200 μg / mL FD10 and different concentrations (0, 1, 2, 5, 10, 20 mM) of LATNa were prepared. Simultaneously, FD10 solutions containing 10 mM C10, SNAC, or sodium lipoic acid (LANa) were prepared as positive osmosis-enhancing controls. All solutions were filtered through a 0.22 µm microporous membrane and preheated to 37°C before use.

[0081] (3) Two-way drug administration and sampling Discard the original Transwell medium and wash twice with HBSS at 37°C. Add 500 μL of drug-containing mixed solution to the AP side and 1.5 mL of blank HBSS to the BL side. Incubate at 37°C in a 5% CO2 incubator. At 120 min, accurately pipette 100 μL from the BL side. Centrifuge the sample and collect the supernatant for analysis.

[0082] (4) FD10 concentration determination FD10 concentration was determined using a fluorescence microplate reader: excitation wavelength 488 nm, emission wavelength 530 nm, slit width 5 nm; a standard curve was plotted using a series of FD10 standard solutions (linear range 0.1 ~ 400 μg / mL, R0). 2 ≥ 0.999), quantified using the external standard method. Calculated according to the formula Papp = (dQ / dt) / (A·C0) in Example 5.

[0083] (5) Experimental results like Figure 8 As shown: The Papp value of FD10 in the blank group (0 mM LATNa) was (0.53 ± 0.27) × 10⁻⁶. -8 The permeability of LATNa is consistent with the low permeability characteristics of high molecular weight markers. LATNa concentration-dependently improves the permeability of FD10. At 1 mM, Papp is increased by about 6 times, at 10 mM it is increased by about 16 times, and at 20 mM LATNa the permeability enhancement ratio is greater than 55 times, which is significantly better than the C10, SNAC and LANa groups at the same concentration (about 7, 3 and 11 times, respectively, p < 0.001).

[0084] (6) Conclusion LATNa significantly enhanced the trans-monolayer transport rate of the protein model molecule FD10 within 120 min in a concentration-dependent manner, with an enhancement factor greater than 50 at 20 mM. This indicates that LATNa has excellent penetration-enhancing effects on protein and peptide drugs (such as protamine, adrenocorticotropic hormone, and insulin), making it suitable for the development of oral protein drug delivery systems.

[0085] Example 7 This example compares the differences in enhancing transmembrane uptake of drugs in a biomolecular model by LATNa and LANa. The specific experimental steps are as follows: (1) Cell seeding and pretreatment Following the method in Example 4, Caco-2 cells were seeded into 12-well plates and cultured until the confluence was approximately 50%. The old culture medium was then discarded, and the cells were gently washed twice with PBS at 37°C.

[0086] (2) Preparation and dosing of test solution Using serum-free DMEM as a solvent, the following mixed solutions were prepared respectively: LATNa 5 mM + FD4 / FD10 200 μg / mL; LANa 5 mM + FD4 / FD10 200 μg / mL; LANa 10 mM + FD4 / FD10 200 μg / mL.

[0087] Add 1 mL of the above mixture to each well. The blank control does not contain any osmosis enhancer. All groups are incubated at 37°C and 5% CO2 for 1 h.

[0088] (3) Cell fixation and nuclear staining After incubation, discard the drug solution and wash three times with PBS; add 500 μL of 4% (w / v) paraformaldehyde per well and fix at room temperature for 15 min; wash three more times with PBS, add 200 μL of DAPI staining solution per well and stain at room temperature in the dark for 10 min; finally wash three times with PBS and retain an appropriate amount of PBS to cover the cells.

[0089] (4) Fluorescence microscopy and semi-quantitative imaging Five fields of view were randomly acquired using an inverted fluorescence microscope (FITC and DAPI channels). ImageJ was used to calculate the intracellular fluorescence integral density, and the relative uptake fold was calculated based on the blank control group.

[0090] (5) Experimental results like Figure 9 As shown, the relative uptake multiples of FD4 and FD10 in the 5 mM LATNa group were 2.0 times and 2.8 times that in the LANa group at the same concentration, respectively; even when the LANa concentration was doubled to 10 mM, its uptake capacity was still significantly lower than that of 5 mM LATNa (p < 0.05).

