WO1995021380A1 - Dosage servant au criblage d'antagonistes d'un recepteur - Google Patents

Dosage servant au criblage d'antagonistes d'un recepteur Download PDF

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WO1995021380A1
WO1995021380A1 PCT/US1995/001375 US9501375W WO9521380A1 WO 1995021380 A1 WO1995021380 A1 WO 1995021380A1 US 9501375 W US9501375 W US 9501375W WO 9521380 A1 WO9521380 A1 WO 9521380A1
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gastrin
binding
cck
compound
gbp
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PCT/US1995/001375
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Graham Sherard Baldwin
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Ludwig Institute For Cancer Research
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Priority claimed from AUPM3650A external-priority patent/AUPM365094A0/en
Priority claimed from AUPM6638A external-priority patent/AUPM663894A0/en
Priority claimed from AUPM7779A external-priority patent/AUPM777994A0/en
Application filed by Ludwig Institute For Cancer Research filed Critical Ludwig Institute For Cancer Research
Priority to AU17405/95A priority Critical patent/AU1740595A/en
Publication of WO1995021380A1 publication Critical patent/WO1995021380A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/60—Salicylic acid; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/595—Gastrins; Cholecystokinins [CCK]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • This invention relates to a method for screening of putative antagonists of binding of gastrin to its receptor, in particular the low-affinity gastrin-cholecystokinin C receptor, more particularly the gastrin-binding protein (GBP), and also of inhibitors of the enzyme activities associated with the GBP.
  • the low-affinity gastrin-cholecystokinin C receptor more particularly the gastrin-binding protein (GBP)
  • GBP gastrin-binding protein
  • Compounds having the ability to block binding of gastrin to GBP or to block the enzyme activities of the GBP are useful in control of cellular proliferation, especially in treatment of neoplastic disease, and are also useful in the control of acid production in the stomach.
  • gastrin has been known for over 80 years to be a stimulant of acid secretion by the parietal cells of the stomach.
  • Gastrin is a small polypeptide hormone which participates in a feedback system for regulating the environment in the lumen of the alimentary tract, and is thought to affect other target organs, including the brain and pancreas. More recently, it has been recognised that gastrin also acts as a growth factor for the gastric mucosa. Both of these functions are presumably mediated by the binding of gastrin to specific receptors on the target cells in the gastrointestinal mucosa. A variety of methods for detection of such
  • Gastrin/CCK-C receptors have been identified on cell lines of a variety of tumour cell lines of epithelial and non-epithelial origin, and on several cell lines of lymphocytic origin.
  • significant levels of gastrin/CCK-C receptors have been observed with cell lines established from carcinomas of the stomach, colon, breast, and vulva, and from malignant melanoma: in addition, lower levels of binding were observed with T- and B-cell lymphoma cell lines, while still smaller levels of binding were observed with promyelocyti ⁇ and myeloid leukaemia cell lines (3).
  • CCK cholecystokinin
  • Progastrin but not mature amidated gastrin, was also produced by all (5/5) colonic carcinoma cell lines tested. Exogenous gastrin 17 or its derivatives enhanced the growth of xenografts of 60% of colon
  • colon carcinoma cell lines do not increase either DNA or protein synthesis in vitro in response to exogenous gastrin, perhaps because they are already maximally stimulated by an autocrine gastrin-like peptide. In some cases previously
  • the antagonist proglumide which does not discriminate clearly between the three known classes of gastrin/CCK receptors, increases the survival time of mice bearing tumours derived from the mouse colon carcinoma line MC26 (1). Proglumide also reverses the increase in tumour volume observed in tumour-bearing mice after treatment with pentagastrin. Proliferation of 6 colon carcinoma cell lines in vitro is inhibited by the non-selective antagonists proglumide and benzotript, with IC 50 values in the mM range (2). Furthermore, in the case of the HCT 116 cell line proliferation is also inhibited by an anti-gastrin antiserum (2).
  • the high concentrations of gastrin required to reverse inhibition by both antagonists and antibodies suggest that neither pancreatic CCK-A nor gastric gastrin/CCK-B receptors are involved, but are consistent with the involvement of the low affinity
  • gastrin/CCK-C receptor which we have identified on the surface of several gastric and colonic carcinoma cell lines (2).
  • the failure of the selective antagonists 1.364,718 and L365,260 to inhibit proliferation of HCT 116 cells at concentrations as high as 1 ⁇ M confirms that neither CCK-A nor gastrin/CCK-B receptors participate in the autocrine loop (4).
  • GBP gastrin-binding protein
  • sequences of the naturall.v occurring protein (Baldwin et al: Int. J. Biochem 26 529-538). These sequences have been used to generate probes which have enabled cloning of the cDNA encoding the porcine GBP from both c-DNA and genomic libraries.
