WO1992020781A1 - Atp extraction reagent containing ammonium vanadate - Google Patents
Atp extraction reagent containing ammonium vanadate Download PDFInfo
- Publication number
- WO1992020781A1 WO1992020781A1 PCT/US1992/004101 US9204101W WO9220781A1 WO 1992020781 A1 WO1992020781 A1 WO 1992020781A1 US 9204101 W US9204101 W US 9204101W WO 9220781 A1 WO9220781 A1 WO 9220781A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- atp
- reagent
- cells
- ammonium vanadate
- tumor
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- bioluminescent assays of somatic tumor cells through measurements made of ATP extracted from the cells.
- This invention also relates to an improved method for screening tumor cells to ascertain their sensitivity to chemotherapeutic agents.
- the selective determination of nucleotides in a sample through the firefly bioluminescent measurement of the adenosine triphosphate (ATP) content in a sample is a rapid and sensitive method for ascertaining the viability and number of cells in the sample.
- This bioluminescent measurement has been applied in the past to analytical test measurements that determine the number and viability of bacterial cells in a test sample, see U.S. Patent No. 3,745,090 and more recently to test measurements for the number and viability of either or both of somatic and microbial cells in a test sample, see U.S. Patent No. 4,303,752.
- these ATP release reagents include a (non-toxic) wetting agent, e.g., non-ionic surface agents whose purpose is to render the cell membrane permeable to whatever relatively small molecules are present among the (cellular) constituents inside of the cell membrane. Thereafter, the small molecules diffuse out past the cell membrane barrier.
- ATP diffuses out past the cell membrane barrier so that the ATP content still inside of the cells and the ATP content in the aqueous medium surrounding the cells approaches an equilibrium that is in proportion to the number of (viable) cells suspended in the medium.
- non-ionic surface agents are the ethoxylated phenols and the fatty acid polygycol ethers.
- Preferred for the surface active agent are TRITON-X or NONIDENT P-40. It is important to note that the surface active agent is present in relatively low proportions, say 0.5 - 0.1%.
- these ATP release reagent compositions known to the art also include a buffer composition whose purpose is to adjust the pH of the cell suspension in the (diluted) release reagent to some desired level.
- a buffer composition whose purpose is to adjust the pH of the cell suspension in the (diluted) release reagent to some desired level.
- any of the usual buffers e.g., tris may be employed but Hepes at pH 7.7 - pH 7.9 is a preferred buffer.
- Hepes which is N-[2-hydroxyethyl]piperazine N' -[2-ethanesulfonic acid] does not interfere with the firefly bioluminescent reaction measurement of the ATP content in the buffered solution and, desirably, is adapted to buffer the suspension at a pH of about 7.8, which is an optimum level for the firefly bioluminescent reaction.
- the method for ascertaining the sensitivity of tumor cells to chemotherapeutic agents to which the practice of this invention pertains comprises
- the ATP release reagent of the present invention comprises a solution of known in the art components for ATP release reagents in art recognized proportions to render cell membranes permeable without destruction of the cell membrane. Also present is NH 4 VO 3 (Ammonium meta Vanadate) in proportions effective to inhibit any cellular ATPase enzyme(s) that might otherwise react away extracted ATP in the solution. In the absence of Ammonium Vanadate the level of ATP in the suspension of somatic tumor cells in the ATP release reagent composition rapidly decreases.
- NH 4 VO 3 Ammonium meta Vanadate
- Vanadate contemplated for the ATP release reagent composition of this invention is about 8-14 mM so that a proper concentration of the Ammonium meta Vanadate preferably in the range of about 2.6 to 4.6 mM
- a separate aspect of this invention is an improved assay method for determining the sensitivity of tumor cells to chemotherapeutic agents
- improvement features comprising incorporating Ammonium Vanadate in ATP release reagent compositions that are adapted to release ATP from human tumor (somatic) cells without totally disrupting the cell membranes.
- extracting ATP from the tumor cells is done by a cell lysis technique using a strong acid such as trichloroacetic acid (TCA).
- TCA trichloroacetic acid
- ATP is not readily detectable in the somatic cell suspension release reagent solution within only about 15 minutes after extraction of the ATP into the ATP release reagent composition. It is believed that cellular ATPase enzyme(s) degrade the ATP.
