Patent Application
20250093329
While advancements have been made in the treatment of various cancers, most treatments have been developed for wide application to all patients. A targeted approach, wherein anti-cancer compounds or any FDA approved drugs are selected for an individual's unique cancer and unique body chemistry remains a challenge. To achieve this goal will require the ability to use cancer cells from the patient in conjunction with new screening methods to identify those compounds capable of killing the cancer without damaging the patient's healthy, normal cells. Further, the ability to transport the patient's cells to the testing lab must be improved to ensure cell viability for a period of time to carry out the necessary tests. Finally, the screening methods need to occur in a rapid manner to permit treatment of the patient as soon as possible.
Disclosed is a method for assessing the in vitro effectiveness of a compound, or a mixture of compounds, against a specific cancer type. The method includes the steps of:
Also disclosed is a method for preparing patient derived cancer cells. The method includes the steps of:
Further disclosed is a method for controlling the metabolic pathways used by cells during in vitro testing. The method includes the steps of:
Throughout this disclosure, the terms “about”, “approximate”, and variations thereof, are used to indicate that a value includes the inherent variation or error for the device, system, the method being employed to determine the value, or the variation that exists among the study subjects.
The following examples demonstrate the use of a variety of heteroaromatic compounds as the R3 scaffold material for production of N-benzyl-sulfonamides and a variety of N-substrates with functional groups R1. The final products are depicted as compounds 1-30 in
The present disclosure provides a method for determining whether or not a variety of compounds will likely have pharmacological activity against a patient's specific cancer cells. The following methods were developed to assess the in vitro effectiveness of the compounds alone, when combined with a metabolic inhibitor or in media formulated to promote use of specific metabolic pathways. However, prior to carrying out the screening method to determine the pharmacological effectiveness of a compound, patient cancer cells must first be obtained and transported to the testing facility.
Cancer cells must acquire nutrients, growth factors, and other components from the patient's own circulatory system, and so have evolved and are adapted to the unique blood serum in which they grow. Therefore, to provide the most accurate assessment of the potential for a compound to treat cancer, the compounds must be tested on cancerous cells obtained from the patient. Cancer cells can be obtained from the patient through conventional biopsy practices, including but not limited to resected tumor, needle biopsy and blood in the case of hematopoietic cancers. These cells are added to a patient derived serum and/or plasma. The patient derived serum and/or plasma having previously been prepared. As used herein, the terms “patient serum” and “patient plasma” indicates that the serum and/or plasma was obtained from the patient. Serum and plasma both come from the liquid portion of the blood that remains once the cells are removed. However, serum is the liquid that remains after the blood has clotted, while plasma is the liquid that remains when clotting is prevented with the addition of an anticoagulant. The following methods may use patient serum, patient plasma or a combination of both. The final choice will be made by the individual conducting the screening method based on the patient status and the cancer type.
The patient derived serum and/or plasma is one optional base media use for preparing patient derived cancer cells in individualized media. In this option, 100% of the base media is patient derived serum and/or plasma. Another optional base media is a conventional cell base media designed to mimic the composition and in vivo environment of the organ in which the cancer appears. The conventional base media is supplemented with between about 5% to about 25% of the patient derived serum and/or plasma.
The method of preparing the patient derived cancer cells includes the use of individualized media specifically formulated to correspond to the location of the cancer within the patient's body, i.e. individualized cancer cell support media. The individualized cancer cell support media includes the patient's cancer cells and either a base media of 100% patient derived serum and/or plasma or the above described conventional cell base media with about 5% to about 25% of the patient derived serum and/or plasma. The individualized cancer cell support media simulates the in vivo environment by inclusion of amino acids, vitamins, inorganic salts and glucose in concentrations corresponding to the concentrations found in the organ in which the cancer appears. One skilled in the art will be readily able to determine the particular amino acids required and the concentrations of the amino acids and other constituents needed for the individualized media solution to which the cancer cells and patient derived serum and/or plasma will be added.