  • porcine GBP The sequence of porcine GBP, deduced from the cDNA sequence, is related to those of enzymes having enoyl CoA hydratase activity and to enzymes having 3-hydroxy-acyl-CoA dehydrogenase activity. These enzymes are
  • the N-terminal half of the GBP is related to the enoyl-CoA hydratase family, while the C-terminal half is related to the 3-hydroxy-acyl-CoA dehydrogenase family (6).
  • the sequence of the porcine GBP is closely related to the sequence of the ⁇ -subunit of a rat
  • MTP mitochondrial trifunctional protein
  • affinities of antagonists for the GBP correlate well with their affinities for the gastrin/CCK-C receptor, and with their potencies for inhibition of colon carcinoma cell growth.
  • gastrin/CCK receptor antagonists we have found that other classes of compounds inhibit the cross-linking.
  • non-steroidal anti-inflammatory drugs inhibit not only the cross-linking, but also proliferation of a human colon carcinoma cell line.
  • gastrin/CCK receptor antagonists and compounds such as non-steroidal anti- inflammatory drugs;
  • the present invention provides a method of identifying a compound having the ability to block the low-affinity gastrin-cholecystokinin type C receptor, comprising the step of measuring the ability of said compound to block the binding to gastrin-binding protein of a compound selected from the group consisting of a gastrin-related peptide, a cholecystokinin-related peptide, an antagonist of gastrin or of cholecystokinin, an acyl CoA, an enoyl CoA, an antibody to gastrin, and an antibody to cholecystokinin.
  • the binding may be reversible or irreversible, but is preferably irreversible.
  • inhibition of irreversible binding is assessed by measuring the ability of the compound to block cross-linking of a gastrin
  • Cross-linking may be effected by an agent which is homobifunctional, heterobifunctional, or photoreactive.
  • agent which is homobifunctional, heterobifunctional, or photoreactive.
  • disuccinimidyl suberate disulfosuccinimidyl suberate, and ethylene glycol bis-(succinimidyl)-succinate.
  • Heterobi-functional cross-linking reagents include succinimidyl-4-(p-maleimidophenyl) butyrate, which has been found to be equally effective in cross-linking the gastrin peptide to the gastrin binding protein.
  • Suitable photoreactive reagents include N-hydroxysuccinimidyl-4-azidobenzoate.
  • the gastrin peptide is labelled with a detectable marker, such as a radioactive label, a fluorescent label, or biotin.
  • a detectable marker such as a radioactive label, a fluorescent label, or biotin.
  • the gastrin peptide may be
  • iodinated or may be synthesised using one or more amino acids comprising a radioactive marker such as 14 C or 3 H
  • Native gastrin gastrin 17 , contains no free amino groups, and is therefore incapable of reacting with the cross-linking reagent.
  • the minimum gastrin sequence required for binding to the gastrin/CCK-C receptor is known to be the C-terminal tetrapeptide, so N-truncated peptides are expected to be suitable.
  • [Leu 15 ] gastrin 17 and [Nle 15 ] gastrin 2,17 have equal ability to bind to isolated canine parietal cells or parietal cell plasma membranes: substitution of methionine by leucine isomers does not interfere with the binding or biological activity of gastrin.
  • a particularly preferred gastrin peptide is [Nle 15 ] gastrin 2,17 , a derivative which lacks the amino-terminal pyroglutamate residue. Substitution of nor-leucine for the naturally occurring methionine at position 15 prevents formation of oxidised methionine derivatives during iodination. Although [Met 15 ]-gastrin 2,17 could be used, there would be some loss of activity for this reason. It is contemplated that gastrin peptides extended at the C-terminus could also be used.
  • a displacement-type assay using reversible binding is employed.
  • the ability of the putative antagonist to prevent binding of a known agonist or antagonist of very high affinity is measured, said known agonist or antagonist being labelled with a detectable marker.
  • the known agonist or antagonist has at least the binding affinity of benzotript.
  • the known antagonist is suitably labelled with 3 H.
  • the method of the invention provides a general screening method for identifying antagonists of the
  • compounds are useful for treatment of diseases, especially neoplastic diseases, involving rapidly proliferating cells. Such antagonists are also useful for controlling gastric acid secretion.
  • Non- steroidal anti-inflammatory drugs which are especially useful for treatment of neoplastic diseases of the gastrointestinal tract, in particular colorectal cancer.
  • NSAIDS non- steroidal anti-inflammatory drugs
  • GBP is widespread in the body, and consequently treatment of cancers of non-gastrointestinal origin is also within the scope of this invention.
  • the NSAID is an inhibitor of prostaglandin activity.
