Systems, methods, and applications for continuous drainage of mesenteric lymph

Abstract

The application belongs to the technical field of biology, and discloses a system for continuous drainage of mesenteric lymph fluid and application thereof, which comprises a mesenteric lymph vessel catheter for being inserted into a mesenteric lymph vessel to drain mesenteric lymph fluid, a jugular vein catheter for being inserted into a jugular vein, a collector for collecting lymph fluid, and a conscious activity device comprising a quick connection button VAB and a mesenteric lymph fluid collecting tube, wherein the outlet end of the mesenteric lymph fluid collecting tube is in the same height as the inlet end of the mesenteric lymph vessel catheter. The method can realize real-time and long-term collection of lymph fluid under the conditions of free activity, unrestricted diet and water and normal physiological state of rats, can avoid the influence of surgical stress, general anesthesia or animal restriction on experimental results, can reflect the real state of lymph fluid, and can be applied to the fields of research on lymph vessel absorption capacity and functional state and explanation of disease mechanism.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to systems, methods and applications for continuous drainage of mesenteric lymph fluid, specifically to systems, methods and applications for continuous drainage of rat mesenteric lymph fluid through jugular vein-assisted reflux. Background Technology

[0002] The intestinal lymphatic system is involved in the transport of immune cells, fluid balance, homeostasis, transmission of intestinal satiety signals, and transport of dietary lipids. Mesenteric lymph is widely used to evaluate the transport and metabolism of lipid-soluble drugs and other lipophilic molecules in the intestine.

[0003] Oral absorption of lipid-soluble drugs has always been a challenge in drug formulation development. After oral administration, drugs undergo first-pass metabolism in the liver before entering the systemic circulation, further reducing their bioavailability. Unlike the traditional portal vein absorption pathway, mesenteric lymphatic absorption is considered an important absorption route for lipids and lipid-soluble drugs. This is because lipid products pass through the mesenteric lymphatic vessels and thoracic duct, ultimately draining into the anterior vena cava, thus avoiding first-pass metabolism in the liver. Analysis of mesenteric lymph can assess the role of nanoparticles, liposomes, and nanoemulsions as drug delivery carriers in improving drug bioavailability. Studies have found that the specific distribution of lipid-soluble drugs in mesenteric lymph is related to chylomicrons; the greater the drug distribution in chylomicrons, the higher the proportion of drug absorbed via the mesenteric lymph, showing a clear linear correlation. Therefore, dissolving the drug in an oil adjuvant before administration can increase its distribution ratio in chylomicrons, thereby improving the transport capacity of the mesenteric lymphatic system. Probucol, a lipid-soluble drug, is one of the strongest synthetic antioxidants currently available for clinical use. It has broad application prospects in lowering blood lipids, preventing atherosclerosis, and treating diseases such as diabetes. In vitro studies have shown that probucol can be distributed in chylomicrons at a rate of up to 38%.

[0004] The rat lymphatic cannulation model is currently the most widely used in vivo model for studying drug lymphatic absorption. However, the animals in this model are often under anesthesia or restraint, making it impossible to simulate the lymphatic transport effect under normal physiological conditions. Furthermore, the surgery is difficult and has a low success rate. Since Bollman (1948) first established the rat mesenteric lymph drainage technique, more and more researchers have used this technique to conduct various related studies. During mesenteric lymph drainage, animals are under anesthesia or restraint for extended periods, which leads to a decrease in intestinal lymph flow and lymphatic transport capacity. In addition, some experiments involve gastric or duodenal cannulation to supplement nutrients, which adds extra trauma to the animals. Current techniques select to collect lymph fluid while the animal is under anesthesia or restraint after surgery (…). Figure 1D), while shortening the experimental time and ensuring a high success rate in sample collection, introduces several problems, such as: a) Reduced gastric emptying, altered small intestinal capillary permeability, decreased lymphatic flow, and reduced lymphatic transport capacity of proteins and lymphocytes under anesthesia, as evidenced by the difference in lymphatic flow between the control and experimental groups in this invention; b) Intermittent lymphatic collection prevents accurate calculation of the total amount of drug transported via lymph, limiting the model's application; c) For convenient drug administration, existing techniques require duodenal cannulation in rats to continuously infuse lipids and drugs via catheter to evaluate their lymphatic transport capacity. However, gastric secretions and nerve conduction play crucial roles in the entire digestive process, so data obtained under these abnormal physiological conditions are highly susceptible to bias; d) Due to restricted food and water intake, subcutaneous injection or gastrointestinal intubation is necessary to maintain fluid balance and energy levels. Therefore, existing methods limit in-depth research on mesenteric lymph. Summary of the Invention

[0005] This invention employs an assisted reflux system to establish a rat mesenteric lymphatic vessel-jugular vein assisted reflux method, enabling real-time and long-term collection of lymph fluid from rats in an awake and free state. This method avoids the influence of surgical stress, general anesthesia, or animal restriction on the formation and composition of mesenteric lymph fluid, providing an innovative technical means for in-depth research on the function of the mesenteric lymphatic system.

[0006] One objective of this invention is to provide a system for continuous drainage of mesenteric lymph, the system comprising:

[0007] 1) Mesenteric lymphatic duct: used to insert into the mesenteric lymphatic vessels and drain mesenteric lymph fluid;

[0008] 2) Jugular vein catheter: used for insertion into the jugular vein;

[0009] 3) Collector, used to collect lymph fluid;

[0010] 4) A two-channel awake activity device, including a first channel for collecting lymph fluid and a second channel for injecting isotonic solutions or for drawing blood.

[0011] One end of the first passage is connected to the mesenteric lymphatic duct via a quick-connect button VAB, and the other end extends into the collector, with the other end at the same height as the inlet end of the mesenteric lymphatic duct.

[0012] One end of the second pathway is connected to the jugular vein catheter via a quick-connect button VAB, and the other end is a port for injecting isotonic ion solution or for drawing blood.

[0013] Another object of the present invention is to provide the application of the system described above in continuous drainage of mesenteric lymph.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) After inserting the mesenteric lymphatic catheter and the jugular vein catheter, a new lymphatic circulation pathway (mesenteric lymphatic - left jugular superficial vein) is established using the VAB circuit connector. On the one hand, the animal can move freely without the need for lymph or blood collection, and its health status is not affected. On the other hand, by connecting the VAB to the two-channel awake activity device, experiments can be carried out in the post-recovery healthy state of the animal (free movement, no restriction on food and water), avoiding interference from factors such as anesthesia, trauma, and infection. For example, more accurate and longer-term measurement of drug transport via lymph can be achieved.

[0016] (2) In terms of anatomical location, the existing technique selects to insert a cannula at the lymphatic vessel parallel to the right renal artery. However, this is the confluence of mesenteric lymphatic vessels and auxiliary lymphatic vessels. Therefore, the lymph fluid collected at this location cannot accurately assess the actual situation of lipid drug transport via the intestinal lymphatic system. Some reports also select to cut off the pathway of this auxiliary lymphatic vessel, which further increases the difficulty and risk of the operation.

[0017] This invention selects lymphatic vessels parallel to the mesenteric artery, which reduces the difficulty of the surgery and allows for precise collection of mesenteric lymph fluid.

[0018] (3) The walls of the mesenteric lymphatic vessels are composed of only a few layers of epithelial cells, making them extremely easy to tear; at the same time, the surrounding lymphatic tissue is easily damaged during blunt dissection, which leads to obstruction of lymphatic return. Avoiding tearing of the mesenteric lymphatic vessel walls and damage to the surrounding lymphatic tissue can significantly improve the success rate of the model.

