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WO1997009038A1 - Diagnostic et traitement d'affection neurologique - Google Patents

Diagnostic et traitement d'affection neurologique Download PDF

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Publication number
WO1997009038A1
WO1997009038A1 PCT/US1996/013506 US9613506W WO9709038A1 WO 1997009038 A1 WO1997009038 A1 WO 1997009038A1 US 9613506 W US9613506 W US 9613506W WO 9709038 A1 WO9709038 A1 WO 9709038A1
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Prior art keywords
gelatinase
mammal
multiple sclerosis
treatment
blood
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PCT/US1996/013506
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English (en)
Inventor
Gary A. Rosenberg
Lance Liotta
William G. Stetler-Stevenson
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University Of New Mexico
The Government Of The United States Of America, Re
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Application filed by University Of New Mexico, The Government Of The United States Of America, Re filed Critical University Of New Mexico
Priority to AU68528/96A priority Critical patent/AU6852896A/en
Publication of WO1997009038A1 publication Critical patent/WO1997009038A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)

Definitions

  • the invention relates to the diagnosis and treatment of neuropathological conditions based on the quantitation of specific types of metalloproteinases present in the cerebrospinal fluid (CSF) .
  • CSF cerebrospinal fluid
  • Disruption of the blood/brain barrier has been for some time associated with observed neuropathologies in neurological diseases and disorders such as multiple sclerosis (MS) , meningi ⁇ tis, HIV encephalopathy, and cerebrovascular disease (Bradbury, "The Concept of a Blood-Brain Barrier", Chichester: John Wiley and Sons, 1979) .
  • This disruption has been recently attributed at least in part to pathological increases in permeability of brain capillary vessels resulting from abnormal activity of certain type IV collagenases which degrade type IV collagens present in the capillary basal lamina and brain cell membranes (see, e.g., Brain Res . 5Z£:203-207, 1992) .
  • MMP extracellular matrix-degrading proteolytic enzymes
  • This class of type IV collagenases includes certain zinc-containing calcium-dependent proteinases which are secreted by brain cells into the extracellular matrix in zymogen (proenzyme or inactive) form following stimulation of an AP-1 promoter region of the controlling MMP gene by, for example, cytokines, TNF- ⁇ , or IL-l ⁇ .
  • the zymogen is activated by cleavage of a pepcide sequence of the zymogen to the corresponding proteolytic enzyme which hydrolyzes matrix molecules such as type IV collagens and fibronectin.
  • Activators such as urokinase plasminogen activator
  • uPA metalloproteinase zymogens
  • Potential treatments to block MMP action include the administration of peptides corresponding to cleavage by-products to inhibit activation of zymogen by feedback mechanisms (U.S. Patent 5,280,106 to Liotta, et al, issued January 18, 1994, incorporated herein by reference) , as well as tissue inhibitors to metalloproteinases (TIMPS) , as described in J_--Bi -.C_r ⁇ em.. 2-6-5 : 13933-13938, 1990. To avoid a shotgun approach to therapy, however, these latter treatments are dependent upon accurate characterization of pathogenic enzymes responsible, and such characterizations have not been heretofore possible.
  • FIGURE 1 A) Gelatin-substrate zymograms of CSF from the MS patients with enhancing lesions (ENH1-ENH4) on MRI. Pre- and post-treatment CSF samples for individual patients are shown. Molecular weights shown by the arrows were determined from human HT1080 cells which constituently express gelatinase A and B. B) Zymograms from patients without enhancement (NEN1-NEN3) on MRI. Low levels of gelatinase B were seen in both the pre- and post- treatment gels.
  • FIGURE 2 Relative lysis zones for gelatinase A for pre- and post-treatment values. Those patients with enhancing lesions showed a significant increase in gelatinase A after treatment. No differences between the pre- and post-treatment values were seen in the CSF of those without enhancement .
  • FIGURE 3 Relative lysis zones for gelatinase B on zymograms analyzed quantitatively. Levels of gelatinase B before (Pre-MP) and after treatment with high-dose methylprednisolone (Post-MP) are shown as means and standard errors. The asterisks shows the statistically significant differences between the before and after values with the significance level given in parentheses.
  • FIGURE 4 A) Western immunoblot with a monoclonal antibody to gelatinase B in the patients with enhancement on MRI. Paired CSF samples from the same patients before and after treatment with 1 gm/kg methylprednisolone for 3 days. Samples from the patients with enhancement on the MRI are run since they had elevated levels of gelatinase B prior to treatment. A band at about 117- kDa is seen, representing a complex of gelatinase B and one or more TIMPs . B) A reduced western blot showing several lower molecular weight forms of gelatinase B. C) The protein antigen detected by the antibody to human gelatinase B as measured by computer image analysis, using NIH Image (infra) . Values shown are for the 117-kDa molecular weight complex. A significant increase in post-treatment (POST) values compared to pre ⁇ treatment (PRE) values was found, using a paired Student t-test as shown by asterisks .