The individualized media will further include antibiotics commonly added to cell supporting individualized media, such as penicillin and streptomycin, in concentrations suitable to ensure the viability of the cells during the time necessary for transport and testing. Finally, the individualized media will include a buffering system suitable for maintaining pH of the individualized media in the range of 7.2 to 7.4. One common buffering system suitable for use in the individualized media is zwitterion HEPES. Alternatively, for long term storage or shipping, the buffering system may rely upon sodium bicarbonate and the final formulated individualized media maintained under a carbon dioxide atmosphere. Thus, the resulting individualized media corresponds closely to the patient's body chemistry, i.e. the environment and metabolic conditions of the cancer. Thus, use of the individualized media will likely result in the cultured patient-derived cancer cell metabolism remaining similar to that of cancer cells in the patient.
The patient derived serum and/or plasma and specially formulated individualized media enhance the likelihood that the harvested cancer cells will continue to use the same metabolic pathways used during cancer growth in the patient. Thus, screening of potential compounds for treatment of the cancer has a greater likelihood of identifying those compounds which will take advantage of the same metabolic pathways. Further, use of the patient derived serum and/or plasma and specially formulated individualized media enhances cell growth of the harvested cancer cells. In general, upon addition of the cancer cells to the patient derived serum and/or plasma and specially formulated individualized media, the cancer cells will immediately begin cellular reproduction. Thus, the cancer cells should not be frozen prior to shipment and/or testing. The disclosed method avoids the need to culture the cells long tenn, which has the potential to alter their metabolism such that it becomes different than that of the tumor. Additionally, the probability of cell contamination during shipping will be reduced.
By providing an environment for cancer cell growth which mimics growth within the patient, the screening method can take advantage of the same metabolic pathways. Hence the screening test will determine those compounds capable of blocking those metabolic pathways which permit cell growth of the cancer cells. Alternatively, the screening method can include steps which simulate diet and environment (e.g. sleep cycles or lack of sleep, body temperature) induced changes in the patient's body chemistry thereby resulting in a change of metabolic pathways used by the cancer cells. Following creation of the change in metabolic conditions, testing may be carried out to determine if a synergistic effect can be achieved by the combination induced metabolic changes and treatment with target compounds.
To demonstrate the ability to identify compounds active against cancer cells in vitro the N-benzyl sulfonamide library of compounds of
The cytotoxicity tests were carried out in the following manner. Living cells are known to convert resazurin to the fluorescent compound resorufin. Test systems which rely upon this reaction are commercially available. One such test is known at the Cell Titer Blue Cell Viability test assay from Promega. Cell cultures for each of the identified cancer lines were obtained from ATCC and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and pen/strep. As known to those skilled in the art, DMEM typically includes the components identified below.
As known to those skilled in the art, pen/strep is a combination of penicillin and streptomycin used to prevent bacterial and fungal contamination of mammalian cell cultures. The pen/strep solution contains 5,000 Units of Penicillin G (sodium salt) which acts as the active base, and 5,000 micrograms of Streptomycin (sulfate) (base per milliliter), formulated in 0.85% saline.
The test method provides for incubating the cell cultures at temperatures which correspond to the range of body temperatures experienced by the patient from which the cancer cells were obtained. Typically, incubation temperatures will be between the temperatures of 36.1° C. and 37.2° C. In most cases incubation will occur at 37° C. Additionally, the cell cultures are kept under an atmosphere which mimics cell conditions of the tumor within the patient's body. In most cases, the tumor microenvironment (TME) is characterized by hypoxia (low oxygen) and may also be characterized by hypercapnia (increased CO2). Hypercapnia results from decreased blood flow which limits CO2 elimination. Restricted CO2 elimination leads to increased HCO3− conversion to CO2 in order to neutralize lactate as altered tumor cell metabolism produces more CO2 than normal. Normal tissue averages about 5% oxygen with oxygen ranges from about 3% to 7.4%. However, median oxygenation in untreated tumors is significantly lower, falling between approximately 0.3% and 4.2% oxygen, with most tumors exhibiting median oxygen levels<2%. In the case of CO2, normocapnic is around 5% CO2, while hypercapnic can rise to 10% CO2. Therefore, to mimic the environment of the tumor the method utilizes an incubator designed to provide the optimal gaseous environment corresponding to that of the tumor. Thus, the incubator will be capable of maintaining an atmosphere having an oxygen content in the range of 0.1% to 10% to 0.1% and a CO2 in the range of 1% to 10%.