[0019] Existing technology involves beveling the tip of the catheter to create a sharp angle. While this facilitates insertion, it also greatly increases the risk of scratching or puncturing the blood vessel wall. Furthermore, during fluid aspiration, the negative pressure generated at the catheter tip causes the blood vessel wall to easily adhere to the beveled tip, leading to catheter blockage. The catheter tip of this invention is designed with a rounded shape, effectively avoiding the problems of blood vessel puncture or blockage.

[0020] This invention uses appropriate microsurgical instruments and catheters to precisely separate lymphatic vessels. Incisions are made in the lymphatic vessels using surgical scissors. Even when using catheters with rounded tips, insertion is convenient. At the same time, the rounded tips can reduce damage to the muscles, connective tissue and adipose tissue around the blood vessels and lymphatic vessels.

[0021] (4) Regarding the handling of intraperitoneal catheters, some existing techniques choose to leave part of the catheter in the abdominal cavity to prevent it from being pulled out of the lymphatic vessels. However, this can easily cause intestinal obstruction in rats during intestinal peristalsis, and may even lead to the death of the animal. Some existing techniques also choose to pass the lymphatic vessels directly out of the body from the dorsal side of the inferior vena cava. This method is extremely difficult to handle and causes significant damage to the animal. In addition, existing techniques use medical glue to fix the inserted catheter. This operation makes it impossible to determine the firmness of the catheter, and the biological glue can severely damage connective tissue or adipose tissue, and may even affect local microcirculation.

[0022] This invention fixes the catheter in a C-shaped position to the inner wall of the abdominal cavity to avoid entanglement with the intestines, and sutures the catheter into the mesenteric lymphatic vessels, which can be well fixed without biological glue. This treatment method can maintain a high survival rate of animals.

[0023] (5) Since lymph and lymphatic vessels are colorless and transparent, the present invention allows rats to be orally fed corn oil before surgery to generate milky white chylomicrons, which can more accurately locate mesenteric lymphatic vessels (this is mainly because long-chain fatty acids (14C-24C) formed by lipid degradation and other lipid products are assembled into milky white milk-like chylomicrons (CM)).

[0024] (6) Animals’ gastrointestinal motility changes rhythmically, and the ability of gastrointestinal motility is weakened when animals are under anesthesia. If organ displacement (ectopic displacement) occurs during surgery, it is very easy to cause death. Therefore, the present invention restores the position of abdominal organs during abdominal surgery.

[0025] (7) The present invention uses medical grade material for the catheter, so that there will be no obvious tissue rejection. Attached Figure Description

[0026] Figure 1 This diagram illustrates the collection of intestinal lymph fluid from rats; where A represents a self-made catheter, B represents the lymph fluid collection system for the experimental group, C represents the structure of the lymph fluid collection system for the experimental group, and D represents the collection system for the control group.

[0027] Figure 2 A schematic diagram of the establishment of a rat mesenteric lymphatic-jugular vein auxiliary return model; where A shows the location of the intestinal lymphatic vessels, B shows the fixation of the intraperitoneal catheter, C shows the location of the jugular vein, D shows the fixation of the jugular vein catheter, E shows the catheter connected to the VAB, and F shows the structure of the lymphatic-jugular vein auxiliary return model.

[0028] Figure 3 The graph shows the lymph flow rate results of the control group and experimental group rats in Example 1.

[0029] Figure 4 The figure shows the average blood drug concentration-time curves for each group of rats in Example 2.

[0030] Figure 5 The curves show the average dose percentage of lymph fluid from each group of rats in Example 2. Detailed Implementation

[0031] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0032] The lymphatic collection system used in this invention enables rats to collect lymph, blood, or administer medication in real time and over a long period of time while they are fully awake, free to move, and without restrictions on food and water. This avoids the effects of surgical stress, general anesthesia, or animal restriction on the formation and composition of mesenteric lymph, ensuring more accurate and complete examination, intervention, and treatment of animals under conditions closer to physiological conditions.

[0033] First, after cannulation, a new lymphatic circulation pathway (mesenteric lymphatic vessel - left superficial jugular vein) is established using a VAB circuit connector. On the one hand, this allows the animal to move freely without the need for lymph or blood collection, and its health status remains unaffected. On the other hand, experiments can be conducted in the animal's post-recovery health condition (free movement, no restriction on food or water), avoiding interference from factors such as anesthesia, trauma, and infection, and enabling more accurate and longer-term measurement of drug transport via the lymphatic system.

[0034] This invention uses a fully automated blood collection and drug delivery device ( Figure 1 (D) This invention enables fully automated lymphatic and blood collection and administration. The system establishes a lymphatic-blood circulation pathway, allowing for immediate blocking of mesenteric lymphatic fluid as needed for experiments. This facilitates research into the pathogenesis of diseases such as sepsis through the lymphatic system. Secondly, utilizing the principle of equal pressure at moderate liquid levels in physics, the mesenteric lymphatic collection tube (collection catheter) is positioned at the height of the mesenteric lymphatic duct catheter (lymphatic cannula). The lymphatic flow rate collected at the end of the collection catheter represents the actual flow rate within the body. Simultaneously, during lymphatic drainage, an isotonic ion exchange solution is continuously infused into the jugular vein, and the flow rate is adjusted in real-time based on lymphatic and blood loss to maintain fluid balance and homeostasis.

[0035] This invention establishes a rat mesenteric lymphatic-jugular vein-assisted return technique to achieve continuous drainage of intestinal lymph fluid. Control group rats underwent double cannulation of the duodenum and mesenteric lymphatic vessels, and intestinal lymph fluid was collected postoperatively. Experimental group rats underwent double cannulation of the jugular vein and mesenteric lymphatic vessels to establish a mesenteric lymphatic-jugular vein return model. On the 7th postoperative day, intestinal lymph fluid was collected using a wakefulness-based device, and the flow rate, cellular components, and various biochemical indicators were measured. This invention successfully established a rat mesenteric lymphatic-jugular vein-assisted return technique, maintaining the model for more than 7 days. This method enables real-time and long-term lymph fluid collection from rats under awake, free, unrestricted food and water intake and normal physiological conditions, avoiding the influence of surgical stress, general anesthesia, or animal restriction on experimental results.

[0036] This invention provides a system for continuous drainage of mesenteric lymph, the system comprising:

[0037] 1) Mesenteric lymphatic duct: used to insert into the mesenteric lymphatic vessels and drain mesenteric lymph fluid;

[0038] 2) Jugular vein catheter: used for insertion into the jugular vein;

[0039] 3) Collector, used to collect lymph fluid;

[0040] 4) A two-channel awake activity device, including a first channel for collecting lymph fluid and a second channel for injecting isotonic solutions or for drawing blood.

[0041] One end of the first passage is connected to the mesenteric lymphatic duct via a quick-connect button VAB, and the other end extends into the collector, with the other end at the same height as the inlet end of the mesenteric lymphatic duct.

[0042] One end of the second pathway is connected to the jugular vein catheter via a quick-connect button VAB, and the other end is a port for injecting isotonic ion solution or for drawing blood.

[0043] In some embodiments, the awakening activity device includes a quick-connect button VAB, a spring tube, a rotor, and a balance arm; the spring tube contains a mesenteric lymph fluid collection tube and a rehydration tube; the mesenteric lymph fluid collection tube passes through the rotor and extends into the collector.