[0091] (6) Conclusion Under the same concentration conditions, LATNa significantly enhanced the cellular uptake of the two biomacromolecules FD4 / FD10 compared to LANa. Even when the concentration of LANa was increased to 10 mM, its uptake-enhancing effect was still less than that of 5 mM LATNa, indicating that LATNa has a superior ability to promote transmembrane transport of macromolecules and can be given priority as a candidate for oral biomacromolecule permeation enhancer.

[0092] Example 8 This embodiment investigated the dose-dependent enhancement effect of LATNa on the transmembrane uptake of negatively charged nanomedicines, and the specific scheme is as follows: (1) Cell seeding and pretreatment Caco-2 cells in good condition and in the logarithmic growth phase were seeded into 12-well plates, DMEM complete medium was added, and the plates were incubated at 37°C in a 5% CO2 incubator for 48 h. The old culture medium was discarded, and the cells were gently washed twice with 37°C PBS to remove residual serum and metabolites.

[0093] (2) Preparation and dosing of test solution Using serum-free DMEM as solvent, mixed solutions containing 1000 μg / mL coumarin-6-labeled lipoic acid nanoparticles (C6@cLANs) with LATNa concentrations of 0, 1, 2, 5, 10, and 20 mM were prepared; the blank control group contained no LATNa. 1 mL of the above mixed solution was added to each well, and the wells were incubated for 1 h.

[0094] (3) Cell fixation and nuclear staining Discard the drug solution in the wells and wash three times with PBS; add 500 μL of 4% (w / v) paraformaldehyde per well and fix at room temperature for 15 min; wash three times with PBS, add 200 μL of DAPI staining solution per well and stain at room temperature in the dark for 10 min; finally wash three times with PBS and leave an appropriate amount of PBS to cover the cells.

[0095] (4) Fluorescence microscopy and semi-quantitative imaging Five fields of view were randomly acquired using an inverted fluorescence microscope (FITC channel and DAPI channel) to observe the fluorescence intensity.

[0096] (5) Experimental results As shown in Figure 10: only weak intracellular fluorescence was detected in the blank group within 1 h. When the LATNa concentration was ≥ 2 mM, the intracellular fluorescence signal was significantly enhanced, showing a typical dose-response relationship.

[0097] (6) Conclusion LATNa significantly and dose-dependently increased the uptake of negatively charged nanomedicine / carrier C6@cLANs by Caco-2 cells, with a significant uptake-promoting effect observed at 2 mM, demonstrating its excellent ability to promote transmembrane transport of negatively charged nanomedicines. This suggests its potential for further development of oral negatively charged nanodelivery systems.

[0098] Example 9 This embodiment investigated the regulatory role of LATNa on the expression levels of key proteins in Caco-2 monolayer tight junctions (TJs) and efflux pumps (P-gp). The specific steps are as follows: (1) Cell processing and protein sample preparation Caco-2 cells were loaded at a rate of 2 × 10⁻⁶. 6 Cells were seeded in 6 cm culture dishes and cultured at 37°C with 5% CO2 until confluence ≥ 90%. The culture medium was discarded, and blank DMEM-prepared LATNa solution (final concentrations of 0, 1, 2, 5, 8, 10, and 20 mM) was added. The cells were incubated at 37°C for 2 h. The solution was discarded, and the cells were washed three times with PBS. Cells were collected using a cell scraper, centrifuged at 1200 rpm for 5 min at 4°C, and the precipitate was used to extract total protein using the RIPA (containing 1% PMSF) method. Quantification was performed using the BCA method. Store at 80℃ until use.

[0099] (2) Western blot detection 20 μg of total protein was subjected to 10% SDS-PAGE electrophoresis, followed by wet transfer to a membrane (PVDF, 0.45 μm), and then blocked with 5% skim milk at room temperature for 1 h. The following primary antibodies were added: ZO-1 (#AF5145, Affinity Biosciences), Claudin-5 (#AF5216, Affinity Biosciences), and P-gp (A27707, ABclonal Biotechnology), and incubated overnight at 4°C. The membrane was washed 3 times with TBST, and HRP-labeled goat anti-rabbit IgG (1:5000) was added and incubated at room temperature for 2 h. After washing again, the membrane was developed using an ECL chemiluminescence system, and grayscale analysis was performed using ImageJ software. The relative expression level was calculated using GAPDH (#AF7021, Affinity) as an internal control.