- Ammonium Vanadate seems to have a molecular structure similar to ATP, and apparently the Ammonium Vanadate binds with ATPase enzymes at an active site thereon. Whatever the reaction mechanism may be.
- Ammonium Vanadate inhibits the catalytic activity of ATPase enzymes on ATP, an inhibition which is known in the art, see Cantley et al., J. Bio. Chem.. 252:7421 (1977).
- the great advantage to stabilizing the ATP which has diffused from the tumor cells into solution in the ATP release reagent should be self-evident.
- the assay method of this invention is adapted to an
- essentially concurrent evaluation of a great many tumor cell samples so as to test varying amounts of different chemotherapeutic agents on the tumor Preferably extraction is performed in all samples. Then the ATP in the samples are measured one by one. Stabilizing the ATP in each sample allows time for extraction and measuring the ATP from tumor cells from each sample, in proportion to the number of cells in the sample. despite the widely differing tumor cells content in some of the multitude of samples. In addition, stabilizing the ATP content allows the time needed to manipulate the multitude of samples however necessary up to introduction of the luciferase-luciferin reagents into each sample, one by one for conduct of the firefly luminescence reaction assay.
- the ATP in solution in the particular tumor samples measured by the firefly bioluminescence reaction should be sufficiently stable to allow up to about 2.5 hours of delay between the addition of the NH 4 VO 3 extraction reagent into the cell suspension medium and conduct of the firefly bioluminescent reaction.
- a principal aspect of the invention is the presence of effective amounts of NH 4 VO 3 in the ATP releast reagent.
- the Ammonium Vanadate content is in the range of about 2-6-4-6 millimolar and preferably in the range of about 3.0-4.0 millimolar for achieving an optimum stability of ATP counts. At concentrations about about 4.0 millimolar, a discernible decrease in counts has been observed. This is believed to be due to inhibition of the luciferase enzyme caused by excess NH 4 VO 3 in the extracted sample. As expected, counts decrease as a function of decreasing NH 4 VO 3 concentration and such occurs below the 3.6 millimolar
- release reagent of this invention is particularly adapted for the release of ATP from human tumor cells without total disruption of the tumor cell membranes.
- a second aspect of the present invention resides in an improved method for evaluating the sensitivity of somatic tumor cells to chemotherapeutic agents.
- the early steps in the process generally follow the
- the use of the ATP bioluminescence reaction to measure the effect of chemotherapeutic drugs against tumor cells cultured in vitro has been so simplified by practice of this invention, that tumor chemosensitivity assays can be performed rapidly and easily in a standardized format.
- the improved method of this invention provides an objective measurement determinative of sensitivity of the tumor cells to various drugs, using relatively few tumor cells in each sample.
- Figure 1 is a flow sheet representation of the method
- Figure 2 is a graph showing luminometer counts 15 minutes after extraction.
- Figure 3 is a graph showing luminometer counts 30 minutes after extraction.
- the first step of the method transforms a solid tumor specimen into a suspension of single cells or multi-cell aggregates of less than about 30 cells per aggregate.
- this transformation is performed as described by Sevin et al., supra.
- the solid tumor specimen is minced into 1-5 mm pieces.
- the minced tumor pieces are disassociated enzymatically into single cells and small size multi-cell aggregates by dispersing about 1 gram of minced tumor in sterile culture medium to which had been added about 1500 units per ml of DNase 1, about 2 mg/ml of collaganase and about 1 mg/ml of Dispase enzymes.
- (tiny) cell aggregates containing less than about 30 cells per aggregate.
- the second step of the method comprises culturing a multiplicity of samples of the disassociated tumor cells in a standard nutrient medium for from 4-7 days.
- the growth medium and cultivation conditions is suitably described by Sevin et al. (McCoy's enriched media with 15% fetal calf serum at 37°C at 95% humidity for 6-7 days) in the presence, sample to sample, of varying concentrations of different chemotherapeutic agents.
- Sevin et al. McCoy's enriched media with 15% fetal calf serum at 37°C at 95% humidity for 6-7 days
- an appropriate number of control samples of the tumor cells are also cultured.
- chemotherapeutic drugs is that frequently normal cells will be present along with the tumor cells in some or all of the samples. Normal cells are an interfering substance in the assay method. ATP measurements derived from a normal cell's content in the samples are not desired. To the extent reasonably possible, normal cells should be eliminated from the test samples of tumor cells.