  • the invention comprises a method for the clinical detection of elevated CSF levels of a non-specific matrix metalloproteinase non-specific zymogen marker for a neurological disease or disorder characterized by blood/brain barrier disruption.
  • the invention further comprises a method for diagnosing a neuropatholdgical condition accompanied by blood/brain barrier disruption including detection and quantitation of marker zymogen with correlation of the results against standards for normal and abnormal patients, herein also referred to as "quantitative zymography" .
  • the methods of the invention are contemplated as useful in the diagnosis and treatment of various neurological diseases or disorders of both spinal cord and brain involving disruption of the blood/brain barrier, including multiple sclerosis, Alzheimer's disease, and bacterial meningitis, especially for the prediction of acute phases of such diseases or disorders.
  • the invention provides a non ⁇ specific marker for multiple sclerosis comprising the 92 kDa zymogen of gelatinase B, and a method for predicting an incipient acute attack of this disease by quantitative zymography.
  • the invention further provides a method for clinical intervention in a progression of neurological diseases or disorders such as MS from latent to acute stage, comorisincr administering to the patient an inhibitor for inhibiting cleavage and/or activation of this zymogen.
  • the invention is predicated on the inventors ' concept that steroid efficacy in the clinical treatment of blood/brain barrier disruption typically-associated with inflammation and/or infection of the cerebrospinal system in active neuropathologies is attributable to steroid interference with gelatinase transcription (from zymogen to active form and induction of an inhibitor) .
  • gelatinase transcription from zymogen to active form and induction of an inhibitor
  • gelatinase A While gelatinase A is involved normally in tissue remodeling, and levels thereof increase in response to normal need, it has been found that gelatinase B is elevated only under pathological conditions. Based on the present findings, it further appears that gelatinase B initiates pathological processes . Steroids suppress binding of early response gene products to the AP-1 promoter site of some gelatinases in cell cultures (see, e.g., Cell £2: 1189-1204, 1990) , but not gelatinase A promoter sites. In fact, elevated levels of gelatinase A have been observed in post-steroid treated afflicted patients (see Examples) .
  • gelatinase A most likely performs a reparative function for damaged tissues, rather than participating in tissue destruction as does gelatinase B.
  • gelatinase A in either latent or active form is not a marker of neuropathologies involving blood/brain barrier disruption.
  • the methods of the invention include diagnosis of blood/brain barrier disruption predicated on abnormally high levels of gelatinase B or its zymogen in the CSF of patients.
  • the methods of the invention further include treatment of this condition by inhibiting cleavage of gelatinase B zymogen to active form to inhibit tissue-destructive activity; by inhibiting activity of the active enzyme, including methods for inhibiting production of urokinase tissue plasminogen activator or other agents which enhance gelatinase B activity; or by increasing complexing of gelatinase
  • gB tissue inhibitors such as TIMPs .
  • Gelatinase B (gB) in latent form has a known amino acid sequence, as do the active cleavage products.
  • the latent zymogen form comprises a 92kDa amino acid sequence, which is cleaved in v vo into an active 84 kDa sequence and byproduct peptide.
  • Gelatinase A in active form comprises a 62-kDa peptide, as is also known in the art (see, e.g., Oncogene 8 : 395-405, 1993) .
  • cerebro- spinal fluid obtained by lumbar puncture is subjected to gel electrophoresis to assess for the presence of gelatinase B zymogen (92 kDa) and/or active gelatinase B (84 kDa) .
  • Any conventional electrophoretic gel is useful, such as SDS (sodium dodecylsulfate) /polyacrylamide gel) ; the selected gel is copolymerized with gelatin to provide a substrate for active gelatinases present.
  • Zy ographic analysis may be conducted according to the published procedures in Ana-L. Biochem. 2---8:325-
  • SDS is used to inactivate activated zymogens to allow them to migrate through the gel without disrupting the gelatin.
  • Triton-X is added to remove the SDS and reactivate the enzymes, allowing them to be seen as clear bands in the Coumassie blue stained gels.
  • gelatinase 3 Since the active form of gelatinase 3 is often present in vivo as a complex with a tissue inhibitor for metalloproteinase (TIMP) which is released along with the enzyme, the analysis must account for this complex.
  • Potential complexes include gB/TIMP-1 and gB/TIMP-3, having, respectively, kDas of 120 and 117. The latter complex was prevalent in the experiments described below; also, the identity of the tissue inhibitor of metalloproteinases complexed to gelatinase B in CSF has been previously shown to be primarily TIMP-3 using a method for detection of TIMP referred to as reverse zymography (J. Biol. Chem. 269: 9352-9360, 1994) .
  • TIMP-complexed gelatinase B When TIMP-complexed gelatinase B is incorporated into SDS-gel along with gelatin, the presence of TIMP blocks the digestion of the gelatin by the gelatinase, and TIMPs are seen as undigested or dark bands in the gel.
