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WO1992022817A1 - Procede de diagnostic des mastocytoses systemiques - Google Patents

Procede de diagnostic des mastocytoses systemiques Download PDF

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Publication number
WO1992022817A1
WO1992022817A1 PCT/US1992/002368 US9202368W WO9222817A1 WO 1992022817 A1 WO1992022817 A1 WO 1992022817A1 US 9202368 W US9202368 W US 9202368W WO 9222817 A1 WO9222817 A1 WO 9222817A1
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metabolite
prostaglandin
histamine
mast cell
urine
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L. Jackson Roberts
John A. Oates
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Vanderbilt University
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Vanderbilt University
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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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors

Definitions

  • the present invention relates to an improved method to diagnose systemic mast cell disorders by detecting the presence or concentration of certain metabolites of prostaglandin D 2 .
  • this invention relates to a more accurate method to diagnose systemic mast cell disorders than the conventional methods.
  • This invention also relates to new compositions of matter comprising substantially pure metabolites of prostaglandin D 2 .
  • mast cell mediators are usually normal between episodes of mast cell activation.
  • the mediators responsible for the vasodilation that occurs during episodes of systemic mast cell activation are histamine and prostaglandin D 2 .
  • the doted horizontal line indicates upper limits of normal for both histamine and N r -methylhistamine.
  • levels of histamine ranged from normal to tenfold greater than normal, but N ⁇ -methylhistamine levels were normal in all urines.
  • Both histamine and its metabolite, N ⁇ -methylhistamine were quantified by highly accurate mass spectrometric assays. Roberts, L.J. II, Oates, J.A. : Accurate and Efficient Method for Quantification of Urinary Histamine by Gas Chromatography Negative Ion Chemical Ionization Mass Spectrometry. Anal. Biochem. 136:258-63 (1984). Morrow, J.D., Parsons, W.G.
  • FIG. 2 shows levels of urinary histamine excretion in a patient measured during two asymptomatic periods (time zero) and following an episode of intense flushing. The dotted line indicated upper limit of normal.
  • simultaneous quantification of histamine metabolites virtually eliminates "false-positive" results.
  • histamine levels as a diagnostic indicator of systemic mast cell disease.
  • Systemic mastocytosis is characterized by an abnormal proliferation of tissue mast cells. Symptoms of mastocytosis are primarily attributed to the release of mast cell mediators during episodes of systemic activation of the excessive numbers of mast cells. Thus, biochemical evidence for the release of increased quantities of mast cell secretory products can suggest or confirm, depending on the clinical situation, a diagnosis of systemic mastocytosis.
  • a major advantage of the biochemical approach to the diagnosis of systemic mast cell disease is that it has allowed the recognition of a class of patients in whom episodes of systemic mastocyte activation can be unequivocally documented biochemically, but in whom clear-cut evidence of abnormal mast cell proliferation is lacking by current histologic criteria.
  • mast cell mediators Although the release of increased quantities of mast cell mediators can be demonstrated during episodes of mast cell activation in such patients, mediator levels are usually normal at quiescent times. By contrast, patients with proliferative mast cell disease (mastocytosis) usually exhibit chronic overproduction of mast cell mediators. Thus, to facilitate the accurate diagnosis of systemic mast cell activation disorders, a method to detect or determine chemical markers of this disease has been developed.
  • the present invention relates to an improved method to diagnose systemic mast cell disorders by detecting the presence or determining the concentration of metabolites of prostaglandin D 2 present in a sample of bodily fluid.
  • the metabolites can be detected by mass spectroscopy.
  • an object of this invention is to provide a method to immunologically detect metabolites of prostaglandin D or antibodies to these metabolites.
  • the metabolites of prostaglandin D 2 detected in biological fluid include: 9 ⁇ -hydroxy-11.15-dioxo- 2,3,18,19,-tetranorprost-5-ene-l,20-dioic acid and 9 ⁇ ,llp-dihydroxy-15-oxo-2,3,18,19-tetranor prost-5-ene-l,20-dioic acid.
  • Figure 1 shows levels of histamine and N- methylhistamine measured in consecutive 24-hour urine collections from a patient.
  • Figure 2 shows levels of urinary histamine excretion in a patient measured following an episode of flushing.
  • Figure 3 shows fold increase above normal in the urinary excretion of histamine and N ⁇ -methylhistamine in eight 24 hour urine collections from a patient with histologically documented systemic mastocytosis.