[0044] The VAB is used for implantation under the skin. The VAB includes a lymphatic vessel port and a jugular vein port; one end of the lymphatic vessel port is used to communicate with the outlet of the mesenteric lymphatic duct, and the other end is used to communicate with the inlet of the first pathway (mesenteric lymph fluid collection tube); one end of the jugular vein port is used to communicate with the outlet of the jugular vein duct, and the other end is used to communicate with the inlet of the second pathway (infusion tube).

[0045] The mesenteric lymphatic duct and the mesenteric lymph collection tube are connected in a U-shape. During use, the liquid levels at both ends of the U-shape are the same (equal liquid levels mean equal pressure), and at this time, the lymph flow rate in the duct is equal to the actual flow rate in the mesenteric lymphatic vessels.

[0046] The mesenteric lymphatic duct is a cannula. The cannula can be made of any material suitable for lymphatic drainage. Preferably, the cannula includes a PU tube and a retaining buckle formed from a silicone tube.

[0047] The silicone tube can be made of any material suitable for lymphatic drainage. Preferably, the silicone tube has a size of ID 0.51 × OD 0.94 mm.

[0048] The PU tube can be made of any material suitable for lymphatic drainage. Preferably, the PU tube has a size of ID 0.4 × OD 0.7 mm.

[0049] The silicone tube forms a retaining clip on the PU tube; the method by which the silicone tube forms the retaining clip on the PU tube is not limited. Preferably, the silicone tube is fixed to the PU tube with adhesive to form the retaining clip.

[0050] The fixing buckle is used to secure the mesenteric lymphatic duct to the abdominal wall muscles during use, preventing displacement within the abdominal cavity. The position of the fixing buckle on the PU tube is not limited; preferably, the fixing buckle is positioned such that the mesenteric lymphatic duct forms a C-shape within the animal's abdominal cavity during use. Preferably, the silicone tube is fixed with adhesive at 1cm, 4cm, 5.5cm, and 7cm from the tip of the PU tube to form fixing buckles.

[0051] The tip of the PU tube is arc-shaped. This design can avoid scratching or puncturing the inner wall of the blood vessel. Furthermore, when drawing liquid, it will not cause the inner wall of the blood vessel to stick to the tip due to negative pressure generated at the tip of the catheter, thus preventing the catheter from becoming blocked.

[0052] The jugular vein catheter is a cannula. The cannula can be made of any material suitable for lymphatic drainage. Preferably, the cannula includes a PU tube and a retaining buckle formed from a silicone tube.

[0053] The silicone tube can be made of any material suitable for lymphatic drainage. Preferably, the silicone tube has a size of ID 0.76 × OD 1.65 mm.

[0054] The PU tube can be made of any material suitable for lymphatic drainage. Preferably, the PU tube has an ID of 0.6 × OD of 1.0 mm.

[0055] The silicone tube forms a retaining clip on the PU tube; the method by which the silicone tube forms the retaining clip on the PU tube is not limited. Preferably, the silicone tube is fixed to the PU tube with adhesive to form the retaining clip.

[0056] The fixing buckle is used to secure the jugular vein catheter to the abdominal wall muscles during use, preventing displacement within the abdominal cavity. The position of the fixing buckle on the PU tube is not limited; preferably, the fixing buckle is positioned such that the mesenteric lymphatic catheter can form a C-shape within the animal's abdominal cavity during use. Preferably, the silicone tube is fixed with adhesive at 3.5cm and 4.5cm from the tip of the PU tube to form a fixing buckle.

[0057] The tip of the PU tube is arc-shaped. This design can avoid scratching or puncturing the inner wall of the blood vessel. Furthermore, when drawing liquid, it will not cause the inner wall of the blood vessel to stick to the tip due to negative pressure generated at the tip of the catheter, thus preventing the catheter from becoming blocked.

[0058] The fixing buckle is used to fix the jugular vein catheter to the abdominal wall muscles to prevent displacement within the abdominal cavity.

[0059] The system also includes a VAB loop connector; the VAB loop connector includes two ports, one for connecting to the lymphatic vessel port and the other for connecting to the jugular vein port.

[0060] When collecting lymph fluid, the VAB and the mesenteric lymph fluid collection tube and the infusion tube are connected to form a collection pathway in the animal's body, such that the infusion tube, jugular venous catheter, jugular vein, mesenteric lymphatic vessels, mesenteric lymphatic vessel catheter, VAB and the mesenteric lymph fluid collection tube form a collection pathway in the animal's body.

[0061] When lymph fluid is not collected, the VAB and VAB circuit connector are connected, so that the mesenteric lymphatic vessels, mesenteric lymphatic ducts, VAB and VAB circuit connectors, jugular vein, and jugular vein duct form a circulatory loop in the animal's body.

[0062] The infusion tubing is used to replenish fluids to the animal. Preferably, the infusion tubing is used to replenish an isotonic solution, which is preferably Ringer's solution. In one specific embodiment, during lymphatic drainage, the infusion tubing is used to continuously infuse an isotonic solution, such as Ringer's solution, into the jugular vein. In one specific embodiment, because the blood extraction time is short, the infusion tubing can be used to extract blood during lymphatic drainage.

[0063] The system also includes a flow regulating component for adjusting the flow rate of the infusion tube; the flow regulating component is used to adjust the flow rate of the isotonic ion solution in real time according to the lymph flow rate in order to maintain fluid balance and homeostasis of the internal environment.

[0064] The system also includes a component for restoring organs to their original positions. The form of this component is not limited; it can be forceps, pry bars, etc. Animals exhibit rhythmic peristalsis, and if organ displacement occurs during surgery, it can easily lead to death. Therefore, restoring organs to their original positions is crucial.

[0065] The system also includes a data analysis module, which is used to analyze the collected lymph fluid. By measuring the cellular components or biochemical indicators of the lymph fluid, it can help to study the absorption capacity and functional status of capillary lymphatic vessels, study specific biomarkers in lymph fluid, and explain disease mechanisms.

[0066] The system also includes microscissors for making incisions in separated lymphatic vessels and / or jugular veins;

[0067] The system also includes arterial clamps and ligation sutures. During insertion of the mesenteric lymphatic vessel, the arterial clamps and ligation sutures are used to temporarily block the lymphatic pathway; during insertion of the jugular vein catheter, the arterial clamps and ligation sutures are used to temporarily block the proximal end of the external jugular vein. Through the catheter insertion method of this invention,

[0068] It can effectively fix mesenteric lymphatic vessels and / or jugular venous catheters, avoiding the use of reagents that have side effects on animals, such as avoiding the use of biological adhesives.

[0069] The system also includes a component for feeding edible oil. The form of this component is not limited; it can be a feeder, funnel, etc. The edible oil is used to generate milky-white chylomicrons in the body to help locate the mesenteric lymphatic vessels. The edible oil is preferably corn oil.

[0070] In some preferred embodiments, the mesenteric lymphatic vessels, jugular vein catheters, mesenteric lymph fluid collection tubes, and infusion tubes are made of medical-grade materials.

[0071] The mesenteric lymphatic vessels are lymphatic vessels running parallel to the mesenteric arteries. The selection of the opening location of the lymphatic vessels that drain lymph is very important. By using the lymphatic vessels running parallel to the mesenteric arteries as the target for lymph drainage, the collected lymph can be used to accurately assess the actual transport of lipid drugs through the intestinal lymphatic system.