After allowing for proliferation of the cells, the cells were distributed across a plurality of test wells containing from 100 μL DMEM plus 10% FBS and allowed to attach to the surface of the test wells. Typically, the time for attachment will require about 12 hours to about 18 hours. Following attachment, the cells were treated with either a solvent control or the N-benzyl sulfonamide compound of interest dissolved in a suitable solvent such as but not limited to DMSO. Typically, about 18 hours to about 36 hours are required to determine the effect of the N-benzyl sulfonamide compound of interest on cell viability. Following treatment of the cells with the N-benzyl sulfonamide or control, the toxicity of the compound to the cells will be determined by addition of a luminescing agent. For example, 10 μl of CellTiter-Blue reagent, i.e. resazurin may be added. Typically, the resazurin is added between about 18 hours to about 36 hours after treating the cells with the N-benzyl sulfonamides or the control. The cells are allowed to consume and convert the resazurin to resorufin for about one to four hours. Subsequently, the fluorescence of resazurin is measured by excitation at 560 nm and recording the emission at 590 nm within an instrument configured to measure fluorescence intensity. Two commercially available systems are the BioTek Cytation 5 plate reader and the Promega Glomax Multi+detection system. For compounds exhibiting cytotoxicity, the half maximal inhibitory concentration (IC50) values were determined using non-linear regression analysis in Graph-Prism software. The method for determining IC50 values is well known in the art and will not be discussed further. As known to those skilled in the art, IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%. Therefore, each N-benzyl sulfonamide will be tested over a range of concentrations. Typically, the concentration ranges will be: 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM. The control in this method is normally dimethyl sulfonamide (DMSO) or other solvent suitable for dissolving the compounds to be tested.
Method for Determining ATP Levels Following Treatment with Two Component Compositions
The conventional cytotoxicity screening method described above can identify compounds that on their own reduce cell viability; however, the above method will miss biologically active compounds that are targeting redundant pathways, pathways that are not being utilized, or pathways the cancer cells can bypass. The above method does not provide any information about a compound's biological targets or mechanism of action. Therefore, one aspect of the present invention includes a screening method suitable for identifying compounds which directly inhibit ATP metabolism by select pathways. Additionally, the following method permits identification of the pathway inhibited. Further, the rapid testing method does not require cell death during the exposure of the cells to the compound of interest.
The improved method for identifying such compounds measures ATP levels as a function of light emitted by any ATP dependent luciferase or modified luciferases, i.e. luciferase derivatives. The method uses a conventional luminescent assay to determine the number of viable cells in a culture. The improvement provided by the present method results from pre-treating cells used in the assays with a metabolic inhibitor prior to treatment with the compound of interest, or switching the cells to a media formulated to force use of a specific metabolic pathway. Luminescent assays for determining cytotoxicity are well known in the art. Assays, kits and methods for measuring ATP levels are disclosed by U.S. Pat. Nos. 7,741,067 and 7,083,911, incorporated herein by reference. Commercially available assays, marketed as the CellTiter-Glo® and CellTiter-Blue® from Promega Corporation, are particularly suited for carrying out the method described below; however, other similar luminescent or fluorescent assays will perform equally well in the described method.
