CANCER TREATMENT BY IN VITRO AMINO ACID DEPRIVATION

Abstract

Provided is a method for cancer treatment in a patient comprising extracorporeal dialysis of blood, plasma or peritoneal fluid of the patient with a dialysis system for removing a target amino acid, and the dialysis system comprising: a dialysis machine, a dialyzer having a dialysis membrane, and a dialysate; wherein the dialyzer is connected to the dialysis machine and the dialysate flows within the dialysis machine and the dialyzer; and an enzyme for degrading the target amino acid is provided to the dialysis membrane and/or the dialysate, and the target amino acid includes asparagine, glutamine, arginine, serine, methionine or any combination thereof. By modifying dialysis to achieve in vitro amino acid deprivation and incorporating personalized diagnosis, the present invention not only provides a novel precision medicine with better anticancer efficacy and less side effects, but also requires less time and cost for development.

Claims

1. A method for cancer treatment in a patient comprising extracorporeal dialysis of blood, plasma or peritoneal fluid of the patient with a dialysis system for removing a target amino acid, and the dialysis system comprising: a dialysis machine, a dialyzer having a dialysis membrane, and a dialysate; wherein the dialyzer is connected to the dialysis machine and the dialysate flows within the dialysis machine and the dialyzer; and an enzyme for degrading the target amino acid is provided to the dialysis membrane and/or the dialysate, and the target amino acid includes asparagine, glutamine, arginine, serine, methionine or any combination thereof.

2. The method as claimed in claim 1, wherein the dialysate is in an amount of 1 time to 9 times of the amount of the blood, the plasma or the peritoneal fluid of the patient.

3. The method as claimed in claim 1, wherein the flow rate or circulation speed of the dialysate is 200 ml/min to 600 ml/min.

4. The method as claimed in claim 1, wherein the concentration of the enzyme for degrading the target amino acid in the dialysate is more than 0.1 K.U./ml.

5. The method as claimed in claim 1, wherein the dialyzer has an effective membrane area more than 0.5 m.sup.2.

6. The method as claimed in claim 1, wherein the extracorporeal dialysis requires 3 minutes to 1 hour for each time.

7. The method as claimed in claim 1, wherein the extracorporeal dialysis is carried out periodically or irregularly and lasts for 3 days to 2 weeks.

8. The method as claimed in claim 1, wherein the cancer is selected from a group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), small cell lung cancer, lung mesothelioma, chest wall tumor, abdomen-pelvis tumor, small intestinal tumor, thymoma, mediastinal tumor, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma blood cancer, lymphoma, pancreatic cancer, sarcoma, brain cancer, low-grade astrocytoma, high-grade astrocytoma, pituitary adenoma, meningioma, CNS lymphoma, oligodendroglioma, brain stem tumor, ependymoma, breast cancer, colorectal cancer, liver cancer, liver adenocarcinoma, gallbladder cancer, biliary tract cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, craniopharyngioma, laryngeal cancer, oropharyngeal cancer, salivary gland tumor, hypopharyngeal cancer, thyroid cancer, oral cavity tumor, kidney cancer, bladder cancer, anal cancer and skin cancer.

9. A method for personalized cancer treatment by removing a target amino acid in a patient, which comprises: a tumor analysis comprising the steps of: (A1) obtaining a cancer cell from the patient; (A2) cultivating the cancer cell to obtain a cell culture; and (A3) conducting an amino acid deprivation test to obtain a profile indicating the type of the target amino acid along with the corresponding concentration and period for cell death of the cell culture, wherein the target amino acid includes asparagine, glutamine, arginine, serine, methionine or any combination thereof; a pattern analysis of blood concentration fluctuation of the target amino acid; extracorporeal dialysis of blood, plasma or peritoneal fluid of the patient with a dialysis system for removing the target amino acid, and the dialysis system comprising: a dialysis machine, a dialyzer having a dialysis membrane, and a dialysate; wherein the dialyzer is connected to the dialysis machine and the dialysate flows within the dialysis machine and the dialyzer; and an enzyme for degrading the target amino acid is provided to the dialysis membrane and/or the dialysate, and the dialyzer is a high flux dialyzer; a blood monitoring process for the concentration of the target amino acid; nutrition management during the cancer treatment of the patient; wherein the pattern analysis is carried out before the extracorporeal dialysis, and the extracorporeal dialysis is carried out stage by stage; and a tumor tracking process during the personalized cancer treatment of the patient.