[0100] (3) Experimental results like Figure 11 As shown: Compared with the 0 mM group, the grayscale of ZO-1 and Claudin-5 protein bands was significantly weakened when LATNa concentration was ≥ 2 mM. In the 20 mM group, the relative expression of ZO-1 decreased by 62% and Claudin-5 decreased by 91%, confirming that LATNa can dose-dependently downregulate the level of tight junction proteins. Under the same conditions, P-gp protein expression decreased by 75% and 85% in the 10 mM and 20 mM groups, respectively, suggesting that LATNa also has a novel function of inhibiting drug efflux. The protein downregulation effect showed a significant concentration-dependent relationship, providing molecular-level evidence for LATNa to open the epithelial barrier and reduce efflux.

[0101] (4) Conclusion LATNa reversibly opens intercellular connections by downregulating the expression of tight junction proteins ZO-1 and Claudin-5; simultaneously, it significantly inhibits the expression of the efflux pump P-gp, with these two mechanisms synergistically increasing drug transmembrane uptake. Given that ZO-1, Claudin-5, and P-gp are also highly expressed in similar epithelial structures such as the blood-brain barrier, blood-retinal barrier, and oral mucosa, LATNa also has potential value in enhancing the penetration of these barriers.

[0102] Example 10 This example investigated the in vivo osmotic-enhancing effect of LATNa on oral administration of a mixed FD4 solution to rats. The specific protocol is as follows: (1) Preparation of the drug administration composition Using physiological saline as a solvent, FD4 at a fixed dose of 10 mg / kg was mixed with LATNa at doses of 50 mg / kg, 200 mg / kg, and 400 mg / kg to prepare oral administration combinations with a total volume of 2 mL; at the same time, an FD4 monotherapy group without LATNa was set up as a blank control.

[0103] (2) Animal grouping and administration Twenty healthy adult SD rats, half male and half female, weighing 220 ± 20 g, were randomly divided into four groups of five rats each. Rats were fasted for 24 hours prior to the experiment but had free access to water. Each group was administered 2 mL of the above-mentioned drug combination orally via gavage. A blank blood sample was collected the day before administration and recorded as the 0 min blood sample.

[0104] (3) Blood sample collection and processing At 15, 30, 60, 120, 240, 480, 720, and 1440 min after drug administration, approximately 400 µL of whole blood was collected via the orbital venous plexus and placed in centrifuge tubes containing EDTA-K2. The tubes were immediately centrifuged at 3000 rpm for 10 min at 4°C. The supernatant plasma was separated, and 100 µL was transferred to a black 96-well plate and stored in the dark for later analysis.

[0105] (4) Determination of plasma FD4 concentration The drug was detected using a fluorescence microplate reader with an excitation wavelength of 488 nm, an emission wavelength of 530 nm, and a slit width of 5 nm. The plasma drug concentration at each time point was calculated using the external standard method with a series of FD4 plasma standard curves (R²≥0.999).

[0106] (5) Results Analysis The experimental results are shown in the table below: Table 1. Results of LATNa's effect on the osmotic enhancement of oral mixed solutions in rats.

[0107] (6) Conclusion As shown in the table above, LATNa can dose-dependently increase the plasma exposure level of FD4 under oral administration conditions in rats, indicating that the addition of LATNa can significantly increase the oral bioavailability of FD4, demonstrating an excellent effect in promoting the absorption of low-permeability / poor-permeability drug molecules.

[0108] Example 11 This embodiment investigated the intestinal permeation enhancement effect of potassium lipoic acid trisulfide (LATK) on the high molecular weight model drug FD10 after oral administration of enteric-coated capsules to rats. The specific protocol is as follows: (1) Preparation of the drug administration composition Experimental group: FD1040 mg / kg and LATK300 mg / kg were thoroughly mixed and filled into rat enteric-coated capsules; Negative control group: FD10 40 mg / kg and mannitol 300 mg / kg were physically mixed and then filled into enteric-coated capsules using the same method; Positive control group: FD10 4 mg / kg was administered via intraperitoneal injection to correct for absolute bioavailability.