  • Figure 4 shows the metabolism of prostaglandin D 2 to a biologically active metabolite 9 ⁇ ,ll)3-prostaglandin F 2 .
  • Figure 5 shows the metabolism of prostaglandin D 2 to metabolite A and metabolite B.
  • Figure 6 shows the level of urinary excretion of Metabolite A of a prostaglandin D 2 in patients with suspected systematic mast cell disease.
  • Figure 7 shows a comparison of urinary levels of Me-histamine with metabolite B of prostaglandin D 2 in patients with documented mastocytosis.
  • Figure 8 shows cyclization and derivatization scheme for GC NICI/MS analysis of the major urinary prostaglandin D 2 metabolite (metabolite B) .
  • Figure 9 shows analysis of urine from a normal human volunteer for prostaglandin D metabolite (metabolite B) .
  • Figure 10 shows analysis of urine from a normal human volunteer for prostaglandin D metabolite (metabolite B) which illustrate skewing in the relative ratio of chromatographic peaks I and II compared to that of the internal standard.
  • Figure 11 shows standard curve for the analysis of prostaglandin D metabolite (metabolite B) by NICI/MS. Varying quantities of unlabelled prostaglandin D metabolite (metabolite B) were added to 4.14 ng of ( 18 0 4 -prostaglandin D metabolite (metabolite B) ) to give the ratios noted on the vertical axis. On the horizontal axis are plotted the actual ratios of m/z 514 (unlabelled prostaglandin D metabolite (metabolite B) to m/z 522 (labelled prostaglandin D metabolite (metabolite B) ) measured by selected ion monitoring analysis.
  • Figure 12 shows analysis of prostaglandin D metabolite, i.e. metabolite B in urine from a patient with systemic mastocytosis.
  • prostaglandin D 2 is a vasodilator. Roberts, L.J. II, Sweet an, B.J., Lewis, R.A. , Austen, K.F., Oates, J.A. : Increased Production of Prostaglandin D 2 in Patients with Systemic Mastocytosis. N. Engl. J. Med. 303:1400-4 (198 . 0). However, prostaglandin D 2 is initially metabolized in vivo by 11-ketoreductase to the metabolite,
  • Fig. 7 shows a comparison in the fold increase above the upper normal limit of urinary methylhistamine and the major urinary metabolite of prostaglandin D 2 in patients confirmed to have mastocytosis. Note that almost all of the dots lie to the right of the line of equality in the fold-increase in methylhistamine and prostaglandin D metabolite.
  • the solid circled dots have normal methylhistamine levels, but substantially elevated prostaglandin metabolite. In such patients, a biochemical diagnosis of mastocytosis could not have been made by measuring methylhistamine but could be established by measuring the prostaglandin metabolite. Additionally the dots circled in a broken line show only minimally elevated methylhistamine levels in comparison to levels of the prostaglandin metabolite several-fold above the normal upper limit.
  • prostaglandin D 2 metabolites are contemplated by this invention.
  • Substantially, pure prostaglandin D 2 metabolites can be used as an antigen in the process disclosed by Kohler and
  • the prostaglandin-like compound can be assayed using immuno or nucleic assays.
  • Immunoassay is a suitable method for the detection of small amounts of specific fatty acid derivatives (5-500 picogram can be readily detected) .
  • antibodies can be raised to these prostanoid-like compounds using conventional techniques. The antibodies can then be used in immunoassays to quantitate the amount of prostaglandin-like compounds in the biological fluid.
  • a small molecule (less than 5-10 kilodaltons) will usually not elicit the production of antibodies in experimental animals unless covalently linked to large immunogenic molecules prior to immunization.
  • Prostaglandin metabolites can be coupled via its carboxyl group to a carrier protein by the dicyclohexyl-carbodiimide method. Rich, D.H., etal.. The Peptides, 1:241-61 (1979); U.S. Patent No. 4,859,613. These compounds can be coupled to keyhole limpet hemocyanin (KLH) by the DCC method disclosed by Levine et al. Prostaglandines 20: 923-34 (1980) . For example, 9 ⁇ ,ll3-dihydroxy-15-oxo-2,3,18,19- tetranorprost-5-ene-l,20-dioic acid is dissolved in 100 ⁇ 1 of N,N-dimethyl formamide.
  • KLH keyhole limpet hemocyanin
  • the conjugate is then extensively dialyzed against phosphate-buffered saline, pH 7.5 (0.15MNaCl, 0.005M NaHP0 4 ) .
  • the conjugate is aliquoted and stored at -20 ⁇ C. It should be noted that many unsaturated fatty acids and their derivatives, are sensitive to oxidation, this does not hold for prostaglandins which have only a single double bond on each side of the chain.
  • myeloma that secrete monoclonal antibodies can be prepared following the techniques described by Kohler & Milstein (1975) . These antibodies can be used in immunoassays for prostaglandin metabolites. Typical assays for these types of compounds include enzyme-linked immunoassays, fluorescent immunoassays, and radioimmunoassays. The assays can be in the sandwich or competitive formats. See e.g. , David, U.S. Patent Nos. 4,736,110 and 4,486,530. In particular, a solid phase radioimmunoassay for the arachidonate derivatives 12(S)-HETE developed by Oxford Biomedical Research, Inc. could serve as a model for an immunoassay to prostaglandin metabolites.