10. A hemodialysis system comprising: a dialysis machine, a dialyzer having a dialysis membrane, and a dialysate; wherein the dialyzer is connected to the dialysis machine and the dialysate flows within the dialysis machine and the dialyzer; and an enzyme for degrading a target amino acid is provided to the dialysis membrane and/or the dialysate, and the target amino acid includes asparagine, glutamine, arginine, serine, methionine, or any combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG. 1 is a schematic diagram showing the extracorporeal dialysis system of the present invention.

[0070] FIG. 2 is a schematic diagram showing the fluctuation of blood amino acid concentration of the patient.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] In the following, several examples are showed to demonstrate the present invention. One person skilled in the art can easily understand the merits and effects through these examples. It should be understood that the examples in the specification are only for the purpose of illustrating the implementation of the present invention, but shall not be used to limit the scope of the present invention. One person skilled in the art can make necessary changes or modifications to implement or apply the content of the present invention without departing from the spirit of the present invention.

EXAMPLE 1

Modified Hemodialysis

[0072] FIG. 1 shows an extracorporeal dialysis system 10 of one embodiment of the present invention in a simple configuration whereby the blood of a patient is taken out of the body through a blood line 100, pumped by the pump (not shown) through a dialyzer 110 and back into the patient through another blood line 120. Dialysates 130 are pumped to flow in the direction opposite to that of the blood in the dialyzer 110. The dialyzer 110 has a dialysis membrane 111, and the dialysis membrane 111 has two surfaces 1111 and 1112 opposite to each other. The surface 1111 contacts the blood and the surface 1112 contacts the dialysates 130. Further, enzymes 112 for amino acid degradation are immobilized or coated on the surface 1112 that contacts the dialysates 130 to refrain the enzymes 112 for amino acid degradation from entering the blood of the patient.

[0073] In the alternative, the enzymes 112 for amino acid degradation could be added in the dialysates 130 in advance or added by a supplement line 140 during the operation of the dialysis system 10.

[0074] All amino acids in the patient's blood could pass the dialysis membrane 111 and only the target amino acid will be decomposed by the enzymes 112 for amino acid degradation. As mass transport across the dialysis membrane 111 will tend to equilibrate concentrations, the concentration of any molecular species in the patient's blood will approach the same concentration in the dialysates 130 of the same species. Hence, while the concentration of the target amino acid in the patient's blood is higher than that in the dialysates 130 in the dialyzer 110, the target amino acid in the patient's blood will keep moving to the dialysates 130 and be removed from the patient's blood so as to reduce the concentration of the target amino acid in the patient's blood.

[0075] The dialysates 130 are provided to the dialyzer 110 by the circulation pipeline 150 of a dialysis machine (not shown). The dialysates 130 could be further filtered to remove the wastes derived from the degradation of amino acid and from the patient's blood. In the alternative, the dialysates 130 are non-recyclable and discarded after leaving the dialyzer 110.

[0076] The dialysis membrane 111 is a flat membrane type. Preferably, the dialyzer 110 is a high flux dialyzer and the dialysis membrane 111 is a hollow fiber type with the patient's blood moving through the fibers, and the extrafibrilar space between the hollow fibers in the dialyzer 110 is filled with and flushed by the dialysates 130. The flow of the dialysates 130 in the extrafibrilar space is in the opposite direction from that of the patient's blood in the fiber. The wall of the fiber allows passage of substances with low molecular weight only, e.g. water and amino acids.

[0077] In the present embodiment, the dialysates 130 are a normal saline. Preferably, the dialysates 130 are further supplemented with other ingredients or nutrients. More preferably, the dialysates 130 are added with amino acids except the target amino acid.

EXAMPLE 2

Modified Hemodialysis

[0078] Example 2 is carried out as follows:

[0079] 1. Simulated blood:

[0080] 900 ml of pig's blood and 100 ml of Gibco Roswell Park Memorial Institute (RPMI) 1640 Medium (RPMI 1640 Medium) were mixed to obtain a diluted pig's blood. The diluted pig's blood was further added with 1.8 mg/ml EDTA-K.sup.2+, 0.05% sodium azide; and 132 μl of Heparin with the concentration of 5000 U/ml to obtain a simulated blood, wherein the concentration of EDTA-K.sup.2+ of the simulated blood was 16 mg/ml.

[0081] 2. Setting for hemodialysis:

[0082] The dialysis machine is Toray TR-321. The dialysate for extracorporeal circulation was triple the amount of the simulated blood and was 3000 ml, and the flow rate of dialysate was 500 ml/min. Before the simulated blood entered the dialysis machine, 1X PBS was used to clean the circulation pipeline of the dialysis machine, to achieve the required balance of the artificial kidney (Polyflux 17L), and to get rid of all bubbles in the artificial kidney, and the circulation speed was 250 ml/min. Besides, the effective membrane area of the artificial kidney is 1.7 m.sup.2.

