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WO1994006014A1 - Procede diagnostique aidant a la detection precoce du cancer et du diabete - Google Patents

Procede diagnostique aidant a la detection precoce du cancer et du diabete Download PDF

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Publication number
WO1994006014A1
WO1994006014A1 PCT/US1992/007415 US9207415W WO9406014A1 WO 1994006014 A1 WO1994006014 A1 WO 1994006014A1 US 9207415 W US9207415 W US 9207415W WO 9406014 A1 WO9406014 A1 WO 9406014A1
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Prior art keywords
ffa
cancer
fluorescence
levels
fatty acid
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PCT/US1992/007415
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English (en)
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Alan M. Kleinfeld
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

  • This invention relates to research and clinical biochemical assays for small hydrophobic molecules, wherein the analyte is caused to react with a fluorescently modified, small-molecular-weight-binding protein, and thereby causes a detectable fluorescence signal.
  • This invention is specifically directed to diagnostic methods to aid in the early detection of cancer and/or diabetes.
  • FFA free fatty acids
  • In vitro studies indicate that elevated levels of free fatty acids (FFA) inhibit immune mediated destruction of tumor cells.
  • FFA free fatty acids
  • FFA levels may be a genera.! signature of cancer. That hypothesis has been tested and found to be valid at least in certain instances.
  • the present invention provides a diagnostic tool to aid the physician in the early detection of cancer.
  • FA Fatty acids
  • OA oleic acid
  • LA ⁇ -linoleic acid
  • CTL Cytotoxic T lymphocytes
  • TCR T cell receptor
  • FAFBSA FA free BSA
  • [Ca 2+ ]i response also inhibits the release of /tic granules from CTL, following tumor target ceD recognition (Richieri, G. V., M. F. Mescher, and A. M. Kleinfeld. 1990. Short term exposure of cytotoxic-T-lymphocytes to cis unsaturated free fatty acids inhibits antigen stimulation of cytosolic calcium increases and degranulation. /. Immunol. 144:671). Most importantly, short term treatment with cis unsaturated but not saturated FA inhibits killing of tumor-target cells by allo reactive CTL clones as well as by in vivo generated effector cells (Samlaska, C. P. 1979. Linoleic acid inhibition of naturally occurring lymphocytotoxicity to breast cancer- derived cells measured by a chromium-51 release assay. JNCL 62:1427;
  • Free fatty acids inhibit cytotoxic-T-lymphocyte mediated lysis of allogeneic target cells. J. Immunol. 145:1074). In contrast to the continuous nature of the inhibitory effect of FA on the [Ca 2+ ]i response and degranulation, inhibition of killing by FFA is only effective in the specific 10 minute window required for lethal hit delivery. Thus, addition of cis FA before, 2-5 minutes after, and 15 minutes after conjugation, inhibits lysis completely, 50%, and not at all, respectively (Richieri, G. V., and A. M. Kleinfeld. 1990. Free fatty acids inhibit cytotoxic-T-lymphocyte mediated lysis of allogeneic target cells. /. Immunol. 145:1074). These results indicate that FA inhibit an event specifically related to lethal hit delivery.
  • CTL stimulated tumor-target cells release of FA occurs early and this release is a general property of the CTL interaction.
  • FA release has been measured from a variety of tumor-target cells bearing various class I allo- antigens (Richieri, G. V., and A. M. Kleinfeld, 1991. Free fatty acids are produced in and secreted from target cells very early in cytotoxic T lymphocyte-mediated killing. J. Immunol. 147:2809-2815). Conjugation of tumor cells with cognate effector cells results in an increase in supernatant FFA within minutes after conjugation and the supernatant is eventually enriched nearly 100 fold in FFA.
  • FA release does not occur simply because the cell membranes fragment as the cells are dying (Richieri, G. N., and A. M. Kleinfeld, 1991. Free fatty acids are produced in and secreted from target cells very early in cytotoxic T lymphocyte-mediated killing. J. Immunol. 147:2809-2815).
  • the target cells themselves contain very httle FA before conjugation so that production of FA requires activation of specific lipolytic enzymes. These enzymes are specifically activated by CTL; other means of cell disruption, including cycles of freeze/thaw, detergent solubilization, or massive ionophore treatment, do not result in FA production.
  • treatment of target cells with lytic granules sufficient to completely lyse cells, also does not generate FA.
  • CTL stimulation of tumor cell release of FA may have two roles: production of FA within the target cell may be crucial for lethal hit delivery while secretion of FFA from the tumor cell may be immunosuppressive.