The commercially available assays are configured for the purposes of determining cell viability. In normal usage, the test determines ATP levels in untreated cells, i.e. the control. Corresponding cells are treated with an agent of interest that is suspected of reducing cell viability. In the common practice, resulting data is presented as a comparison of the ATP levels in the treated and untreated cells with the decrease in ATP levels indicative of the effectiveness of the agent. The data may be presented as a direct comparison of the assay output levels or as a percentage using the untreated control cells ATP level as 100%.
In most instances, following the treatment period, the commercially available assays use a lysis buffer to break the cells apart and release ATP. The releasing agent also contains an enzyme which catalyzes a light emitting reaction. Typically, the enzyme is luciferase and its substrate D-luciferin. In the presence of ATP, luciferase catalyzes a reaction that emits light (see
In the present method, the method of using the available assays has been modified to measure the short-term effect of compounds of interest on ATP levels in the cells. The present method does not result in cell death and does not measure cell viability. In the modified method, the control and test cells are initially treated with the metabolic inhibitor for a period of about thirty minutes to about four hours. Typically, the treatment of the cells with the metabolic inhibitor or modified media is for one hour prior to adding the compound to be screened for anti-cancer properties. However, simultaneous treatment with the metabolic inhibitor and the compound being screened should provide satisfactory results. Thus, the modified luminescent assays have been adapted to screen for compounds that directly inhibit ATP metabolism. ATP synthesis in cells occurs over multiple biochemical pathways. These pathways are very responsive to metabolic inhibitors and/or changes in available nutrients. By incorporating a metabolic inhibitor or specially formulated media which is known to impact certain pathways, the disclosed method provides for measurement of a compound's direct effects on the remaining metabolic pathways available for ATP synthesis in the cell.
Because cancers exhibit dysregulation of metabolic pathways, compounds identified in the disclosed method are potential anti-cancer therapeutics. In this screening methodology, cells used in the assays are pretreated with a metabolic inhibitor such as 2-deoxyglucose (2-DG) (inhibits ATP production via glycolysis) or rotenone (inhibits ATP production by mitochondria). As a consequence of the pre-treatment with a metabolic inhibitor, the cell must utilize the remaining uninhibited pathways to maintain cellular ATP levels.
Following treatment of the control and test cells with the metabolic inhibitor (or switching to a new media), the method calls for addition of the compound to be screened for anti-cancer properties to the cells. Following addition of the compound of interest, the assay is allowed to continue for about one hour to about four hours or depending on the luminescing agent up to eighteen hours. However, approximately 60 minutes will be sufficient to determine the ATP inhibiting effect of the compound to be screened on the cells. Following completion of the selected time period, the ATP level within the cell is determined. ATP levels can be determined by luminescence according to standard measuring procedures, i.e. the luminescence level of the assay from the living cells treated with the two component composition is compared to the luminescence level of the assay from the living cells without the two component composition, i.e., the control experiment. Thus, the ATP levels are determined without killing the cells during the incubation of the cells in the presence of the compound of interest. As a result, the measured reduction in ATP level corresponds directly to the inhibition of the metabolic pathways uninhibited by the metabolic blocker.
Thus, the method provides the ability to screen compounds for the ability to specifically target the uninhibited pathway being used to generate ATP. Furthermore, because cells pre-treated with a metabolic inhibitor targeting a first known pathway are forced to use an alternative known pathway that is not inhibited to maintain ATP levels, the screening method provides immediate mechanistic information about the active compound mechanism of action. These results are provided in a relatively short time period of about ninety minutes to about five hours.