- normal cells are generally anchorage dependent cells. Therefore, culturing.
- a preferred embodiment culture period is 4-7 days.
- Agarose coated microtiter plates prepared as described in copending application, S.N. 07/651,940, filed February 7, 1991, is preferred for culturing the tumor cell samples.
- the agarose coated well bottom will prevent proliferation of normal cells therein.
- other techniques for placing an agarose coating on the well bottom of the commercially available polystyrene microtiter plate than through practice according to S.N. 07/651,940 may be used.
- polypropylene microtiter plates may be employed for culturing the tumor cell samples according to the method of this invention.
- the third step in the bioluminescent method of this invention is extraction of the tumor cell cultures (without total disruption of the cell
- the ATP releasing reagent is comprised of known in the art ingredients.
- one of the commercially available ATP release reagents FL-SARTM, SOMALIGHTTM or PICOEXTM may have a concentrated solution of Ammonium Vanadate added thereto generate 2.6-4.6 millimolar NH 4 VO 3 in the cell suspension medium and then the solution is used to release ATP from the somatic tumor cell suspension following the details of the procedure set out in U.S. Patent No. 4,303,752.
- the ATP release reagent has been buffered with 0.1 - 0.35 M Hepes which is a relatively high buffer content.
- the pH of the tumor cells sample drops during cultivation to about pH 6.5, and the final pH (for the bioluminescent reaction) is pH 7.7 - 7.9.
- the Hepes concentration in the cell suspension is about 0.125 M.
- Triton X-100 or NP-40 seems to be a reasonable optimum and such constitute preferred embodiment wetting agents and the content thereof .
- extracted into the ATP release reagent should be relatively stable and not be degraded by ATPase enzymes, indigenous to the tumor cells. Stabilizing the ATP dissolved in the medium in which the cells are
- the ATP content in the cell suspension medium should be stable enough to allow for quantitative reproductability of the ATP measurements made with the firefly bioluminescence reaction throughout about a 2 1/2 hour period following
- ATP extraction reagents containing 0.32 M Hepes buffer pH 7.8, varying amounts of Triton X-100 or NP-40 in the range of from 0.5 to 0.03% by wt with and without the inclusion of 10.86 mM NH 4 VO 3 in the reagent were prepared and then used to extract ATP from ME180 ovarian tumor cells.
- the tumor cell extracts were counted by conducting the firefly bioluminescence reaction on Sample 15, 30 and 60 minutes after the ME180 tumor cells suspended in a fetal calf serum culture medium were first mixed with the ATP extraction reagent. In the absence of Ammonium Vanadate the ATP extracted from the tumor cells became rapidly
- the optimum cell concentrations for being assayed with the preferred tumor cell extraction reagent is 10,000-80,000
- the work up procedure is to disassociate the solid tumor as is illustrated in Figure 1 herein by mincing the same while the tumor cells are being bathed in a nutrient medium containing fetal calf serum, sufficient antibiotic to control possible microbial contamination and a tumor
- the minced tumor pieces are enzymatically cultured in more of the same nutrient solution mixture at 37°C until the tumor cells have visually been disassociated or as convenient, say for 8-16 hours.
- ficoll-hypaque density gradient separation is used to reduce dead cell and erythrocyte contamination.
- the disassociated tumor cells are washed, then resuspended in the fetal calf serum nutrient medium at a cell concentration of about 4 ⁇ 10 5 cells per ml. About 0.1 ml of this suspension is added to each well of a 96 well agarose coated polystyrene culture plate. Parenthetically, it is noted that the best range of tumor cell concentration per well is from
- PPC Peak Plasma Concentration
- the drugs in appropriate concentration dissolved in the fetal calf serum nutrient medium are applied from a multi-channel pipette to the wells in the culture plates and then the microwell culture plates containing 0.2 ml per well of the drug dosed tumor cell culture samples are incubated for seven days in a humidified, 37°C, 5% CO 2 incubator. If rapid cell proliferation is evidenced, the incubation period should be decreased to 4-6 days.
- the Ammonium Vanadate stabilized tumor cell extraction reagent formulated as described herein, is then added to each well in the culture plate at the rate of 0.1 ml per well and thoroughly mixed in to form a cell suspension in the mixture of culture medium and ATP extraction reagent that constitutes a Hepes
- luciferin-luciferase counting reagent for ATP measurement is injected into the aliquots one by one and the bioluminescent counts are measured over a 20 second period in a luminometer (a count integration time in the range of 15-30 seconds is recommended).