[0072] The jugular vein is preferably the superficial jugular vein; using the method of the present invention, the superficial jugular vein and the mesenteric lymphatic vessels form a lymph collection pathway, and the outlet end of the mesenteric lymph fluid collection tube is set at the same height as the inlet end of the mesenteric lymphatic vessel conduit, so that the lymph fluid flow rate collected through the end of the mesenteric lymph fluid collection tube represents the actual flow rate in the animal's body, and the system is a system for studying the corresponding functions of the lymphatic system.

[0073] Possible causes of catheter blockage include: a) the catheter is not inserted into the tubing; b) blood clots block the catheter; c) the inner wall of a blood vessel or lymphatic vessel blocks the catheter tip; d) air bubbles are present in the catheter; e) the catheter is too shallow or too deep. Therefore, during the construction of an animal model for lymph fluid collection, it is necessary to continuously check the patency of the catheter and avoid introducing air into the tubing.

[0074] Another objective of this invention is to provide the application of the system described above in continuous mesenteric lymph drainage, through which the absorptive capacity and functional status of intestinal capillary lymphatic vessels can be assessed, specific biomarkers in the lymph can be studied, and mechanisms of diseases related to lymph or lymphatic transport can be explained.

[0075] Another object of the present invention is to provide the use of olive oil in the preparation of adjuvants that improve the bioavailability of probucol. Adding olive oil to probucol can effectively improve the bioavailability of probucol and facilitate its absorption in the body.

[0076] Another object of the present invention is to provide a pharmaceutical composition comprising probucol and olive oil.

[0077] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0078] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0079] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0080] Example 1 Model Construction and Index Measurement

[0081] 1. Materials and Methods

[0082] 1.1 Experimental Animals

[0083] Sixteen SPF-grade male SD rats, 8 weeks old, with a body weight of (250 ± 9.33) g, were all purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. [Certificate No. 20180004058749]. All rats were housed in the experimental animal barrier facility of the Center for Excellence in Molecular Cell Sciences, Chinese Academy of Sciences [License No. SYXK (Shanghai) 2018-0007], with free access to drinking water and food, at a temperature of 20°C - 25°C, a relative humidity of 40% - 70%, a noise level of ≤60 dB, and a living illumination of 20 lx (12 / 12 h light / dark cycle). All rats were housed 3 per cage and started the experiment after 7 days of adaptive feeding. All experimental procedures in this invention were reviewed and approved by the Experimental Animal Use and Management Committee of the Center for Excellence in Molecular Cell Sciences, Chinese Academy of Sciences (IACUC No. SIBCB-S118340-2112-045), and the experimental procedures were all carried out under sterile conditions in accordance with the Guide for the Care and Use of Laboratory Animals.

[0084] 1.2 Instruments and Reagents

[0085] Stereomicroscope (SZX16; OLYMPUS), microinjection pump (XFP01-BD; Suzhou讯飞), rat 2-channel VAB tether (VABR2T / 25; Instech), rat 2-channel vascular access button (VAB) (VABR2B / 25R22; Instech), rat 2-channel VAB loop connector (VABR2L; Instech), PinPort injector (PNP3M; Instech), 2-channel rotor (375 / D / 25LT; Instech), multi-axis balance arm (MCLA / MED; Instech), rat sampling cage appliance (MTANK / WF; Instech), polyurethane (PU) catheter (BTPU-027, BTPU-040; Instech), silicone catheter (3Fr, 5Fr; SAI), medical tissue glue (1469SB; 3M), whole blood diluent CELLPACK DCL, hemolysin (WNR, WDF), staining solution (WNR, WDF, PLT), cleaning solution and calibrator (XN CAL / XN CAL PF) from Sysmex Corporation (Japan), in vitro diagnostic reagent dry slides from Ortho-Clinical Diagnostics (USA).

[0086] 1.3 Experimental Grouping and Model Preparation

[0087] Sixteen male SD rats were randomly divided into two groups (n=8 per group): an experimental group (intestinal lymphatic vessel-jugular vein cannulation reflux model) and a control group (duodenal-mesenteric lymphatic vessel cannulation model). In the experimental group, intestinal lymphatic vessel cannulation and jugular vein cannulation were performed to establish the reflux model. In the control group, mesenteric lymphatic vessel cannulation and double duodenal cannulation were performed to establish the conventional drainage model.

[0088] 1.4 Measurement of lymph flow rate

[0089] In the control group, intestinal lymph fluid was collected from rats continuously for 1 hour after surgery. During the collection process, Ringer's solution was injected into the duodenum at a rate of 3 ml / h to rehydrate the rats.

[0090] Seven days after surgery, intestinal lymph fluid was collected from rats in the experimental group for 1 hour each time. During the collection process, Ringer's solution was injected into the jugular vein at a rate of 3 ml / h to rehydrate the rats.

[0091] For both the experimental and control groups, the volume of lymph fluid collected per hour was accurately measured using a pipette, and the intestinal lymph fluid flow rate was calculated.

[0092] 1.5 Observation of Lymphatic Fluid Cell Components

[0093] Quantitatively extract 20 μL of lymph fluid from both the experimental and control groups, and mix each extract with 120 μL of CELLPACK DCL diluent, then store at 4°C. White blood cell count (WBC), NEUT#, LYMPH#, MONO#, EO#, BASO#, NEUT%, LYMPH%, MONO%, EO%, BASO%, RBC, HGB, HCT, MCV, MCH, MCHC, and RDW-SD were measured using a fully automated hematology analyzer XN-1000 (Sysmex).

[0094] 1.6 Determination of biochemical components in urinary tract samples

[0095] Lymph fluid from the experimental and control groups was collected quantitatively, allowed to stand for 30 minutes, and then centrifuged (5 minutes, 3000g) to obtain the supernatant, which was then stored at 4°C. Electrolyte levels (K+, Na+, Cl-, Ca2+, Mg2+, P3+), metabolic indicators (urea, creatinine, uric acid, glucose, triglycerides (TG), total cholesterol (CHO), high-density lipoprotein (HDL), protein levels (total protein (TP), albumin (Alb),) and acid-base balance (carbon dioxide (CO2)) were measured using a V4600 (Ortho) fully automated biochemical analyzer.

[0096] 2. Model Construction

[0097] 2.1 Preoperative preparation

[0098] (1) Animal preoperative preparation

[0099] Rats were given soybean oil (5 ml / kg) orally 0.5 hours before surgery. As the lipids were absorbed in the small intestine, milky white chylomicrons were produced, which were located in the mesenteric lymphatic vessels.

[0100] (2) Sleeve preparation

[0101] Preparation of rat jugular vein cannulas: A 1 mm silicone tube (ID 0.76 × OD 1.65 mm) was fitted onto the tip of a PU tube (ID 0.6 × OD 1.0 mm) at 3.5 cm and 4.5 cm respectively, and secured with glue to form a fixing buckle. The tip of the PU tube was heated with an alcohol lamp to make it arc-shaped, increasing the smoothness of insertion into the blood vessel and avoiding damage to the inner wall of the blood vessel (Figure 1A).

[0102] Preparation of rat mesenteric lymphatic cannulas: A 1mm silicone catheter (ID 0.51×OD 0.94mm) was fitted onto the tip of a PU tube (ID 0.4×OD 0.7mm) at 1cm, 4cm, 5.5cm, and 7cm respectively. Figure 1 A).