  • an increase in TIMP-3 was observed after steroid treatment in patients with acute MS, which explains the increase in the gB/TIMP complex seen in Western immunoblots using antibody to gelatinase B.
  • steroids may improve blood-brain barrier integrity by blocking gelatinase B production, interfering with gB activation, or by increasing TIMP-3.
  • zymogenic gelatinase B is conveniently confirmed by western immunoblot techniques well-known in the art, employing, for example, monoclonal antibodies to gB which recognize only the latent form of the enzyme, available from Oncogene Science, Inc., Uniondale, NY, USA (see for techniques, Cancer Re-a--. 5-3: 140-146, 1993; HybridQma 12: 349-363, 1993) .
  • Monoclonal antibodies to Tl P-1 are described in C.c_llagen---and Related Research 2:341-350, 1987.
  • lysis zones above about 200 on the above-described gel electrophoresis may indicate that therapy should be undertaken.
  • the lysis zone size is determined by the amount of gelatinase present, as known in the art. Determination of urokinase tissue plasminogen activator is described below.
  • Protein electrophoresis of the CSF and serum was performed. Subjects were treated for three days with methylprednisolone (1 gm/ day) . After treatment, clinical grading (EDSS) and lumbar puncture were repeated. Eight patients entered into the study and seven completed the entire protocol. Data from one patient was removed because one of the paired CSF samples were los . Ail were experiencing an increase in clinical symptoms due to an exacerbation of multiple sclerosis that was considered sufficiently severe to warrant treatment with high-dose methylprednisolone .
  • Matrix metalloproteinases were measured in the CSF of the above patients by substrate-gel electrophoresis with gelatin- containing gels (Ana3-. -_-i_--------em._L02:196-202, 1980) .
  • the zymographic technique employed is described in l_-a-- ⁇ o----a_t--o-z-y- Investigation, o-p--_c-i_-----) Briefly, CSF was analyzed by substrate- gel sodium-dodecylsulfate polyacrylamide gels with gelatin copolymerized into the gels.
  • Polyacrylamide minigels (10%) were prepared (1.5 ml distilled water; 1.18 ml Tris-HCl (1.5M, pH 8.8) ; 45 ⁇ l 10% sodium dodecylsulfate (SDS) ; 1.5 ml N',N'-bis- methylene-acrylamide (30% total monomer concentrations and 2.67% crosslinking monomer concentrations) ; 2.2 ⁇ l N,N,N' ,N' -tetra- methyl ethylenediamine (TEMED) ; and 22 ⁇ l ammonium per sulfate solution (APS, 100 mg/ml) ) , with gelatin (300 ⁇ l , 15 mg/ml solution) copolymerized into the gels (Sigma #G-2500) .
  • GIBCO 26041-20 and HT1080 fibrosarcoma human cell line culture media conditions were used as protein standards.
  • HT1080 contains 72- kDa and 92-kDa type IV collagenases, and was run against every gel in order to determine molecular weights of collagenases observed in CSF. Following electrophoresis, gels were agitated in 2.5% Triton X-100 to remove the SDS and restore enzymatic activity.
  • Power PC 7100 Power PC 7100.
  • a computer imaging system (NIH Image SoftwareTM) was used to determine relative lysis zone areas. The paired samples were run on the same gel to reduce errors introduced by differences between gels.
  • Nonspecific binding sites on the PVDF-Plus membranes were blocked overnight at 4°C with 10% bovine serum albumin (BSA) in Tris-buffered saline (0.02M Tris-HCl and 0.5M NaCl,pH 7.5 ) . Overnight blocked membranes were washed with 50 ml of wash buffer (0.5% BSA, 0.001% Tween 20, and TBS) for 1 hour with four changes of buffer.
  • BSA bovine serum albumin
  • Immunoblots were performed using monoclonal antibodies to human gelatinase B (Oncogene, supra) that recognize only the latent form of gelatinase B, or monoclonal antibodies to TIMP-1, supra.
  • Primary antibodies were diluted to 0.002mg/ml or 0.01 mg/mi in wash buffer and blotted membranes were shaken in primary antibodies at 4°C overnight for 15 hours. Membranes were then washed at room temperature for 1 hour in 50 ml wash buffer with four changes of buffer.
  • results were statistically analyzed by a paired t- test (SAS Statistical Analysis System; Cary, NC) . Significance was taken as p ⁇ 0.05. Values are given as mean ⁇ standard error of the mean.
  • Table 1 shows the results of the CSF studies before and after treatment.
  • the changes in CSF protein, IgG index, and myelin basic protein were not significant.
  • the average disability score (EDSS) showed a small, but significant, decrease from 4.4 ⁇ 0.4 before treatment to 4.1 ⁇ 0.4 (p ⁇ 0.047) .
  • Oligoclonal bands were present in six of the seven patients.
  • Gelatin-substrate zymograms of paired CSF samples before and after steroid treatment are shown in Figure 1.
  • 72-kDa type IV collagenase (gelatinase A) was seen in all samples.
  • Several patients had low levels of 84-kD gelatinase, which is the molecular weight of the active form of gelatinase B. All except one of the patients had detectable levels of gelatinase B.
  • Gelatinase A however, increased slightly for the entire group.
  • Urokinase-type plasminogen activator decreased significantly (Table 2) .
  • the molecular species at 117- kDa detected by the antibody to gelatinase B, represents a complex of gelatinase B with one or more TIMP molecules. Subtracting 92-kDa from the complex molecular weight gave an estimated TIMP molecular weight of 25t3-kDa. There was a significant increase in the complex after treatment with steroids in patients with contrast enhancement a d elevated levels of gelatinase B ( Figure C) .