[0083] The dialysate consisted of Buffer A (hemodialysis concentrate No. 16, purchased from Taiwan Biotech Co., LTD), Buffer B (Sodium Bicarbonate Concentrate 8.4%, purchased from Fressenius Medical Care Ag, 61348 Bad Homburg, Germany) and R.O. water, wherein the volume ratio of Buffer A, Buffer B and R.O. water is 1:1.225:32.775.

[0084] The target amino acid is Asparagine (Asn), and the rest are non-target amino acids. The 3000 ml dialysate was added with 3 ml of Asparaginase with the concentration of 1000 K.U./ml (LEUNASE®) in the Experimental group 1, so the concentration of Asparaginase in the dialysate is 1 K.U./ml. No Asparaginase was added in the Control group 1.

[0085] The conductivity of the dialysate was adjusted to about 14 mS/cm. The dialysis temperature was set to be 37° C. When the pressure of the circulation pipeline, the temperature and the conductivity of the dialysate reached the desirable value and were stable, a green light signaled to indicate that hemodialysis was ready. The simulated blood was pumped into the circulation pipeline and entered the artificial kidney. Besides, the simulated blood was further added with Heparin with the concentration of 5000 U/ml at the flow rate of 4.4 ml/hour before the simulated blood entered the artificial kidney. The concentrations of amino acids in the simulated blood before and after dialysis were detected by ultra performance liquid chromatography (UPLC) and the results were shown in Table 1 and Table 2.

TABLE-US-00001 TABLE 1 The concentrations (μM) of amino acids of the simulated blood of the Control group 1 Retention Dialysis time (min) types Time 0 10 20 30 Asn 2.14 22.3 9.9 N/A N/A Ser 2.854 147.8 43.1 27.2 25.3 Gln 3.006 204.5 49.5 30.9 22.2 Arg 3.119 111.0 27.2 16.4 13.8 Met 7.373 33.3 9.7 5.8 5.0

[0086] According to Table 1, the concentrations of all amino acids were lowered due to the equilibrium between the dialysate and the simulated blood. Further, Asparagine (Asn) is undetectable within 20 minutes of dialysis, and the limit of detection (LOD) of Asparagine was 5 μM.

TABLE-US-00002 TABLE 2 The concentrations (μM) of amino acids of the simulated blood of the Experimental group 1 Retention Dialysis time (min) types Time 0 3 5 10 20 30 Asn 2.1 41.8 32.3 24.2 N/A N/A N/A Ser 2.9 159.8 67.7 63.2 17.5 20.0 17.8 Gln 3.0 381.8 147.5 131.5 24.2 18.2 18.3 Arg 3.1 212.4 83.0 74.6 16.9 14.7 14.3 Met 7.4 38.5 16.0 14.4 3.7 3.8 3.5

[0087] According to Table 2, due to the addition of Asparaginase, Asparagine (Asn) is undetectable within 10 minutes of dialysis, even the original concentration of Asparagine in the Experimental group 1 is higher than that in the Control group 1. Hence, by adding an enzyme for degrading the target amino acid, the removal of the target amino acid could be speeded up. Further, as the extracorporeal dialysis could be completed earlier after adding the enzyme for degrading the target amino acid, the reduction of non-target amino acids in the blood by equilibrium will be alleviated so as to lower the risk of incurring side effect derived from the removal of non-target amino acids.

EXAMPLE 3

Modified Peritoneal Dialysis

[0088] Example 3 is carried out as follows:

[0089] 1. Preparation of peritoneal fluid:

[0090] RPMI 1640 Medium was ten times diluted to obtain 1 L of RPMI 1640 diluent with Dianeal PD-2 (Peritoneal Dialysis Solution) and to serve as a peritoneal fluid for the control group 2. 1 L and 2 L of the same RPMI 1640 diluent was prepared for the Experimental group 2 and Experimental group 3, respectively.

[0091] 2. Setting for peritoneal dialysis:

[0092] The dialysis machine (Toray TR-321) had a circulation volume for a dialysate which was the same amount of the RPMI 1640 diluent, and the volume ratio for the peritoneal fluid and dialysate is 1:1 for the control group 2 and the experimental groups 2 & 3. The flow rate or circulation speed of the dialysate was 250 ml/min. Before the peritoneal fluid (RPMI 1640 diluent) entered the dialysis machine, RPMI 1640 diluent was also used to clean the circulation pipeline of the dialysis machine, to achieve the required equilibrium of the artificial kidney, and to get rid of all bubbles in the artificial kidney. Besides, the artificial kidneys of the control group 2 and the experimental group 2 were NV-15U with the effective membrane area of 1.5 m.sup.2; and the artificial kidney of the experimental group 3 was NV-21U with the effective membrane area of 2.1 m.sup.2.