  • Example 1 Quantification of major urinary metabolites in urine samples.
  • 6-keto-Prostaglandin F l ⁇ (m/z 614) .
  • standardization by GC/MS against 6-keto-prostoglandin F l ⁇ was chosen because both it and prostaglandin D metabolite (metabolite B) are stable PGF-ring compounds of similar polarity which require methoximation for analysis. Since the two compounds were co-derivatized for anlysis in the same vial, any derivatization losses that occur would be expected to be similar for both compounds. This standardization was carried out six times with a precision of ⁇ 12%.
  • Urinary PGD-M To one ml of urine is added 2.07 ng of the [ 0 ]-prostaglandin D metabolite (metabolite B) internal standard. The sample is then acidified to pH 3 with 1M HCL and allowed to stand at room temperature for 30 minutes. This quantitatively results in cyclization of the molecule to Compound C in Figure 8. Confirmation that cyclization under these conditions is quantitative was established upon analysis of uncyclized standard subjected to the same treatment by thin layer chromatography which effectively separates the cyclized and uncyclized forms of the metabolite. Following this, the sample is applied to a C-18 SEP-PAK preconditioned with 10 ml methanol and 10 ml pH 3 water.
  • the SEP-PAK is then washed with 10 ml pH three water followed by 10 ml heptane.
  • the extracted compounds are then eluted with 10 ml of ethyl acetate: eptane (50:50) into a scintillation vial.
  • the ethyl acetate:heptane is dried over 10 grams anhydrous Na 2 S0 4 and then applied to a silica SEP-PAK prewashed with 10 ml methanol and 10 ml ethyl acetate.
  • the SEP-PAK is subsequently washed with 5 ml ethyl acetate and compounds are then eluted into a reactivial with 5 ml ethyl acetate: ethanol (50:50) .
  • the ethyl acetate: ethanol is then dried under a stream of nitrogen and the residue reconstituted in 50 ul methanol.
  • the upper side chain carboxyl of prostaglandin D metabolite (metabolite B) is a free acid ( Figure 8, Compound C) .
  • the upper side chain carboxyl is then converted to a methyl ester by adding excess ethereal diazomethane and allowing the mixture to stand at 20°C for five minutes.
  • the lower side chain carboxyl is not methylated by this treatment because it is in the form of a lactone ( Figure 8, Compound D) .
  • the methylation solvents are evaporated under nitrogen and the residue is resuspended in 50 ul of acetonitrile.
  • the upper side chain carboxyl of the prostaglandin D metabolite (metabolite B) molecule is methylated whereas the lower side chain carboxyl is a free acid.
  • other acidic lipids which contain carboxyl groups that cannot undergo cyclization would have been methylated by the previous treatment with diazomethane. Therefore, considerable purification of prostaglandin D metabolite (metabolite B) from such potentially interfering lipids can be achieved at this point by incorporating an extraction step at neutral pH.
  • the lower carboxyl group on the prostaglandin D metabolite (metabolite B) molecule is converted to a pentafluorobenzyl ester ( Figure 8, Compound F) by adding 40 ul of a 10% solution of pentaflurobenzyl bromide in acetonitrile and 20 ul of a 10% solution of diisopropylethanolamine in acetonitrile. The mixture is incubated for 20 minutes at 37 ⁇ C and then dried under nitrogen. This procedure is then repeated a second time. Two cycles of esterification are performed since we have found that two cycles achieves more quantitative esterification of prostaglandins compared to one cycle, even if the length of the derivatization is allowed to proceed for a longer period of time with excess reagents.
  • PGF 2 ⁇ methyl ester is used as a thin layer chromatography marker because of the unavailability of sufficient quantities of synthetic unlabelled prostaglandin D metabolite (metabolite B) for this purpose.
  • Prostaglandin D metabolite (metabolite B) is then converted to a trimethylsilyl ether derivative
  • the major ion generated in the NICI mass spectrum of the O-methyloxime, mono-methyl ester, mono-pentabluorbenzyl ester, bis-trimethylsilyl ether derivative of prostaglandin D metabolite (metabolite B) is m/z 514 which represents the M-181 (M-CH 2 C g F 5 ) carboxylate anion.
  • the r L 18 n ⁇ 4]-internal standard generates an anlogous ion at m/z 522.
  • Quantification of endogenous prostaglandin D metabolite (metabolite B) is accomplished by selected ion monitoring analysis of the ratio of intensities of m/z 514 to m/z 522.
  • the recovery of prostaglandin D metabolite (metabolite B) through the assay is in the range of 10-20%.