- RESISTANT or STIMULATORY Mean inhibition for 6.25% - 25% PPC drug concentrations is less than 50%. Inhibition of less than 15% may reflect a stimulatory effect of the test drug.
- An assay as described above was made on a patient's Sigmoid Colon primary tumor using disassociated tumor cells at 1, 2 and 4xl0 5 cells ml/for the samples.
- the cell samples were continuously exposed to test drugs in vitro for seven days. Drugs were tested at 200, 100, 50, 25 12.5, 6.25, 3.13 and 1.56 percent of their estimated Peak Plasma Concentration (PPC) in vivo.
- PPC Peak Plasma Concentration
- Tumor growth inhibition was quantitated by ATP luminometry. Interpretations are based on mean percent inhibition at 25, 12.5, 6.25 and 3.13 percent of PPC. The results are tabulated below.
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Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US70187091A | 1991-05-17 | 1991-05-17 | |
US701,870 | 1991-05-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1992020781A1 true WO1992020781A1 (en) | 1992-11-26 |
Family
ID=24819011
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1992/004101 WO1992020781A1 (en) | 1991-05-17 | 1992-05-15 | Atp extraction reagent containing ammonium vanadate |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0585367A4 (en) |
AU (1) | AU2140892A (en) |
WO (1) | WO1992020781A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2071567A1 (en) * | 1993-05-21 | 1995-06-16 | Inia | Method for toxicological evaluation of an aquatic medium |
US7132249B1 (en) | 2003-05-12 | 2006-11-07 | Charm Sciences, Inc. | Method of determining allergenic food on surfaces |
US7494781B1 (en) | 2003-05-12 | 2009-02-24 | Charm Sciences, Inc. | Sensitive method for detecting low levels of ATP |
US11959838B2 (en) | 2015-11-06 | 2024-04-16 | Ventana Medical Systems, Inc. | Representative diagnostics |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4303752A (en) * | 1977-05-31 | 1981-12-01 | Kolehmainen Seppo E | Selective determination of nucleotides in viable somatic and microbial cells |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57162797A (en) * | 1981-03-31 | 1982-10-06 | Lion Corp | Anionic surfactant |
-
1992
- 1992-05-15 AU AU21408/92A patent/AU2140892A/en not_active Abandoned
- 1992-05-15 EP EP92912569A patent/EP0585367A4/en not_active Withdrawn
- 1992-05-15 WO PCT/US1992/004101 patent/WO1992020781A1/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4303752A (en) * | 1977-05-31 | 1981-12-01 | Kolehmainen Seppo E | Selective determination of nucleotides in viable somatic and microbial cells |
Non-Patent Citations (3)
Title |
---|
Gyneacological Oncology, Volume 31, issued 1988, SEVIN et al., "Application of an ATP-Bioluminescence Assay in Human Tissue Chemo-sensitivity Testing", pages 191-204, see entire document. * |
Journal of Biological Chemistry, Volume 252, No. 21, issued 10 November 1977, CANTLEY, Jr. et al., "Vanadate Is a Potent (Na,K) -ATPase Inhibitor Found in ATP Derived From Muscle", pages 7421-7423, see page 7422, column 2, paragraph 3. * |
See also references of EP0585367A4 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2071567A1 (en) * | 1993-05-21 | 1995-06-16 | Inia | Method for toxicological evaluation of an aquatic medium |
US7132249B1 (en) | 2003-05-12 | 2006-11-07 | Charm Sciences, Inc. | Method of determining allergenic food on surfaces |
US7494781B1 (en) | 2003-05-12 | 2009-02-24 | Charm Sciences, Inc. | Sensitive method for detecting low levels of ATP |
US7824878B2 (en) | 2004-05-11 | 2010-11-02 | Charm Sciences, Inc. | Sensitive method for detecting low levels of ATP |
US11959838B2 (en) | 2015-11-06 | 2024-04-16 | Ventana Medical Systems, Inc. | Representative diagnostics |
Also Published As
Publication number | Publication date |
---|---|
EP0585367A1 (en) | 1994-03-09 |
AU2140892A (en) | 1992-12-30 |
EP0585367A4 (en) | 1994-12-21 |
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