[0109] (2) Animal grouping and administration Fifteen healthy SD rats, weighing 220 ± 20 g, were randomly divided into three groups of five each. Rats were fasted for 24 hours prior to the experiment but allowed free access to water. The experimental and negative control groups were administered the capsules orally via gavage, while the positive control group was administered the capsules intraperitoneally. Blank blood samples were collected one day prior to administration (recorded as 0 min). Free access to food and water was resumed two hours after administration.

[0110] (3) Blood sample collection and processing At 15, 30, 60, 120, 240, 480, 720, and 1440 min after drug administration, approximately 400 μL of whole blood was collected via the orbital venous plexus and placed in EDTA-K2-coated centrifuge tubes. The tubes were immediately centrifuged at 4°C and 3000 rpm for 10 min to separate the plasma. 100 μL of the plasma was transferred to a black 96-well plate and stored in the dark for later analysis.

[0111] (4) Determination of plasma FD10 concentration Detection was performed using a fluorescent microplate reader: excitation wavelength 488 nm, emission wavelength 530 nm; a series of FD10 plasma standard curves (R) were used. 2 Quantitative analysis was performed using the external standard method (≥ 0.999) to calculate plasma drug concentrations at each time point.

[0112] (5) Experimental results The results are shown in the table below: Table 2. Results of LATK's effect on the penetration of enteric-coated capsules in rats.

[0113] The results showed that in the presence of 300 mg / kg LATK, FD 10 The relative bioavailability reached 9.68%. The existing FD... 10 The relative bioavailability was 0.3%. LATK enhanced the uptake of low-permeability molecules with a molecular weight of 10,000 by 32.3 times, indicating that it has an excellent promoting effect on the permeation of hydrophilic macromolecular peptides.

[0114] (6) Conclusion When LATK is administered orally via enteric-coated capsules, it can increase the relative bioavailability of FD10 by more than 30 times, demonstrating its excellent permeation-enhancing effect on high molecular weight, low-permeability hydrophilic peptides. It can be used to improve the bioavailability of oral protein / peptide drugs.

[0115] Example 12 This embodiment investigated the permeation-enhancing effect of LATNa on orally administered semaglutide capsules in rats. The specific experimental protocol is as follows: (1) Preparation of test reagent Experimental group: 1.34 mg / kg semaglutide was physically mixed with 80, 160, and 240 mg / kg LATNa and then filled into ordinary gastric-soluble capsules for rats; Negative control group: An equal amount of semaglutide (1.34 mg / kg) was individually filled into capsules of the same specification; Positive control group: 1.34 mg / kg semaglutide and 280 mg / kg SNAC were mixed and then filled into capsules of the same specification.

[0116] (2) Animal grouping and administration Twenty healthy SD rats, weighing 220 ± 20 g, were randomly divided into 5 groups of 4 rats each. Rats were fasted for 16 hours before the experiment but allowed free access to water. The experimental group, negative control group, and positive control group were administered the corresponding capsules orally by gavage; free access to water and food was resumed 2 hours later.

[0117] (3) Blood sample collection and processing Whole blood (approximately 400 μL) was collected via the orbital venous plexus at 30, 60, 120, 240, 480, and 720 min after drug administration. The blood was placed in EDTA-K2-modified centrifuge tubes and centrifuged at 3000 rpm for 10 min at 4°C to separate the plasma. Store at 80℃ until testing.

[0118] (4) Determination of plasma semaglutide concentration Quantitative analysis was performed using HPLC-MS / MS (refer to Tang Qi et al., Chinese Journal of Clinical Pharmacology, 2023, 39(9):1316-1320): The chromatographic column was C18 (2.1 × 100 mm, 1.7 μm), mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile, with gradient elution. The linear range of the method was 2–500 ng / mL, R0. 2 ≥ 0.99.

[0119] (5) Experimental results The results are shown in the table below: Table 3. Results of LATNa's effect on the permeation-enhancing effect of oral gastric-coated capsules in rats.

[0120] As shown in the table above, semaglutide was not detected in the plasma of the negative control group; significant drug exposure was detected in the low, medium, and high dose groups of LATNa, with the 240 mg / kg group showing the highest levels. maxThe concentration reached 133.83 ± 12.17 ng / mL, which was significantly higher than that of the SNAC positive control group (Cmax 18.28 ± 1.76 ng / mL, p<0.001).