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Abstract

La présente invention concerne un procédé de diagnostic et de traitement de troubles ou d'affections neurologiques caractérisées par la rupture de la barrière hémato-méningée. Le zymogène 92 kDa de la gélatinase B et la gélatinase B active sont des marqueurs des neuropathologies liées à la rupture de la barrière hémato-méningée.
PCT/US1996/013506 1995-09-01 1996-08-29 Diagnostic et traitement d'affection neurologique WO1997009038A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002066057A3 (fr) * 2001-02-23 2003-07-17 Biophage Inc Methodes et compositions de prevention et de traitement de maladies induites par les neutrophiles
CN112451683A (zh) * 2019-09-09 2021-03-09 中国医学科学院药物研究所 Timp-1在制备预防或治疗创伤性脑损伤药物中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5532265A (en) * 1994-11-30 1996-07-02 The Board Of Trustees Of The Leland Stanford Junior University Treatment of central nervous system inflammatory disease with matrix metalloprotease inhibitors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5532265A (en) * 1994-11-30 1996-07-02 The Board Of Trustees Of The Leland Stanford Junior University Treatment of central nervous system inflammatory disease with matrix metalloprotease inhibitors

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
INFLAMM. RES., Volume 44, issued 1995, NORGA et al., "Prevention of Acute Autoimmune Encephalomyelitis and Abrogation of Relapses in Murine Models of Multiple Sclerosis by the Protease Inhibitor D-Penicillamine", pages 529-534. *
J. CLIN. INVEST., Volume 94, issued December 1994, GIJBELS et al., "Reversal of Experimental Autoimmune Encephalomyelitis With a Hydroxamate Inhibitor of Matrix Metalloproteinases", pages 2177-2182. *
JOURNAL OF NEUROIMMUNOLOGY, Volume 41, Number 1, issued 1992, GIJBELS et al., "Gelatinase in the Cerebrospinal Fluid of Patients With Multiple Sclerosis and Other Inflammatory Neurological Disorders", pages 29-43. *
LABORATORY INVESTIGATION, Volume 71, Number 3, issued 1994, ROSENBERG et al., "Injury-Induced 92-Kilodalton Gelatinase and Urokinase Expression in Rat Brain", pages 417-422. *
NEUROLOGY, Volume 46, issued June 1996, ROSENBERG et al., "Effect of Steroids on CSF Matrix Metalloproteinases in Multiple Sclerosis: Relation to Blood-Brain Barrier Injury", pages 1626-1632. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002066057A3 (fr) * 2001-02-23 2003-07-17 Biophage Inc Methodes et compositions de prevention et de traitement de maladies induites par les neutrophiles
CN112451683A (zh) * 2019-09-09 2021-03-09 中国医学科学院药物研究所 Timp-1在制备预防或治疗创伤性脑损伤药物中的应用
CN112451683B (zh) * 2019-09-09 2023-09-26 中国医学科学院药物研究所 Timp-1在制备预防或治疗创伤性脑损伤药物中的应用

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