  • FIG. 9A and B A representative selected ion monitoring chromatogram obtained from analysis of urine from a normal individual is depicted in Figure 9A and B. At the bottom is the m/z 522, selected ion monitoring chromatogram representing the r L18 n u 4]-labelled internal standard. Two peaks are present representing the syn- and anti-methoxime isomers. The m/z 514 chromatogram at the top of the figure representing endogenous compounds reveals four peaks of approximately equal intensity (labelled I-IV) rather than two as is present for the internal standard.
  • Peaks I and II have the same retention time on capillary gas chromatography as the 18 0-labelled prostaglandin D metabolite (metabolite B) internal standard and that their intensity is markedly suppressed by cyclooxygenase inhibitors strongly suggests that they represent the endogenous metabolite of prostaglandin D 2 .
  • the material in peaks I and II may also be comprised, at least in part, by the analogous metabolite of cyclooxygenase derived prostoglandin F 2 ⁇ .
  • the analogous metabolites of prostaglandin D 2 and prostaglandin F 2 ⁇ have the same basic structure and molecular weight and would thus generate the same M-181 ion (m/z 514) , they differ in regards to the stereochemistry of the cyclopentane ring hydroxyls.
  • the cyclopentane ring hydroxyls in the prostaglandin D 2 metabolite are oriented trans due to the fact that 11-ketoreductase stereospecifically transforms prostaglandin D 2 to 9 ⁇ ,110-prostaglandin F 2 . Liston, T.E., Roberts, L.J. II: Proc. Natl. Acad. Sci. USA 82:6030-4 (1985).
  • the cyclopentane ringe hydroxyls of the analogous metabolite derived from prostaglandin F 2 ⁇ are cis.
  • the prostaglandin F 2 ⁇ metabolite is a by-product of the chemical synthesis of the prostaglandin D 2 metabolite. Prakash, C. , Saleh, S., Roberts, L.J., Blair, L.A. , Taber, D.F. J. Chem. Soc. Perkin Trans. 1:2821-6 (1988) . Therefore, we were able to examine the capillary gas chromatography characteristics of this compound. It was found to be separated from peaks I and II, eluting with a longer retention time in region of peaks III and IV.
  • peak II was suppressed by only approximately 40% (Figure 10B) .
  • the second peak is comprised of more than one compound, one of which is derived from cyclooxygenase sources (prostaglandin D 2 ) and the other from noncyclooxygenase sources.
  • peak II is in fact comprised of at least two compounds with slightly different but overlapping R f values.
  • peak II in urines such as shown in Figure 10 is comprised of a mixture of compounds with cis and trans hydroxyls.
  • cyclopentane-ring hydroxyls in the material in peak I are exclusively oriented trans. This provides further evidence that peak I is not a mixture of compounds and that it is comprised entirely by the first methoxime isomer of the endogenous metabolite of prostaglandin D 2 .
  • a standard curve was construced by adding varying amounts of unlabelled prostaglandin D metabolite (metabolite B) to a fixed quantity of 4.14 ng of [ 0 4 ]-prostaglandin D metabolite (metabolite B) and the measured ratio of m/z 514 to m/z 522 to the expected ratio was compared.
  • the standard curve was found to be linear over a concentration range of 16-fold ( Figure 11) .
  • the amount of endogenous prostaglandin D metabolite (metabolite B) measured in the precision experiment was subtracted from the total measured and the accuracy of the assay to measure the added 1.2 ng of prostaglandin D metabolite (metabolite B) was calculated. The accuracy was found to be 96%.
  • Urinary Prostaglandin D Metabolite (metabolite B) Levels in Clinical Situations Associated With Increased Release of PGD 2 We then examined the ability of the assay to assess overproduction of PGD 2 in clinical situations in which increased quantities of prostaglandin D are known to be released in vivo. Prostaglandin D has been shown to be markedly overproduced in patients with systemic mastocytosis. Roberts, L.J. , Sweetman, B.J. , Lewis, R.A. , Austen, K.F., Oates, J.A. : N. Engl. J. Med. 303:1400-4 (1980) .
  • Figure 12 shows the results of urine analyzed for prostaglandin D metabolite (metabolite B) in such a patient. Note in this chromatogram that the ratio of the first two peaks (I and II) relative to the second two (III and IV) is markedly altered opposite to what was found with treatment with indomethacin ( Figure 9B) .
  • the level of prostaglandin D metabolite (metabolite B) in this patient's urine was substantially elevated approximately 12-fold above normal at 12 ng/mg creatinine.
  • niacin Ingestion of the hypolipidemic agent, niacin, evokes intense flushing. Recently we reported that niacin induces the release of large quantities of prostaglandin D 2 assessed by measuring levels of the prostaglandin D 2 metabolite, 9 ⁇ ,113-prostaglandin F 2 , in plasma. Morrow, J.D., Parsons, W.G., Roberts, L.J.: Prostaglandins. 38:263-74 (1989). Analysis of urines from three individuals collected over a seven hour period following ingestion of 500 mg of niacin revealed elevated urinary excretion of prostaglandin D metabolite (metabolite B) ranging from 25-48 fold above normal.