[0103] 2.2 Experimental Group Model Construction

[0104] (1) Mesenteric lymphatic vessel cannulation

[0105] Make a 3-4 cm midline incision in the skin of the abdomen, two-thirds of the way up from the xiphoid process to the pubic symphysis, to open the peritoneal cavity. Using sterile gauze soaked in 37°C saline, fix the gastrointestinal organs to the peritoneum, exposing the left renal vein and inferior vena cava region. At this point, the milky-white mesenteric lymphatic vessels parallel to the mesenteric artery (bright red and pulsating) can be observed. Figure 2 A). Under a stereomicroscope, the mesenteric lymphatic vessels (approximately 0.5-1 mm in diameter) are dissected, and the passage is temporarily blocked using arterial clamps and ligation sutures. An incision is made at a 45° angle above the lymphatic vessels, and a lymphatic duct is inserted and descended 1 cm. At this point, lymphatic fluid flow can be observed in the duct. A 1 cm fixing buckle and the intestinal end of the lymphatic vessel are then secured. The gauze is removed, and the duct is placed in a C-shape against the inner side of the abdominal wall. The 4 cm, 5.5 cm, and 7 cm fixing buckles are secured to the abdominal wall muscles to prevent the duct from shifting within the abdominal cavity, which could affect intestinal peristalsis and cause complications such as intestinal obstruction. Figure 2 (B) Restore the organ to its original position. Guide the other end of the catheter and connect it to the lymphatic port of the neck VAB.

[0106] (2) Jugular vein cannulation

[0107] Make a 0.5cm incision in the skin of the left clavicle region of the neck and abdomen to expose the external jugular vein and superficial jugular vein (the external jugular vein is thicker and located superficially). Temporarily block the proximal and distal ends of the external jugular vein and the distal end of the superficial jugular vein with arterial clamps and ligation sutures. Figure 2 C). Make an incision at a 45° angle above the blood vessel. Following the course of the rat's neck vessels, insert a venous catheter and descend 3.5 cm to the left subclavian vein. Secure the catheter with a fixation buckle at 3.5 cm and to the proximal end of the superficial jugular vein. Figure 2 D). Guide the other end of the catheter into the jugular vein port of the VAB. After cannulation, connect the VAB and the VAB circuit connector. Figure 2 E, Figure 2 F), suture the wound.

[0108] (3) Connection of the conscious activity device

[0109] Connect the VAB, the 2-channel VAB tether, the rotor, the multi-axis balance arm, and the sampling cage. Because the lymphatic system has a U-shaped structure and the fluid levels at both ends of the U are the same (equal pressure at equal fluid levels), the lymph flow rate in the duct is equal to the actual flow rate in the mesenteric lymphatic vessels. Figure 1 B, Figure 1 C).

[0110] 2.3 Construction of the control group model

[0111] (1) Mesenteric lymphatic vessel cannulation

[0112] The surgical site was the same as in the experimental group. A 23G needle was used to puncture the mesenteric lymphatic vessel, and a catheter was inserted into the puncture site. Tissue glue was then dripped in to fix the catheter in place. After successful insertion, the other end of the catheter was protruded from the body to collect lymph fluid.

[0113] (2) Duodenal intubation.

[0114] A 23G needle was used to puncture the duodenum approximately 2 cm below the pylorus. A catheter was inserted into the puncture site, tissue glue was dripped in to fix it, and the other end of the catheter was led out of the body and connected to a microinfusion pump. The muscle layer and skin were continuously sutured, and the rat was placed in a fixation device. Figure 1 D).

[0115] Through the above operations, the present invention successfully established a rat mesenteric lymphatic vessel-jugular vein assisted return model. The rat intestinal lymphatic drainage system composed of this model and the awake activity device can still collect intestinal lymphatic fluid in real time on the 7th day, and the animals showed no abnormalities during this period.

[0116] 3 Experimental Results

[0117] 3.1 Model Preparation Results

[0118] This invention successfully established a rat mesenteric lymphatic vessel-jugular vein assisted return model. The rat intestinal lymph drainage system, consisting of this model and a wakeful activity device, can still collect intestinal lymph fluid in real time on day 7.

[0119] 3.2 Measurement of lymph flow rate

[0120] In this invention, the intestinal lymph flow rate of the experimental group rats (2.01±0.12) ml / h was significantly higher than that of the control group (0.92±0.09) ml / h (P<0.01). Figure 3 This may be related to the lipid intake of the test animals and their physiological state (including whether they were anesthetized, restrained, or infected).

[0121] 3.3 Lymphatic fluid cellular components

[0122] The total number of lymphocytes (LYMPH) and the percentage of lymphocytes (LYMPH%) in the lymph fluid of the experimental group were significantly higher than those in the control group. This may be related to the decreased lymphocyte transport capacity of the thymus, spleen and other central lymphatic organs in animals under anesthesia.

[0123] Secondly, the proportion of neutrophils (NEUT%) and monocytes (MONO%) in the lymph fluid of the experimental group were significantly lower than those of the control group (P < 0.01, mainly because the lymph fluid of the rats in the control group was extracted immediately after the operation, and the body had pain stress and inflammatory response; other indicators were not significantly different from those of the control group (P > 0.05) (Table 1).

[0124] 3.4 Lymphocyte biochemical indicators

[0125] Among the 16 biochemical indicators tested, K in the lymphocytes of the experimental group was [missing information]. + Na + CO2 and Urea were significantly higher than those in the control group, while TG and P were significantly higher. 3+ All were significantly lower than the control group (P<0.01-0.05), while other indicators showed no significant difference from the control group (P>0.05) (Table 2).

[0126] Among them, K + Na + P 3+ The difference was due to the different ways in which the control group and the experimental group absorbed Ringer's fluid during lymphatic drainage.

[0127] In this study, the control group rats did not eat normally during the experiment, resulting in insufficient protein intake compared to the experimental group rats. Urea is an important indicator of changes in kidney function, and insufficient protein intake leads to a decrease in urea levels. Therefore, the difference in protein intake resulted in a significant difference in the Urea index between the experimental and control groups.

[0128] Studies have reported that lipids can promote the transport and absorption capacity of the mesenteric lymphatic system. Therefore, even in pharmacokinetic studies, oral administration of lipids beforehand is not conducive to reflecting the true absorption, distribution, and metabolism of drugs in vivo. However, the use of lipids is unavoidable due to the need for lymphatic vessel labeling. The control group rats showed a significant increase in triglycerides (TG) compared to the experimental group, mainly because a large amount of corn oil was administered orally before surgery, which was digested and broken down into TG and transported to the mesenteric lymphatic system. The physiological states of the control and experimental groups of rats differed significantly, leading to the significant difference in TG levels.

[0129] Among them, the acid-base index CO2 in the lymph fluid of the experimental group was significantly higher than that of the control group, which may be related to the metabolism of tissue cells under anesthesia. On the other hand, it also suggests that since the lymph fluid is an alkaline environment, the alkaline substances in the lymph fluid can counteract the acidosis caused by shock, which may be one of the mechanisms of lymph fluid in resisting shock.

[0130] Furthermore, the cellular components and biochemical indicators of the experimental group model were verified to be consistent with the normal physiological levels of rats, demonstrating reliability and reproducibility, and it can successfully replace existing models.