  • the fact that FA production may be the earliest event reported to occur within the target cell following CTL recognition and that FA releases and target cell lysis are highly correlated suggests that CTL activation of lipolytic enzymes within the target cell plays an important part in kilhng.
  • FA are themselves known to be potent modulators of cellular behavior, inhibition of the CTL response is an example, and have been directly implicated in several different types of cell death (Jones, R. L., J. C. Miller, H. K. Hagler, K. R. Chien, J. T. Willerson, L. M.
  • Plasma [FFA] levels are regulated by the buffering action of albumin (Spector, A. A., and J. E. Fletcher. 1978. Transport of fatty acid in the circulation. In DISTURBANCES IN LIPID AND LIPOPROTEIN METABOLISM (Dietschy, J.M., Gotto, A.M., & Ontko, A. eds) pp 229-249). Thus, while total plasma FA levels are quite high, in healthy individuals between 0.1 and
  • FFA aqueous phase monomers
  • [FFA] levels in a FA - albumin mixture are functions solely of the ratio of total FA to total albumin. If [FFA] is plotted as a function of FA/albumin ( ⁇ , the parameter used in Scatchard analysis) then [FFA] is found to increase approximately linearly with v at low v values ( ⁇ 2) and exponentially at higher values. At low v values albumin is a relatively effective buffer of [FFA] while above this value its buffering capacity is exceeded, indicating that binding affinities of the sites 3-8 are much smaller that the first 2 sites.
  • This invention provides a diagnostic tool to aid in the early detection of cancer and/or diabetes.
  • Serum or plasma from a patient suspected of having cancer, or diabetes is mixed with a reagent comprising a fluorescently modified fatty acid binding protein.
  • a change in fluorescence of the fluorescently modified fatty acid binding protein is detected.
  • the change in fluorescence is related to the amount of free fatty acid in serum or plasma.
  • the level of free fatty acid in the serum or plasma is greater than about 7 nM, a test for diabetes that does not depend on the level of free fatty acid, such as measuring A1C hemoglobin, is run. The absence of diabetes is an indication of possible cancer. If diabetes is found, then the test is non ⁇ specific for cancer.
  • a central element in this invention is the use of a new fluorescent probe of FFA to determine [FFA] in aqueous media, including human serum.
  • This new probe is denoted ADIFAB for AcryloDan Intestinal Fatty Acid Binding protein and is prepared by covalently reacting the fluorescent group Acrylodan with recombinant fatty acid binding protein from rat intestine (I- FABP), as described by Richieri, G. N., R. T. Ogata, and A. M. Kleinfeld, 1991, Quantitation and cellular release of free fatty acids determined with a new fluorescent indicator, J. Biol. Chem. .
  • ADIFAB (FABP) molecule ADIFAB (FABP) molecule
  • the concentrations of FFA and of FA bound to ADIFAB are determined from the ratio of 505/432 emission intensities (R), essentially by the method of Grynkiewicz et al (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca 2 + indicators with greatly improved fluorescence properties. /. Biol. Chem. 260:3440). In this procedure, total intensities at each wavelength are expressed in terms of the contributions from free and bound ADIFAB.
  • R max and and Q were determined as described by Richieri, G.V., et al., supra) and found to similar for all FA studies (11.5 and 19.5, respectively).
  • ADIFAB K- for a normal distribution of FA of mostly PA, OA, and LA
  • the ADIFAB probe will bind ⁇ 5 nM FA or ⁇ 0.1% of the total FA.
  • the presence of ADIFAB will have a negligible affect on the albumin buffering capacity and therefore on [FFA] even when the measurement is done on a 50 fold dilution of total serum ( ⁇ 600 ⁇ M).
  • [FFA] because of the nature of the albumin binding capacity, [FFA] remains a function only of FA/albumin over an extremely wide range of F- ⁇ , concentrations and therefore the same [FFA] is obtained with undiluted and 50 fold diluted serum.
  • ADIFAB can be used to determine [FFA] in human serum. These measurements can be done with great accuracy within a few minutes and can be done with relatively simple instrumentation.
  • FFA FFA was determined in human serum samples from patients with Type II diabetes (NIDDM). This technology has, in accordance with this invention, applied to determine FFA levels in human sera. In these initial measurements sera was obtained from studies whose goals were unrelated to FA levels. Samples were obtained from two donor groups; a normal (healthy) population (15 samples) and six samples from patients with type II or non insulin dependent diabetes mellitus (NIDDM). The normal population was obtained from a cohort of donors chosen because they were HIV and EBV negative. These donors, 14 whites and one Asian, included 11 males and 4 females, 13 of whom were between 25 and 35 and two between 40 and 50 years old with no histories of disease. Of these, 2 (both females) were smokers.