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Abstract

Cette invention concerne un procédé amélioré permettant de diagnostiquer les mastocytoses systémiques en détectant la présence ou la concentration de certains métabolites de la prostaglandine D2. Plus spécifiquement, cette invention concerne un moyen plus précis qui permet de diagnostiquer plus exactement les mastocytoses systémiques que le test normal utilisé actuellement qui détecte l'histamine-Me.
PCT/US1992/002368 1991-06-14 1992-03-25 Procede de diagnostic des mastocytoses systemiques Ceased WO1992022817A1 (fr)

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US715,568 1991-06-14

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CLINICAL RESEARCH, Vol. 38, issued 1990, J.D. MORROW et al., "Correlation of Endogenous Production of Prostaglandin D2 and Histamine in Patients with mastocytosis", page 581A. *
JOUR. CHEM. SOCIETY PERKIN TRANS. I, Vol. 10, issued 1988, C. PRAKASH et al., "Synthesis of the Major Urinary Metabolite of Prostaglandin D2", pages 2821-2826. *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol. 105, No. 6, issued 1983, E.J. COREY et al., "Total Synthesis of the Major Urinary Metabolite of Prostaglandin D2, a Key Diagnostic Indicator", pages 1662-1664. *
METHODS IN ENZYMOLOGY, Vol. 86, issued 1982, L.J. ROBERTS II, "Quantification of the PGD2 Urinary Metabolite 9 alpha-Hydroxy-11,15-Dioxo-2,3,18,19-Tetranorprost-5-Ene-1,20-Dioic Acid by Stable Isotope Dilution Mass Spectrometric Assay", pages 559-570. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 260, No. 24, issued 25 October 1985, T.E. LISTON et al., "Metabolic Fate of Radiolabeled Prostaglandin D2 in a normal Human Male Volunteer", pages 13172-13180. *
THE NEW ENGLAND JOURNAL OF MEDICINE, Vol. 303, No. 24, issued 11 December 1980, L.J. ROBERTS II et al., "Increased Production of Prostaglandin D2 in patients with Systemic Mastocytosis", pages 1400-1404. *

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