[0121] (6) Conclusion LATNa can dose-dependently increase the plasma concentration of semaglutide in rats after oral administration, at 240 mg / kg C max Both the exposure levels and the oral permeability enhancement levels of LATNa are superior to those of the commercial positive stimulant SNAC, indicating that LATNa has excellent oral permeability enhancement effects on peptide drugs and can be used to improve the oral bioavailability of GLP-1 analogs such as semaglutide.

[0122] Example 13 This embodiment investigated the enhancing effect of LATNa on the permeability and bioavailability of orally loaded drug nanoparticles. The specific scheme is as follows: (1) Preparation of test reagent Experimental group: 10 mg / kg of paclitaxel-loaded polylactic-co-glycolic acid copolymer nanoparticles (prepared by nanoprecipitation according to the method of Langer et al., see Farokhzad OC, et al. Proceedings of The National Academy of Sciences of The United States of America. 2006, Vol. 103, No. 16, pp. 6315–6320.) and 300 mg / kg LATNa were dissolved together in 2 mL of physiological saline and vortexed to obtain the gavage solution for the experimental group. Control group: An equal amount of drug-loaded nanoparticles (10 mg / kg) was dissolved separately in 2 mL of physiological saline and mixed in the same way to serve as the control group gavage solution.

[0123] (2) Animal grouping and administration Twelve healthy SD rats, weighing 220 ± 20 g, were randomly divided into two groups of six each. Rats were fasted for 12 hours before the experiment but allowed free access to water. The experimental or control groups were administered the drug solution orally via gavage. Free access to water and food was restored 2 hours later.

[0124] (3) Blood sample collection and processing Whole blood (approximately 400 μL) was collected via the orbital venous plexus at 30, 60, 120, 360, 720, 1440, and 2880 min after drug administration. The blood was placed in EDTA-K2-modified centrifuge tubes and centrifuged at 4 ℃ and 3000 rpm for 10 min to separate the plasma. Store at 80 ℃ until testing.

[0125] (4) Determination of plasma paclitaxel concentration HPLC-MS / MS was used (refer to Song Yining et al., Journal of the Academy of Military Medical Sciences, 2010, 34(2):135-138): chromatographic column was C18 (2.1×100 mm, 1.7 μm), mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile, with gradient elution. The linear range of the method was 5–1000 ng / mL. 2 ≥ 0.99.

[0126] (5) Experimental results The results are shown in the table below: Table 4. Results of LATNa's effect on the penetration of drug-loaded nanoparticles

[0127] As shown in the table above: Control group (without LATNa) AUC 0-48 The concentration was 38.53 μg·h / L; the AUC of the experimental group (with 300 mg / kg LATNa) was 38.53 μg·h / L. 0-48 The concentration increased to 344.32 μg·h / L, with a relative bioavailability increase of 8.94 times (p<0.001).

[0128] (6) Conclusion LATNa can significantly enhance the oral permeability of drug-loaded nanoparticles, increasing paclitaxel exposure by nearly 9 times, indicating its suitability for improving the oral bioavailability of nanoformulations and its potential for development as an oral nanodrug penetration enhancer.

[0129] The mg / kg mentioned in the embodiments of the present invention refers to the dosage per kg of body weight of rats. For example, 50 mg / kg means that the dosage per kg of body weight of rats is 50 mg.

[0130] Example 14 This embodiment compares the regulatory effects of LATNa and LANa on the transcaco-2 monolayer efflux flux of doxorubicin hydrochloride (DOX·HCl). The specific steps are as follows: (1) Preparation and quality control of cell monolayers Caco-2 cells were seeded in 12-well Transwell plates and cultured continuously in DMEM complete medium for 21 days. During this period, TEER was monitored using a Millicell ERS 3.0 impedance meter, and only cells with TEER ≥ 400 Ω·cm were selected. 2 The dense monolayer is used for transport tests.