[0131] Table 1. Lymphocyte composition of rats in control and experimental groups

[0132]

[0133] *p<0.05 *p<0.01 vs blood

[0134] Table 2. Biochemical indicators of lymph fluid in rats of control and experimental groups

[0135]

[0136]

[0137] *p<0.05 **p<0.01 vs serum

[0138] This invention establishes a rat mesenteric lymphatic-jugular vein assisted return technique to achieve continuous drainage of intestinal lymph fluid. Sixteen male SD rats were randomly divided into two groups (n=8 / group). The control group underwent double cannulation of the duodenum and mesenteric lymphatic vessels, and intestinal lymph fluid was collected postoperatively. The experimental group underwent double cannulation of the jugular vein and mesenteric lymphatic vessels to establish a mesenteric lymphatic-jugular vein assisted return model. On the 7th postoperative day, intestinal lymph fluid was collected using a wake-up device, and the flow rate was recorded. Cellular components and various biochemical indicators were analyzed. This invention successfully established a rat mesenteric lymphatic-jugular vein assisted return technique, and the model was maintained for more than 7 days. The intestinal lymph fluid flow rate in the experimental group (2.01±0.12 ml / h) was higher than that in the control group (0.92±0.09 ml / h). The LYMPH and LYMPH% in the lymph fluid of the experimental group were higher than those in the control group; NEUT% and MONO% were lower in the experimental group. K in the lymphocytes of the experimental group + Na + CO2 and Urea were higher than those in the control group, while TG and P were higher. 3+ The lymph flow rate was lower than that of the control group (P<0.01-0.05). That is, in this invention, a control group of rats was constructed using existing methods. Comparison with this control group revealed a significant difference in lymph flow rate between the experimental and control groups. In cellular component analysis, the total number and proportion of lymphocytes in the lymph of the experimental group were significantly higher than those in the control group. Furthermore, the proportions of neutrophils and monocytes in the control group were significantly higher than those in the experimental group, indicating that the control group mice were in an infected state when their lymph was collected. Comparison of the biochemical components of the lymph showed that the electrolyte K in the lymph of the experimental group was significantly higher. + Na + All were significantly higher than the control group, and P 3+ The levels of urate in the experimental group were significantly lower than those in the control group. The metabolic index Urea in the lymphocytes of the experimental group was significantly higher than that of the control group, while TG was significantly lower, and the acid-base index CO2 was significantly higher. Furthermore, the cellular components and biochemical indicators of the experimental model constructed in this invention are consistent with normal physiological levels in rats, demonstrating reliability and reproducibility, and can successfully replace existing models.

[0139] Example 2: Pharmacokinetic study of mesenteric lymphatic transport of probucol oral solution in rats.

[0140] After probucol was dissolved in an oily adjuvant, by constructing an innovative lymphatic vessel-jugular vein auxiliary reflux model, it was observed whether the mesenteric lymphatic absorption of probucol was significantly enhanced. Twelve male SD rats were selected for the experiment and randomly divided into 4 groups (3 rats / group). Among them, single jugular vein intubation surgery was performed on the PO-1 group and the PO-3 group, and double jugular vein intubation and mesenteric lymphatic vessel intubation surgery were performed on the PO-2 group and the PO-4 group. The rats in the PO-1 group and the PO-2 group were orally gavaged with a probucol olive oil preparation (2 mg / ml), and the rats in the PO-3 group and the PO-4 group were orally gavaged with a probucol suspension preparation (2 mg / ml). Whole blood was collected from the jugular vein at corresponding time points (0, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours). After the samples were processed, the whole blood samples were analyzed by high performance liquid chromatography tandem mass spectrometry (LC-MS / MS). The results showed that after probucol was administered by dissolving in olive oil, the drug transport ratio of the mesenteric lymphatic system increased significantly. In terms of the comparison of the overall AUC, the bioavailability of the oral solution of probucol with olive oil as an adjuvant was significantly enhanced compared with that of the tablet (AUC: 12360 vs 3210, P < 0.01). Therefore, for the oral solution of probucol with an oily adjuvant, the drug is not only transported through cell transmembrane transport but also through the mesenteric lymphatic system.

[0141] 1 Materials and Methods

[0142] 1.1 Experimental Animals

[0143] Twelve SPF-grade male SD rats, 9 weeks old, with a body weight of (280 ± 8.96) g, were all purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. [Certificate No. 20211221Aazz0619000169]. All rats were housed in the experimental animal barrier facilities of the Center for Excellence in Molecular Cell Sciences, Chinese Academy of Sciences [License No. SYXK (Shanghai) 2018-0007], with free access to drinking water and food, at a temperature of 20°C - 25°C, a relative humidity of 40% - 70%, a noise level of ≤ 60 dB, and a living illumination of 20 lx (12 / 12 h light / dark cycle). All rats were housed at 3 rats / cage and started the experiment after 7 days of adaptive feeding. All experimental procedures in this study were reviewed and approved by the Experimental Animal Use and Management Committee of the Center for Excellence in Molecular Cell Sciences, Chinese Academy of Sciences (IACUC No. SIBCBS118340-2112-045), and the experimental procedures were carried out under sterile conditions in accordance with the Guidelines for the Care and Use of Laboratory Animals.

[0144] 1.2 Drugs, Reagents and Main Instruments

[0145] Probucol (C31H48O2S2) and succinylphenol (C13H11NO3) were provided by Kaihui Pharmaceutical (Shanghai), with batch numbers 051M14375V and 23288-49-5 respectively; acetonitrile (CH3CN) was an HPLC-grade reagent manufactured by SIGMA-ALDRICH, USA, with batch number S93271; ammonium acetate (NH4OAc) was a chromatographically pure reagent manufactured by TEDIA, USA, with batch number 601058; experimental water was prepared by MillIPORE-Q; and all other chemical reagents were of analytical grade. LC-MS / MS liquid chromatography-mass spectrometry system (LCMSMS-001); Mettler-Toledo XP26 electronic balance; Thermo Fisher -70ºC ultra-low temperature freezer (USA); Eppendorf 5810R high-speed large-capacity low-temperature centrifuge (Germany); IKA Vibrax VXR mini shaker (Germany); IKAVortex shaker (Germany); KQ5200DA ultrasonic cleaner (Kunshan).

[0146] 1.3 Formulation

[0147] (1) Preparation of probucol oil-olive oil solution: 1) Accurately weigh 42.48 mg of probucol powder and place it in a clean glass bottle; 2) Slowly add a total of 21.24 ml of olive oil (ACROS, batch number: A0285378) to the bottle and gently vortex for about 1-2 min; 3) Sonicate for about 10-15 min until the compound is completely dissolved, and obtain a yellow, clear, transparent liquid with a concentration of 2 mg / ml. Store at low temperature for later use.

[0148] (2) Preparation of probucol suspension: 1) Accurately weigh 93.89 mg of probucol tablet powder (purity: 62.55%; batch number: H10980054) and place it in a clean glass bottle; 2) Slowly add a total of 29.364 ml of 10 mM phosphate saline buffer (pH 7.4) to the bottle and gently vortex for about 2-3 min; 3) Sonicate for about 30-40 min to obtain a white homogeneous suspension with a concentration of 2 mg / ml.