[0093] The dialysate is DIANEAL PD-2 Peritoneal Dialysis Solution with 1.5% Dextrose (Baxter). The target amino acid is Asparagine (Asn), and the concentration of Asparaginase in the dialysate is 1 K.U./ml in the Experimental groups 2 & 3. No Asparaginase was added in the control group 2.

[0094] The dialysis temperature was set to be 37° C. The peritoneal fluid (RPMI 1640 diluent) was first pumped into slot A for temporary storage and then entered the artificial kidney. After peritoneal fluid (RPMI 1640 diluent) left the artificial kidney, peritoneal fluid (RPMI 1640 diluent) further entered slot B for temporary storage, and then left the dialysis machine, e.g. the peritoneal fluid (RPMI 1640 diluent) was pumped back to the peritoneal cavity of the patient. The concentrations of amino acids in the peritoneal fluid before and after dialysis were studied by using Amino Acid Analysis Systems (Waters UPLC® Amino Acid Analysis (AAA) Solution) and the result was shown in Table 3 to Table 5.

TABLE-US-00003 TABLE 3 The concentrations (μM) of amino acids of the peritoneal fluid of the control group 2 Dialysis time (min) types 0 3 5 10 20 30 60 Asn 46.276 33.144 27.376 17.28 19.86 16.02 16.036 Arg 101.5 81.1 66.7 39.6 46.9 38.2 38.4

[0095] Arginine (Arg) serves as a reference to know the decreasing rate of a non-target amino acid.

[0096] According to Table 3, the concentration of both amino acids were lowered due to the equilibrium between the dialysate and the peritoneal fluid.

TABLE-US-00004 TABLE 4 The concentrations (μM) of amino acids of the peritoneal fluid of the experimental group 2 Dialysis time (min) types 0 3 5 Asn 27.284 23.254 9.088 Arg 102.6 90.0 70.5

[0097] According to Table 4, due to the addition of Asparaginase, the concentration of Asparagine (Asn) is reduced to be less than 50% within 5 mins. In contrast, according to Table 3, the concentration of Asn is still more than 50% within 5 mins. Hence, by adding an enzyme for degrading the target amino acid, the removal of the target amino acid could be speeded up. Further, the decreasing rates of Arg in Table 3 and Table 4 are similar, which means the side effect derived from the removal of non-target amino acids could be lowered by decreasing the dialysis time.

TABLE-US-00005 TABLE 5 The concentrations (μM) of amino acids of the peritoneal fluid of the experimental group 3 Dialysis time (min) types 0 3 5 10 Asn 30.674 N/A N/A N/A Arg 83.86 70.12 37.31 19.99

[0098] According to Table 5 and compared with Table 4, the artificial kidney of the experimental group 3 has the effective membrane area of 2.1 m.sup.2, which is larger than that of the experimental group 2, and Asparagine (Asn) is undetectable within 3 minutes of dialysis, and the limit of detection (LOD) of Asparagine was 5 μM. Hence, by using the artificial kidney with a larger effective membrane area, the removal of target amino acid could be speeded up.

[0099] Besides, the concentration of Arginine (Arg) is lower to 50% of the original concentration within 5 minutes in experimental group 3, and the concentration of Arginine is lower to 50% of the original concentration within 10 minutes in experimental group 2. Hence, by using the artificial kidney with larger effective membrane area, the removal of non-target amino acids will be speeded up as well. As the removal of non-target amino acids may incur side effect, the artificial kidney with the proper effective membrane area is suggested and further illustrated in Example 4.

EXAMPLE 4

Modified Peritoneal Dialysis

[0100] Example 4 was carried out with the method the same as Example 3 except that the artificial kidneys with different effective membrane areas were adopted. The concentrations of amino acids in the peritoneal fluid before and after dialysis were studied and the result was shown in Table 6.