[0131] (2) Preparation of test solution Preheated at 37 °C and without Ca 2+ / Mg 2+Using HBSS as a solvent, a base-side solution containing 100 µM doxorubicin hydrochloride was prepared, and the following were added: LATNa 0, 1, 5, 10, 20 mM, and LANa 10 mM (control); an equal volume of blank HBSS was added to the top side to ensure that the liquid levels in the two chambers were level.

[0132] (3) Two-way transport and sampling The Transwell was placed in a 37°C, 5% CO2 incubator. 100 µL of sample was taken from the AP side at 10, 30, 60, 90 and 120 min. The sample was placed at 4°C in the dark and an equal amount of preheated HBSS was added immediately to maintain the volume and osmotic pressure.

[0133] (4) Doxorubicin concentration determination and efflux rate calculation Determined by HPLC according to the Chinese Pharmacopoeia (2020 edition, Part II): C18 column (4.6 × 250 mm, 5 µm), mobile phase sodium dodecyl sulfate-methanol-acetonitrile, flow rate 1.0 mL / min, detection wavelength 254 nm; quantification was performed using the external standard method based on peak area. Efflux rate (%) = (cumulative drug amount on the AP side / initial drug amount on the BL side) × 100; the lower the value, the stronger the efflux inhibition.

[0134] (5) Experimental results like Figure 12 As shown, with increasing LATNa concentration, the efflux rate of doxorubicin hydrochloride gradually decreased. When the LATNa concentration reached 20 mM, almost no efflux was observed; while at the same concentration (10 mM), the efflux rate of LANa cells was over 40%. Furthermore, the time progression showed that the inhibitory effect of LATNa on doxorubicin efflux increased with prolonged incubation time, while no significant improvement was observed in LANa cells at any time point.

[0135] (6) Conclusion LATNa can inhibit the efflux of doxorubicin hydrochloride by Caco-2 monolayer in a concentration- and time-dependent manner. At 20 mM, it almost completely blocks the efflux pathway, which is significantly better than LANa at the same concentration. This effectively increases the intracellular accumulation of doxorubicin, providing direct cellular evidence for its use as an oral efflux inhibitor-permeability enhancer.

[0136] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. The application of lipoic acid trisulfide or its salts, isomers, and derivatives, characterized in that, Application in the preparation of drug penetration enhancers and / or efflux inhibitors.

2. The application according to claim 1, characterized in that, Lipoic acid trisulfides and their derivatives include at least one of the compounds with the following structures:

3. The application according to claim 2, characterized in that, The compound shown in Formula I can be freely converted into the compound shown in Formula II or Formula III in vivo, and the compounds shown in Formula IV, Formula V and Formula VI are all homologues of the compound shown in Formula I.

4. The application according to claim 1, characterized in that, The drug includes low-permeability drugs, preferably at least one of low-permeability small molecule chemical drugs, peptide drugs, protein drugs, and nanomedicines.

5. A pharmaceutical preparation, characterized in that, It includes a drug penetration enhancer and / or efflux inhibitor, wherein the drug penetration enhancer and / or efflux inhibitor is lipoic acid trisulfide or its salt or isomer.

6. The pharmaceutical preparation according to claim 5, characterized in that, The drug is a hypotonic drug or includes at least one of hypotonic small molecule chemical drugs, peptide drugs, protein drugs, and nanomedicines.

7. The pharmaceutical preparation according to claim 6, characterized in that, The mass ratio of lipoic acid trisulfide or its salts, isomers to the drug is 1:1 to 1000:1; The preferred ratio is 5:1 to 500:

1.

8. The pharmaceutical preparation according to claim 7, characterized in that, The formulations include those administered orally and those administered via the intestines.

9. The pharmaceutical preparation according to claim 8, characterized in that, The oral administration formulations include gastric-coated formulations, enteric-coated formulations, and oral formulations; Preferably, the gastric-soluble formulation includes gastric-soluble solid dosage forms and gastric-soluble liquid dosage forms; Preferably, the enteric-coated formulation includes enteric-coated solid dosage forms and enteric-coated liquid dosage forms; Preferably, the oral preparations include orally disintegrating tablets, lozenges, sublingual tablets, oral adhesive tablets, oral sprays, and mouthwashes.

10. The pharmaceutical preparation according to claim 8, characterized in that, The enteral administration preparations include suppositories and enemas.

Images