[0149] 1.4 Animal grouping and experimentation

[0150] Rats were divided into four groups according to the experimental design: PO-1, PO-2, PO-3, and PO-4, with three rats in each group. Rats in groups PO-1 and PO-3 underwent single-cannulation of the left superficial jugular vein to establish a jugular vein blood collection model. Rats in groups PO-2 and PO-4 underwent double-cannulation of the left superficial jugular vein and mesenteric lymphatic vessels to establish an intestinal lymphatic-jugular vein auxiliary return system. Figure 1 , Figure 2After establishing the rat model, rats in the PO-1 and PO-2 groups were orally administered probucol olive oil preparation (20 mg / kg) by gavage, while rats in the PO-3 and PO-4 groups were orally administered probucol suspension (20 mg / kg) by gavage. Blood samples were collected before administration (0) and at 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours after administration, while lymph samples were collected at 0–4, 4–8, and 8–24 hours after administration (Table 3).

[0151] Table 3 Animal Experiment Protocol

[0152]

[0153]

[0154] 1.5 Sample Analysis

[0155] Whole blood samples were diluted with deionized water, and lymph samples were anticoagulated with heparin. The concentration of probucol in the test samples was analyzed using LC-MS / MS-001 (Q-trap-3200). Based on the drug mean concentration-time data, the pharmacokinetic parameters of probucol, including the area under the curve (AUC) and elimination half-life (T0.05), were calculated using a non-compartmental model with WinNonlin 6.2 software. 1 / 2 Peak concentration (C) max ) and peak time (T) max Data below the 80% lower limit of quantitation are not included in the calculation of pharmacokinetic parameters.

[0156] 2 Results

[0157] 2.1 Whole blood pharmacokinetic results

[0158] Rats in the PO-1 and PO-2 groups reached peak plasma concentrations at 13.3±9.24 hr and 18.7±9.24 hr, respectively, after oral gavage administration of 20 mg / kg probucol in olive oil. The maximum plasma concentrations were 453±204 ng / ml and 309±177 ng / ml, respectively, with elimination half-lives of 12.8±2.78 hr and 19.0±11.2 hr, respectively. The area under the drug-time curve (AUC) from 0 to the end of the time interval was [not specified in the original text]. last The effective doses were 12360±6629 hr*ng / ml and 8080±3064 hr*ng / ml, respectively. The area under the curve (AUC) from 0 to infinity was... infThe concentrations of probucol in rats in the PO-3 and PO-4 groups were 12623±6650 hr*ng / ml and 8947±2218 hr*ng / ml, respectively. After oral administration of 20 mg / kg probucol suspension to rats in the PO-3 and PO-4 groups, peak concentrations were reached at 10.7±11.5 hr and 5.33±2.31 hr, respectively, with maximum whole blood concentrations of 148±59.8 and 338±207 ng / ml, and elimination half-lives of 12.1±1.20 hr and 16.7±3.34 hr, respectively. The area under the drug-time curve (AUC) from 0 to the end of the time point was [not specified in the original text]. last The effective doses were 3210±885 hr*ng / ml and 3677±2014 hr*ng / ml, respectively. The area under the curve (AUC) from 0 to infinity was... inf The concentrations were 3293±899 hr*ng / ml and 3970±2295 hr*ng / ml, respectively (Table 4). Figure 4 ).

[0159] Table 4. Mean pharmacokinetic parameters of whole blood in rats of each group

[0160]

[0161] 2.2 Pharmacokinetic Results of Intestinal Lymph Fluid

[0162] After oral administration of 20 mg / kg probucol olive oil preparation to rats in group PO-2, the percentage of doses at 0-4 hr, 4-8 hr, and 8-24 hr were 0.161±0.108%, 0.299±0.197%, and 0.408±0.236%, respectively. After oral administration of 20 mg / kg probucol suspension preparation to rats in group PO-4, the percentage of doses at 0-4 hr, 4-8 hr, and 8-24 hr were 0.075±0.041%, 0.201±0.172%, and 0.304±0.231%, respectively. Figure 5 ).

[0163] Oral absorption of lipid-soluble drugs has always been a challenge in formulation development. The lymphatic transport of lipid-soluble drugs via the adjuvant route is primarily influenced by the lipid material itself. Factors considered in lipid material selection include type, chain length, and degree of unsaturation; generally, long-chain lipids are considered superior to short-chain lipids, and monounsaturated lipids are superior to polyunsaturated lipids. After oral absorption, a portion of the lipid-encapsulated drug is absorbed through the small intestinal capillaries into the portal vein, while the remaining portion, along with triglycerides from the breakdown of lipid adjuvants, assembles into chylomicrons. These chylomicrons then flow into the mesenteric lymphatic vessels via the central lacteal duct and ultimately enter the systemic circulation. Therefore, improving the lymphatic absorption of lipid-soluble drugs is considered an effective way to improve their oral bioavailability. Pharmacokinetic analysis of whole blood and intestinal lymph fluid in rats revealed that the bioavailability of the probucol olive oil formulation was significantly higher than that of the suspension (AUC). last 12360 vs 3210 P <0.01). (AUC of PO-1 and PO-2 groups) last The difference was 4543 ng / ml, mainly because after the mesenteric lymph fluid of the PO-2 group rats was collected, the drug transported via the intestinal lymphatic system no longer entered the bloodstream. The proportion of probucol olive oil preparation transported via the intestinal lymphatic system after oral administration was significantly increased compared to the suspension group (37% vs 0%). P <0.01), which is consistent with the results of in vitro chylomicron distribution experiments. The AUC values ​​of the PO-3 and PO-4 groups were <0.01. last The difference between the two groups was very small. Combined with the analysis of lymph fluid from the PO-2 and PO-4 groups, it was found that the total dose percentage of lymph fluid in the PO-2 group was significantly higher than that in the PO-4 group (Dose: 0.868% vs 0.580%). P <0.01). This indicates that the intestinal lymphatic transport capacity of probucol olive oil preparations is higher than that of probucol suspension preparations.

[0164] Because lipid-soluble drugs have poor water solubility, their formulation development is often a significant challenge. One common formulation approach is to prepare the drug as a nanoscale suspension, freeze-dry it, and then form it into tablets or capsules. However, these formulations are often very expensive. Another approach is to prepare the drug as an emulsion; however, because emulsions contain a large amount of active agents on their surface, they cannot be used for long-term administration. Currently, probucol drugs on the market are mainly in tablet form. Some researchers have attempted to formulate probucol into nanoforms, which significantly enhances its oral bioavailability. [6]However, this places high demands on pharmaceutical costs. Since olive oil is rich in long-chain fatty acids and monounsaturated fatty acids, this study is the first to select olive oil as an excipient for probucol formulations. Pharmacokinetic studies confirmed that the oral bioavailability of probucol olive oil formulations is higher than that of suspension formulations, suggesting the potential of olive oil in drug lymphatic transport and providing strong experimental data for the development of new dosage forms of lipid-soluble drugs.

[0165] Drug absorption via the mesenteric lymphatic system offers significant advantages over traditional portal vein absorption. Firstly, drugs in the intestinal lymphatic fluid directly enter the systemic bloodstream through the left jugular angle, avoiding the first-pass effect of the liver, which significantly improves oral bioavailability. Furthermore, some drugs bind to intestinal lymph nodes, making them suitable for targeted tumor therapy and immunotherapy. The rat intestinal lymphatic cannulation model is currently the most widely used in vivo model for studying drug lymphatic absorption. This model, which collects post-administration lymphatic fluid through mesenteric lymphatic cannulation in rats, is uniquely significant for studying the pharmacokinetics of orally administered lipid-soluble drugs. Rat models can be categorized into anesthetized rat models, awake and restricted rat models, and awake and unrestricted rat models. The awake and unrestricted rat model best simulates normal physiological states, but its application is limited due to the difficulty and low success rate of surgical modeling. This study overcame many difficulties in modeling, successfully establishing an awake and unrestricted rat model with assisted reflux of intestinal lymphatic fluid drainage, and conducted pharmacokinetic experiments on the mesenteric lymphatic transport of probucol. This is the first time that this model has been used to conduct pharmacokinetic studies of probucol, both domestically and internationally. The success of this experiment also provides new research methods and ideas for similar pharmacokinetic studies in the future.