TABLE-US-00006 TABLE 6 The concentrations (μM) of amino acids of the peritoneal fluid of Example 4 Dialysis time (min) Dialyzer Types 0 3 5 10 20 polyflux-140H Asn (μM) 36.9 31.6 23.0 N/A N/A (1.4 m.sup.2) Asn (%) 100 85.8 62.3 N/A N/A Arg (μM) 93.7 87.6 79.4 66.9 54.2 Arg (%) 100 93.6 84.8 71.4 57.9 BG-1.6U Asn (μM) 45.5 33.6 N/A N/A N/A (1.6 m.sup.2) Asn (%) 100 73.90 N/A N/A N/A Arg (μM) 95.3 82.2 71.3 62.7 47.4 Arg (%) 100 86.3 74.8 65.8 49.7 BG-1.8U Asn (μM) 36.7 N/A N/A N/A N/A (1.8 m.sup.2) Asn (%) 100 N/A N/A N/A N/A Arg (μM) 89.0 66.8 68.0 54.7 54.4 Arg (%) 100 75.1 76.5 61.5 61.2

[0101] According to Table 6, by using the artificial kidney with a larger effective membrane area, the removal of target amino acid could be speeded up, while the concentration of non-target amino acid could be maintained at about 50% to reduce the risk of side effect.

[0102] Besides, the adaptation of the artificial kidney with the effective membrane area less than 1.4 m.sup.2 requires longer time for dialysis. Hence, the artificial kidney with the effective membrane area within 1.4 m.sup.2 to 1.8 m.sup.2 was suggested.

EXAMPLE 5

Tumor Analysis

[0103] The present invention further comprises a tumor analysis before the extracorporeal dialysis, and the tumor analysis comprises the steps of (A1) to (A3) as follows:

[0104] (A1) obtaining a cancer cell from the patient: The cancer cell could be taken from the biopsy after surgery. Preferably, the cancer cell is a circulating tumor cell (CTC) collected from the blood sample of the patient. The collection method could be isolation by size of epithelial tumor cells (ISET) method or carried out by IsoFlux system of Fluxion Biosciences, Inc. or other commercially available technologies.

[0105] (A2) cultivating the cancer cell to obtain a cell culture: The cell culture could be a traditional two-dimensional (2D) culture. Preferably, the cell culture is a 3D cell culture. For example, the 3D cell culture could be a patient-derived three-dimensional organoid culture by using Matrigel to suspend the cell pellets. In the alternative, 3D scaffold system by the fabrication of 3D polycaprolactone (PCL) porous scaffolds is also available for culturing CTCs.

[0106] (A3) conducting an amino acid deprivation test to obtain a profile indicating the type of the target amino acid along with the corresponding concentration and period for cell death of the cell culture: After the 2D or 3D cell culture of the cancer cell from the patient is available, such cell culture is further divided for subculture in different levels and types of amino acid deprivation to acquire the data for the cell death of the cell culture. The data includes the types of the deprived amino acid, and the lethal low level of concentration and the period for the cell death of the cell culture. By analyzing such data, the vulnerability or sensitivity of the tumor could be utilized for the hemodialysis for removing target amino acid so as to kill the tumor specifically.

EXAMPLE 6

Blood Amino Acid Concentration Management

[0107] The concentration of the target amino acid in blood could be quantified by Ultra Performance Liquid Chromatography (UPLC). After the target amino acid is available from the tumor analysis, the blood amino acid concentration fluctuation as shown in FIG. 2 could be observed and a pattern analysis thereof could be executed before the extracorporeal dialysis. As the target amino acid in blood may increase sharply after meals, the timing to carry out extracorporeal dialysis to timely remove the target amino acid is calculated and decided according to the pattern of blood amino acid concentration fluctuation.

[0108] During the cancer treatment of the patient, continuous blood monitoring process for the concentration of the target amino acid is also required. Preferably, the data of the target amino acid concentration is received in a real time manner during the extracorporeal dialysis and periodically during the intervals of extracorporeal dialysis. In the alternative, the frequency to get the data of the target amino acid concentration could be reduced and said data could be acquired within 2 hours after a meal and whenever the extracorporeal dialysis is completed only.

[0109] Further, the blood monitoring process could be provided to track compensatory effect due to the deprivation of the target amino acid.

[0110] In order to timely remove the target amino acid at an accurate clearance rate, nutrition management for the patients is provided during the cancer treatment, so that the concentration of the target amino acid after meals could be well-controlled to stay within an acceptable fluctuation range. Further, the nutrition management will not remove the food releasing the target amino acid from daily diet of patients completely.

[0111] Preferably, the data to draw the blood amino acid concentration fluctuation as shown in FIG. 2 is acquired under the condition that the patient follows the instruction the same as the nutrition management, so that the pattern analysis aforementioned could be more precise and reliable. To sum up, the present invention modifies dialysis to achieve in vitro amino acid deprivation and further incorporates personalized diagnosis, the present invention not only provides a novel precision medicine with better anticancer efficacy and less side effects, but also requires less time and cost for development. Even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the steps and features of the invention, the disclosure is illustrative only. Changes may be made in the details, especially in matters of shape, size, and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.