  • inhibition of the enoyl CoA hydratase and/or 3-hydroxyacyl CoA is another preferred embodiment, inhibition of the enoyl CoA hydratase and/or 3-hydroxyacyl CoA
  • dehydrogenase activities intrinsic to the GBP is assayed. Either activity can be measured in either direction, but is conventionally assayed spectrophotometrically by the decrease in enoyl CoA absorption on hydration or by the decrease in NADH absorption on oxidation. Alternative assays could employ radiochemically labelled substrates.
  • Gastrin-binding protein suitable for use in the method of the invention may be partially purified or purified naturally occurring GBP, or may be recombinant GBP.
  • a high-affinity labelled antagonist in a reversible binding assay, a high-affinity antibody, such as a monoclonal antibody, labelled with a detectable marker may be used.
  • a detectable marker for example, it is contemplated that an antibody capable of blocking binding of an acyl CoA or of gastrin to GBP may be used.
  • the invention in another aspect therefore provides a method of treating neoplastic disease
  • the neoplastic disease is selected from the group consisting of gastrointestinal cancers such as colon carcinoma and gastric carcinoma, mammary
  • the invention provides a pharmaceutical composition of a compound with the ability to inhibit the binding of a gastrin analogue to the low-affinity gastrin-cholecystokinin type C receptor,
  • the compound is a non-selective antagonist of the gastrin-cholecystokinin receptor.
  • the antagonist will have a IC 50 value" in the millimolar range or lower.
  • compositions of the invention may comprise an antagonist either alone, or in combination with other pharmaceutically-active molecules such as antacid compounds or anti-cancer agents.
  • Anti-cancer agents may include cytokines.
  • the invention provides a method of treatment of a neoplastic disease, comprising the step of inducing production of antisense gastrin mRNA in neoplastic tissue of an animal in need of such treatment.
  • Tissue-specific production of anti-sense mRNA can be achieved by targeting of corresponding DNA to the desired tissue, using tissue-specific promoters.
  • Figure 1 shows gastrin analogues and antagonists inhibit covalent cross-linking of 125 I-[Nle 15 ]-gastrin 2,17 to the 78kDa GBP.
  • Figure 2 shows binding of fatty acyl CoA derivatives to the 78 kDa GBP.
  • Cross-linking of 125 I-[Nle 15 ]-gastrin 2,17 to the 78 kDa GBP was measured by phosphorimager (insets) in the presence of increasing concentrations of crotonyl CoA, a substrate of enoyl CoA hydratase, or of acetoacetyl CoA, a product of 3-hydroxyacyl CoA dehydrogenase, and expressed as a percentage of the value obtained in the absence of competitor. Lines of best fit for inhibition by
  • acetoacetyl CoA and crotonyl CoA were obtained with the program LIGAND. Values for IC 50 and for the predicted ordinate intercept were as follows: Crotonyl CoA (•), 43 ⁇ M, 76%; acetoacetyl CoA ( ⁇ ), 74 ⁇ M, 70%. No inhibition was observed with NADH ( ⁇ ), a cofactor for 3-hydroxyacyl CoA dehydrogenase.
  • Figure 3 shows that anti-proliferative gastrin/CCK receptor antagonists target the 78 kDa GBP.
  • the mean IC 50 values (Table 1) for the inhibition of cross-linking of 125 I-[Nle 15 ]-gastrin 2,17 to the 78 kDa GBP by gastrin analogues (closed symbols as in Figure 2) and antagonists (open symbols as in Figure 2) were determined as described in the legend to Figure 2, and compared with the IC 50 values for (A) inhibition of binding of 125 I-CCK 8 labelled using Bolton-Hunter reagent to the cloned human gastrin/CCK-B receptor expressed in COS cells (analogues) (8) and of 125 I-CCK 33 to the gastrin/CCK-B receptor on guinea pig gastric glands (antagonists) (9), or (B) inhibition of binding of 125 I-gastrin 17 to the gastrin/CCK-C receptor on HCT 116 colon carcinoma cells (2). Lines
  • Figure 4 illustrates processing and sequences of gastrin-related peptides.
  • the C-terminal sequence of human progastrin is shown using the one letter code.
  • the corresponding region of porcine progastrin is only shown where it differs from human progastrin.
  • the indicated peptides (numbered from the N-terminus of gastrin 1-17 ) were synthesised according to the human sequence, with the N-terminal glutamic acid of gastrin 1-4 , gastrin 1-11 , gastrin 1-17 and gastrin 1-17 G cyclized to pyroglutamate.
  • Figure 5 shows binding of gastrin-related peptides to the 78 kDa GBP.