[0166] Studies have shown that probucol primarily undergoes elimination in vivo, a slow elimination process. After absorption into the bloodstream, the drug is distributed to various tissues and organs or excreted. If the elimination rate is slow, the drug remains in tissues for a longer period, resulting in a prolonged duration of action. Because probucol is poorly absorbed orally and accumulates in adipose tissue, it can persist for up to six months after discontinuation. Therefore, pharmacokinetic studies of this drug and its various dosage forms are a long-term and complex undertaking, and no relevant experimental data have been reported domestically or internationally to date. This experiment compared probucol olive oil formulations with probucol suspensions to realistically reflect the drug's basic pharmacokinetic data, directly demonstrating the increased bioavailability of probucol using oil as an adjuvant. This provides strong pharmacokinetic evidence for improving the oral absorption of poorly soluble drugs using oil adjuvants. This suggests that when developing new probucol dosage forms, research on lipid-soluble adjuvant formulations and their pharmacokinetic parameters should be increased.

[0167] In summary, this invention establishes a novel method for continuous drainage of mesenteric lymph fluid in rats via jugular venous reflux. This method allows for repeated and timely collection of intestinal lymph fluid under normal physiological conditions in rats, enabling qualitative and quantitative analysis of lipids, proteins, exosomes, electrolytes, and immune cells. This contributes to a better understanding of the absorptive capacity and functional status of intestinal capillary lymphatic vessels. For example, in pathological conditions (such as cancer, metabolic disorders, acute or chronic inflammatory diseases), the concentration of specific biomarkers (such as tumor cells and viruses) in lymph fluid is significantly enhanced compared to peripheral blood. Analyzing lymph fluid helps researchers interpret disease mechanisms and make early diagnoses. Furthermore, mesenteric lymph fluid is widely used to evaluate the transport and metabolism of lipid-soluble drugs and other lipophilic molecules in the intestine because lipid products flow through the mesenteric lymphatic vessels and thoracic duct, ultimately draining into the anterior vena cava, thus avoiding first-pass metabolism in the liver. Nanoparticles, liposomes, and nanoemulsions enhance drug bioavailability. Therefore, this invention provides an innovative technical means for the collection of mesenteric lymph fluid, and has broad application prospects in the study of physiological functions of intestinal lymph, absorption of lipid-soluble drugs, diagnosis and mechanism of intestinal diseases.

[0168] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A system for continuous drainage of mesenteric lymph, characterized in that, The system includes: 1) Mesenteric lymphatic duct catheter: used to insert into the mesenteric lymphatic vessels to drain mesenteric lymph; the mesenteric lymphatic vessels are lymphatic vessels running parallel to the mesenteric arteries; the mesenteric lymphatic duct catheter is a cannula, including a PU tube and a fixing buckle formed by a silicone tube; the fixing buckle is used to fix the mesenteric lymphatic duct catheter to the abdominal wall muscles to prevent displacement within the abdominal cavity; the tip of the PU tube is arc-shaped; 2) Jugular vein catheter: used for insertion into the jugular vein; 3) Collector, used to collect lymph fluid; 4) A two-channel awake activity device, including a first channel for collecting lymph fluid and a second channel for injecting isotonic solutions or for drawing blood. One end of the first passage is connected to the mesenteric lymphatic duct via a quick-connect button VAB, and the other end extends into the collector, with the other end being at the same height as the inlet end of the mesenteric lymphatic duct. The other end is the outlet end of the mesenteric lymph fluid collection tube. One end of the second pathway is connected to the jugular vein catheter via a quick-connect button VAB, and the other end is a port for injecting isotonic ion solution or for drawing blood.

2. The system as described in claim 1, characterized in that, The VAB is used for implantation under the skin.

3. The system as described in claim 1, characterized in that, Includes at least one of the following: 1) The silicone tube has an ID of 0.51 × an OD of 0.94 mm; 2) The PU tube has an ID of 0.4 × an OD of 0.7 mm; 3) The silicone tube is fixed with glue at 1cm, 4cm, 5.5cm and 7cm from the tip of the PU tube to form a fixing buckle.

4. The system as described in claim 1, characterized in that, The jugular vein catheter is a cannula, including a PU tube and a fixing buckle formed by a silicone tube. The fixing buckle is used to fix the jugular vein catheter to the abdominal wall muscle to prevent displacement within the abdominal cavity.

5. The system as described in claim 4, characterized in that, Includes at least one of the following: 1) The silicone tube has an ID of 0.76 × an OD of 1.65 mm; 2) The PU tube has an ID of 0.6 × an OD of 1.0 mm; 3) The silicone tube is fixed with glue at 3.5cm and 4.5cm from the tip of the PU tube to form a fixing buckle; 4) The tip of the PU tube is arc-shaped.

6. The system as described in claim 1, characterized in that, Including the following: The system also includes a VAB loop connector; When collecting lymph fluid, the VAB and the mesenteric lymph fluid collection tube and the infusion tube are connected, so that the infusion tube, jugular venous catheter, jugular vein, mesenteric lymphatic vessels, mesenteric lymphatic vessel catheter, VAB and mesenteric lymph fluid collection tube form a lymph fluid collection pathway in the animal's body. When lymph fluid is not collected, the VAB and VAB circuit connector are connected, so that the mesenteric lymphatic vessels, mesenteric lymphatic ducts, VAB and VAB circuit connectors, jugular vein, and jugular vein duct form a circulatory loop in the animal's body.

7. The system as described in claim 1, characterized in that, Includes at least one of the following: 1) The system also includes microscissors for making incisions in lymphatic vessels and / or jugular veins; 2) The system also includes arterial clamps and ligation sutures for temporarily blocking lymphatic pathways and / or temporarily blocking the proximal end of the external jugular vein; 3) The system also includes a component for restoring the organ's position to its original state; 4) The system also includes a component for administering edible oil, which is used to generate milky white chylomicrons in the body for locating the mesenteric lymphatic vessels; 5) The mesenteric lymphatic vessels, jugular vein catheters, mesenteric lymph fluid collection tubes, and infusion tubes are made of medical grade materials.

8. The system as described in claim 7, characterized in that, In feature 4), the edible oil is preferably corn oil.

9. The system as described in claim 1, characterized in that, Includes at least one of the following: 1) The jugular vein mentioned is the superficial jugular vein; 2) The flow rate of lymph collected at the end of the mesenteric lymph collection tube represents the actual flow rate in the animal's body, and the system is a system used to study the corresponding functions of the lymphatic system.

10. The system as claimed in claim 1, characterized in that, Includes at least one of the following: 1) The system also includes a flow regulating component for adjusting the flow rate of the infusion tube; the flow regulating component is used to adjust the flow rate of the isotonic ion solution in real time according to the lymph flow rate in order to maintain fluid balance and homeostasis of the internal environment; 2) The system also includes a data analysis module, which is used to analyze the collected lymph fluid.

11. Use of the system according to any one of claims 1-10 in the preparation of products for continuous drainage of mammalian mesenteric lymph.

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