A wide variety of ATP luminescing detection reagents are available commercially. So long as the reagent produces a luminescence in the presence of ATP, the reagent will be suitable for use in the present method. Suitable reagents include but are not limited to any ATP dependent luciferase such as but not limited to firefly luciferase, other modified luciferase based reagents, i.e. luciferase derivatives. According to the rapid method for determining the anti-cancer activity of a compound, a sample of living cells is distributed across a number of testing wells. Typically, 96-well plates are used; however, the number of wells is not critical to the current method. When using a 96-well plate the number of living cells will commonly be about 20,000. However, evidence presented herein shows that as few as 200 cells can be accurately evaluated. The sample wells contain a cell growth medium to promote cell health and growth and an additive to prevent bacterial contamination of the wells. One common example of the cell growth medium is DMEM with 10% FBS as described above. One example of the additive to prevent bacterial contamination is a solution of penicillin G and streptomycin referred to commonly as Pen-Strep. The Pen-Strep solution typically contains 5000 units of penicillin G and 5000 micrograms of streptomycin. Thus, the rapid determination of anti-cancer compounds can be carried out using the following method.
Table 1 reports the POC values for a variety of compounds of interest. The number in the far left column of Table 1 corresponds to the compound number of compounds depicted in
As reported in Table 1 below, one group of assays included only the compound of interest. Another group of assays included the compound of interest in combination with 2-deoxyglucose and a third group of assays included the compound of interest with rotenone. Other metabolic inhibitors suitable for use in the disclosed method include but are not limited to: 2-deoxyglucose, rotenone, Lonidamine, 3-bromopyruvate, imatinib, oxythiamine, and 6-aminonicotinamide Glutaminase Inhibitor 968, 6-Diazo-5-oxo-L-norleucine, Amytal, Antimycin A, Sodium Azide, Cyanides, oligomycin, FCCP, Phloretin, Quercetin, 3BP, 3PO, DCA, NHI-1 and Oxamic acid, Fisetin, myricetin, apigenin, genistein, cyanidin, daidzein, hesperetin, naringenin, and catechin.
In Table 1 below, the cell lines tested are reported across the top row of the table. The POC values are reported for each compound of interest and each combination of interest in combination with 2DG or rotenone. A POC value of less than 50 reflects the likely inhibition of the cell line by the compound of interest or the combination of the compound of interest with the indicated metabolic inhibitor. Compound 2 in particular demonstrated reduction in luminescence, corresponding to reduced ATP activity by the cells, across many of the cell lines. When combined with 2-DG compound, 2 showed effectiveness against each cell line and a remarkable value for BxPC3 the pancreatic cancer cell line. Compound 2 would also be expected to have effectiveness against other pancreatic cancer cell lines.
While the resulting N-benzyl sulfonamides provided by the method discussed above have shown some effectiveness in vitro against select cancer cell lines, further toxicity against cancer cells would be desired. To that end, the present disclosure also provides a two-component composition which has shown a synergistic effect against cancer cells in vitro.
The two-component composition consists of a N-benzyl sulfonamide and a metabolic inhibitor. In one embodiment, the metabolic inhibitor is 2-deoxyglucose (2-DG). In another embodiment, the metabolic inhibitor is rotenone. Other metabolic inhibitors suitable for use in the two-component composition are: Lonidamine, 3-bromopyruvate, imatinib, oxythiamine, and 6-aminonicotinamide Glutaminase Inhibitor 968, 6-Diazo-5-oxo-L-norleucine, Amytal, Antimycin A, Sodium Azide, Cyanides, oligomycin, FCCP, Phloretin, Quercetin, 3BP, 3PO, DCA, NHI-1 and Oxamic acid, Fisetin, myricetin, apigenin, genistein, cyanidin, daidzein, hesperetin, naringenin, and catechin. While subsequent in vivo testing may determine a narrower range for the ratio of the N-benzyl sulfonamide to the metabolic inhibitor, the current ratio that has demonstrated effectiveness against cancer lines, as identified in the table below, is in the range of about 1:50 to about 1:1500. Thus, the metabolic inhibitor may comprise from about 75% by weight to about 99.99% by weight of the composition containing both N-benzyl sulfonamide to the metabolic inhibitor where the N-benzyl sulfonamide has the structure set forth in
The Table of
Table 1 below provides the results of testing 30 different N-benzyl sulfonamides, as depicted in
While the two-component composition of N-benzyl sulfonamide with rotenone showed effectiveness against several cell lines, the combination of 100 μM N-benzyl sulfonamide compound number 2 as identified in
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Having demonstrated the ability to identify compounds effective against cancer cells with and without the use of metabolic inhibitors, the same testing protocols may be used to determine the effectiveness of other compounds in treating cancer cells obtained from the patient and maintained in the previously described patient derived serum and/or plasma and specially formulated individualized cancer cell support media. In particular, by maintaining the cancer cells under conditions which mimic the original growth environment, one can alter the conditions in vitro thereby forcing the cancer cells to alter their metabolic pathways followed by treatment with various compounds to determine if the combination results in cell death. In other words, the changes to the metabolic pathways force the cancer cells into a condition where the remaining available metabolic pathways are potentially blocked by the compounds being screened. When a tested compound blocks cell metabolism, the cancer cells will no longer be able to produce ATP resulting in cell death. As described above, the lack of ATP can be easily identified, using the foregoing testing methods, in which case the compound that produced cell death will also be identified as a potential treatment compound for the patient's particular cancer.