  • ADIFAB emission ratios were measured using 0.2 ⁇ M ADIFAB, a 1:50 dilution of the sera, and total sample volume of 1 ml. The results were compared by plotting the number of samples corresponding to a given [FFA] ⁇ 1 nM, centered around every half integer value. The average [FFA] values for the two groups are ⁇ 5 and 10 nM for normal healthy donors and NIDDM, respectively. Total FA values, determined using the WAKO enzyme assay, within each group, were well correlated with the measured [FFA] values
  • ADIFAB measurements for FFA were done using serum dilutions between 10 and 100:1 and virtually identical results were obtained for all dilutions of a given sample, consistent with the buffering capacity expected from the albumin studies above. It was also demonstrated that addition of excess FAFBSA to the serum sample returned the ADIFAB R value to basal levels (R 0 ), consistent with FFA generating the response.
  • the average [FFA] value of the normal group is about 5 nM while that of the NIDDM patients is 10 nM.
  • the uncertainties in these measurements were approximately ⁇ InM (SD).
  • Average total FA levels were found to be 0.41 mM and 0.79 mM in the normals and NIDDM samples, respectively.
  • Immunosuppression is a common correlate of advanced cancer. The mechanisms for this decrease in immune function are likely multifactorial.
  • the objective of the study was to determine if a significant correlation existed between [FFA] levels as measured by the described procedure and the very limited clinical data regarding FA levels in cancer patients.
  • Such studies have not been done previously since a unifying hypothesis for such measurements was lacking and the technical difficulties of determining accurately total FA would be daunting on a large scale.
  • the newly developed FFA probe technology eliminates the technical barriers to such a study and will allow rapid and accurate measurements of [FFA] levels in a substantial number of serum samples from patients with cancer. If comprehensive data on FFA levels support the data developed to date, the FFA probe technology could become an important adjunct to cancer diagnosis and treatment. Sera of cancer patients could easily be studied and correlated with known prognostic features such as stage of disease, tumor burden, weight loss, performance status, and LDH.
  • FFA levels before and after resection of an apparently localized malignancy might provide an indirect assessment of the presence/absence of residual tumor and serve as a prognostic indictor for recurrence. Alterations in FFA levels in response to immune stimulants could serve to indicate the impact of therapies on tumor cells. Elevated FFA levels would also be significant because of the potential implications for tumorigenesis itself. Thus if tumor generation of FFA is autoprotective, then entirely new strategies, focusing on reduction of FFA levels, could be realized for treatment of cancer.
  • the protocol for measurements in human sera was as follows: 1) serum, diluted 100 fold in aqueous buffer at pH 7.4, is aliquoted into two cuvettes to a volume of 1.5 ml each. 2) ADIFAB is added to one cuvette at a concentration of ⁇ 0.5 ⁇ M. 3) The fluorescence emission intensities at 432 and 505 nm are measured at 37 ° C from each cuvette. 4) The intensities of the blank (serum without ADIFAB) are subtracted from the sample with ADIFAB and the [FFA] values are evaluated from the ratio of intensities as described in hereinafter.
  • the average [FFA] level for the normals was between 4 and 5 nM and all but one of the 11 diabetics had [FFA] levels exceeding this value by an average of more than 100%.
  • the value of 4.5 nM for the normal population is obtained in a solution of total oleic acid and human albumin at a ratio of ⁇ 1 and this is the value reported as the average value of normal individuals.
  • measurements of the total FA concentration of the normals and diabetics using the enzymatic assay for total FA shows parallel tracking of FFA and FA ⁇ levels. Thus the assay correctly reflects the increase in FA. otal levels known to occur in type II diabetics (Reaven et al).
  • an individual whose blood serum has FFA levels exceeding 7 nM is considered to have a high probabihty of having either diabetes or cancer. Since diabetes can be determined by measuring A1C hemoglobin and/or blood glucose, this assay represents a potentially general screen for cancer in non-diabetic patients.
  • the primary application of this technology is as a diagnostic aid in detecting cancers at an early stage.
  • a secondary application of the technology is in early detection of diabetes.