Cancer cells are generally addicted to glucose (Warburg effect). Thus, screening when glucose is present will identify compounds that can inhibit glucose metabolism. The screening process outlined above utilizes glucose as an energy source for the cells. However, cells may substitute other energy sources for glucose. When doing so, the metabolic pathways will be altered. Screening of the library of compounds identified in
To promote the use of different metabolic pathways by the cancer cells, the cultured cells may be transferred to media formulations which differ from the individualized cancer cell support media, i.e. test media formulations. The test media formulations will differ from the individualized cancer cell support media used for cancer cell transport by addition or deletion of compounds thereby resulting in the cancer cells being forced into using different metabolic pathways. As discussed above, the change in metabolic pathways should be a condition which can be replicated in the patient and will result in the cancer cells becoming susceptible to a compound. Following screening of multiple compounds to identify those which produce cell death under those conditions, the identified compounds can be further assessed against the patient derived cancer cells to determine those compounds most likely to provide a positive outcome in the patient with minimal side effects.
As discussed above, selection of the preferred compounds for treating cancer will be based on the ability of a compound to block the metabolic pathways used by the cancer cells. Such ability may be attributable solely to the compound or a result of a determined synergistic effect resulting from the addition of a second compound or from forcing the patient derived cells into a different metabolic state by manipulating the individualized cancer cell support media supporting cell growth to provide test media formulations. The following method bypasses the current limited understanding of metabolic pathways in cancer, by allowing one to compare the outcomes of various compounds to determine efficacy. Due to the rapidity in which the following method may be carried out, the method can screen known chemotherapeutics, as well as any FDA approved drug can be evaluated for efficacy. While such an approach would likely fail to identify relevant compounds in population based studies, on an individual level it may be possible to identify and repurpose previously characterized drugs to treat a specific patient's cancer.
The use of proliferation inhibitors/apoptosis inducers requires precise dosing and of action entails inhibiting proliferation and/or inducing apoptosis, both of which can take significant time to accomplish. The mammalian cell cycle, for example, requires 18-24 hrs. to generate a new cell. Likewise, apoptotic cell death is a relatively slow, tightly controlled process that can take anywhere from 8-24 hrs. Thus, targeting/inducing these molecular events typically requires extended drug exposure because the cancer cells are not synchronized; i.e. they are in different stages of the cell cycle. This means that a significant period of time might need to elapse before a particular cancer cell becomes susceptible to the drug. A further complication is that cellular and genetic heterogeneity within the tumor means that not all cancer cells are proliferating at the same time, nor are they all susceptible to the same apoptotic inducing signals. The concept of dormancy (i.e. non-proliferating cancer cells) has emerged as a major impediment to traditional chemotherapeutic drugs because they escape death and can re-activate at a later date.