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Abstract

Procédé diagnostique aidant à la détection précoce du cancer, et consistant à mesurer des niveaux d'acide gras libres chez des patients soupçonnés de souffrir du cancer, et à déterminer également la présence ou l'absence du diabète.
PCT/US1992/007415 1992-09-02 1992-09-02 Procede diagnostique aidant a la detection precoce du cancer et du diabete Ceased WO1994006014A1 (fr)

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PCT/US1992/007415 WO1994006014A1 (fr) 1992-09-02 1992-09-02 Procede diagnostique aidant a la detection precoce du cancer et du diabete

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PCT/US1992/007415 WO1994006014A1 (fr) 1992-09-02 1992-09-02 Procede diagnostique aidant a la detection precoce du cancer et du diabete

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6750030B2 (en) * 1999-12-08 2004-06-15 Torrey Pines Institute For Molecular Studies Method of detecting cardiac ischemia using fatty acid binding protein
US7202089B2 (en) 2001-09-14 2007-04-10 Alan Kleinfeld Early diagnosis of stroke
US7262017B2 (en) 2001-09-14 2007-08-28 Torrey Pines Institute For Molecular Studies Diagnostic markers for ischemia
US7601510B2 (en) 2004-03-22 2009-10-13 Ffa Sciences Llc Development and use of fluorescent probes of unbound analytes
WO2013166431A1 (fr) * 2012-05-03 2013-11-07 Beth Isreal Deaconess Medical Center, Inc. Lipides augmentant la sensibilité à l'insuline et leurs procédés d'utilisation
US9164109B2 (en) 2006-10-27 2015-10-20 Alan Kleinfeld Use of probes for unbound metabolites
US10604473B2 (en) 2013-03-15 2020-03-31 Beth Israel Deaconess Medical Center, Inc. Lipids that increase insulin sensitivity and methods of using the same
US11013711B2 (en) 2016-06-10 2021-05-25 Beth Israel Deaconess Medical Center, Inc. Fatty acid esters of hydroxy fatty acids (FAHFAs) for use in the treatment of type 1 diabetes
CN112930568A (zh) * 2019-04-23 2021-06-08 艾斯皮雷科技股份有限公司 用于定位或识别恶性肿瘤的装置和方法
WO2024034606A1 (fr) * 2022-08-10 2024-02-15 学校法人日本大学 Procédé d'analyse d'un échantillon, trousse d'analyse pour cancer gynécologique et lésion précancéreuse associées, et médicament
JP2024025634A (ja) * 2022-08-10 2024-02-26 学校法人日本大学 試料の検査方法、婦人科癌及びその前癌病変の検査キット及び医薬

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009310A1 (fr) * 1989-12-12 1991-06-27 Lidak Pharmaceuticals Determination d'acide gras libre

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009310A1 (fr) * 1989-12-12 1991-06-27 Lidak Pharmaceuticals Determination d'acide gras libre

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6750030B2 (en) * 1999-12-08 2004-06-15 Torrey Pines Institute For Molecular Studies Method of detecting cardiac ischemia using fatty acid binding protein
US7202089B2 (en) 2001-09-14 2007-04-10 Alan Kleinfeld Early diagnosis of stroke
US7262017B2 (en) 2001-09-14 2007-08-28 Torrey Pines Institute For Molecular Studies Diagnostic markers for ischemia
US7879558B2 (en) 2001-09-14 2011-02-01 Torrey Pines Institute For Molecular Studies Diagnostic markers for ischemia
US7601510B2 (en) 2004-03-22 2009-10-13 Ffa Sciences Llc Development and use of fluorescent probes of unbound analytes
US8466090B2 (en) 2004-03-22 2013-06-18 Ffa Sciences Llc Development and use of fluorescent probes of unbound analytes
US9164109B2 (en) 2006-10-27 2015-10-20 Alan Kleinfeld Use of probes for unbound metabolites
WO2013166431A1 (fr) * 2012-05-03 2013-11-07 Beth Isreal Deaconess Medical Center, Inc. Lipides augmentant la sensibilité à l'insuline et leurs procédés d'utilisation
US11174216B2 (en) 2012-05-03 2021-11-16 Beth Israel Deaconess Medical Center, Inc. Lipids that increase insulin sensitivity and methods of using the same
US10604473B2 (en) 2013-03-15 2020-03-31 Beth Israel Deaconess Medical Center, Inc. Lipids that increase insulin sensitivity and methods of using the same
US11013711B2 (en) 2016-06-10 2021-05-25 Beth Israel Deaconess Medical Center, Inc. Fatty acid esters of hydroxy fatty acids (FAHFAs) for use in the treatment of type 1 diabetes
CN112930568A (zh) * 2019-04-23 2021-06-08 艾斯皮雷科技股份有限公司 用于定位或识别恶性肿瘤的装置和方法
WO2024034606A1 (fr) * 2022-08-10 2024-02-15 学校法人日本大学 Procédé d'analyse d'un échantillon, trousse d'analyse pour cancer gynécologique et lésion précancéreuse associées, et médicament
JP2024025634A (ja) * 2022-08-10 2024-02-26 学校法人日本大学 試料の検査方法、婦人科癌及びその前癌病変の検査キット及び医薬
JP7716691B2 (ja) 2022-08-10 2025-08-01 学校法人日本大学 試料の検査方法、婦人科癌及びその前癌病変の検査キット及び医薬

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