The following method preferably targets a molecular process that is common, essential, and specific to the entire tumor cell population. The disclosed method quickly disrupts metabolic pathways producing ATP and occurs independent of cell cycle position thereby producing rapid cancer cell death in a manner which decreases the probability of developing drug resistance. As described below, cancer cell metabolism represents an excellent target to accomplish these aims. Although disrupted metabolism is a hallmark of cancer, different types of cancers likely disrupt cell metabolism in a unique way. Thus, screening a wide array of FDA approved compounds for drugs that target metabolism from patient-derived cancer cells represents a unique approach for identifying effective therapeutics specific for that patient's tumor.
The following method uses the ability to measure ATP as a way to rapidly screen for metabolic inhibitors that directly decrease ATP levels. Because ATP turnover is dynamic, this assay technique only requires one to two hours to complete. In contrast, traditional cell viability assays typically require from 24 to 72 hours. Steady state ATP levels are determined by the balance between the synthesis and hydrolysis of ATP; therefore, if ATP production is inhibited then ATP levels will decrease rapidly even though cells are not yet dead. As we have shown, however, if ATP levels are lowered sufficiently and maintained at that low level, the cells eventually die (as revealed by a different cell viability assay). Thus, in the following methodology decreased cellular ATP levels resulting from a short exposure to drug are likely a direct indicator of inhibiting a metabolic pathway required for ATP production. Subsequent cell death occurs later and results from this initial rapid decrease in ATP because the cell does not have energy to perform basic functions.
As a first step, the method screens the compounds of interest to ensure that the selected compounds are not inhibiting the luciferase enzyme responsible for generating light in the presence of ATP. This step is accomplished by evaluating the effect of the target compounds on the luciferase reaction in the absence of cells with exogenously added ATP.
Subsequently, the various individual compounds and combinations of compounds will be tested against a commercially available cell line corresponding to the patient's cancer type. The commercially available cell line will be cultured in the patient's derived serum and tissue specific individualized cancer cell support media prepared as described above. This step will reduce the number of potentially useful compounds and or combinations of compounds.
Following the initial screening step, those compounds which have been identified as likely candidates for treating the patient's specific cancer will be tested against the cells derived from the patient prepared and maintained in the patient derived serum and/or plasma and individualized cancer cell support media selected for the specific organ or tissue location of the cancer. Testing conditions may include: (1) use of solely the identified compounds; (2) use of the identified compounds in combination with metabolic inhibitors; (3) either of the foregoing approaches under conditions where the individualized cancer cell support media supporting the cells has been modified to provide test media formulations designed to induce metabolic changes in the cells. When using metabolic inhibitors, the patient derived cancer cells may be exposed to the metabolic inhibitors prior to treatment with the identified compound or simultaneously with treatment by the identified compound.
The final screening steps are outlined above in detail in the section entitled “Method of in vitro Cytotoxicity Screening.” Thus, as a summary, the method of identified compounds particularly suited to treating the patient's cancer includes the following steps:
The foregoing steps are repeated until a desired number of compounds have been identified for testing against the patient derived cells. The foregoing steps are then repeated using the patient derived cells in patient derived serum and/or plasma and the subsequently prepared individualized cancer cell support media selected to support the patient derived cells. As previously noted, the steps of adding the compound of interest may be preceded by any one of the following additional steps or combination thereof:
The following test results demonstrate the effectiveness of the above describe screening method. The following tests results were obtaining using the above described screening method on different types of commercially available cancer cell lines as a demonstration of the ability to rapidly test and confirm the effectiveness of compounds of interest. When used to identify compounds for specific patients, the following steps will be carried out:
The data presented in
As noted above,
While L15 is a well known media, the following describes the composition of L15 used in the above described tests:
Other embodiments of the present invention will be apparent to one skilled in the art. As such, the foregoing description merely enables and describes the general uses and methods of the present invention. Accordingly, the following claims define the true scope of the present invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/011528 | 1/25/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63303350 | Jan 2022 | US |