  • Figure 6 illustrates how expression of antisense gastrin inhibits cell proliferation.
  • Figure 7 shows the inhibition of YAMC cell proliferation by gastrin/CCK receptor antagonists.
  • Proliferation of YAMC cells in the presence of increasing concentrations of the non-selective gastrin/CCK receptor antagonists proglumide ( ⁇ ) or benzotript ( ⁇ ) was measured with the MTT assay at two days after seeding.
  • Antagonists were added one day after seeding.
  • the means of triplicate absorbance readings are shown as a percentage of the mean absorbance reading obtained on day 1 for the cells grown in 1% fetal calf serum; error bars represent the standard error of the mean. Lines of best fit were
  • FIG. 8 shows the inhibition of growth of
  • Points are the mean values of 5 samples, and lines of best fit were obtained with the program LIGAND. Bars represent 1 SD.
  • FIG. 9 illustrates proliferation of LIM 1215 colon carcinoma cells in the presence of increasing
  • NSAIDS concentrations of NSAIDS, measured with the MTT assay as described for Figure 7.
  • Cells were grown in a medium containing 10% foetal calf serum, and NSAIDS were added one day after seeding. The means of triplicate absorbance readings were expressed as a percentage of the mean
  • Figure 10 shows that the inhibition of proliferation of LIM 1215 cells by NSAIDS is not correlated with the ability of the NSAIDS to inhibit either
  • cyclooxygenase I Panel A
  • cyclooxygenase II Panel B
  • the IC 50 values for inhibition of proliferation of LIM 1215 cells were measured as described for Figure 8, and are summarized in Table 4.
  • IC 50 values for inhibition of cyclooxygenase I and cyclooxygenase II were taken from
  • Figure 11 shows the ability of four different NSAIDS to block cross-linking of gastrin 2,17 to GBP in the assay described above.
  • Figure 12 shows the correlation between the ability of NSAIDS to inhibit proliferation of LIM 1215 cells and their ability to inhibit cross-linking of gastrin 2/17 to GBP.
  • GBP gastrin-binding protein
  • Na 125 I was from NEN, North Ryde, Australia.
  • the 78 kDa GBP was partially purified from detergent extracts of porcine gastric mucosal membranes (5), and crosslinked to 125 I-[Nle] 15 -gastrin 2,17 with
  • protease inhibitors were included in all buffers to prevent proteolysis: pepstatin, 1 ⁇ M; benzamidine, 1 mM; hexamethylphosphoramide, 0.1% (w/v); aprotinin, 500 units/ml.
  • pepstatin 1 ⁇ M
  • benzamidine 1 mM
  • hexamethylphosphoramide 0.1% (w/v)
  • aprotinin 500 units/ml.
  • the products of the cross-linking reaction were separated by polyacrylamide gel electrophoresis, and radioactivity associated with the 78 kDa GBP was detected and quantitated with a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
  • a phosphorimager Molecular Dynamics, Sunnyvale, CA
  • nonpolyposis colorectal cancer (20), were obtained from Dr. R.H. Whitehead, Ludwig Institute for Cancer Research, Melbourne.
  • a colorimetric assay (21) was used to measure cell proliferation. Briefly, 10 4 cells were seeded in a 96 well plate in medium containing 1% fetal calf serum. The medium was replaced the next day with fresh medium
  • LIM 2412 cells were grown in RPM1 1640 medium, and LIM 1215 cells in the same medium containing 1 mM thioglycerol, 25 units/ml insulin, 1 mg/ml hydrocortisone, 5 mg/ml bovine serum albumin, 10 ⁇ g/ml iron-saturated transferrin and 1.5 mg/ml glutamine. Both cell lines were cultured in the absence of serum.
  • Proglumide 200 mM, Sigma, St. Louis, MO was made up in 50 mM Na + HEPES, pH 7.6, readjusted to pH 7.6 with 10 M NaOH, and added at concentrations ranging from 1 to 50 mM.
  • the gastrin concentrations in conditioned media and cell extracts were determined by radioimmunoassay against an amidated gastrin 17 standard curve with 125 I-amidated gastrin 17 as label (22).
  • Levels of amidated gastrin were assayed with antiserum 1296, which detects all amidated carboxyl terminal fragments greater than the pentapeptide, and does not cross-react with glycine-extended forms.
  • Levels of unprocessed, partially processed and mature amidated forms of gastrin i.e. all progastrin-derived peptides
  • were measured with antiserum 8017 which detects the amino terminal portion of
  • Gastrin/CCK-B receptors were measured by the method of Kopin et al (24). 10 4 cells/well were seeded in a 24 well plate and grown for 2 days at 33°C in RPMI medium containing 10 mM thioglycerol, 100 units/ml insulin, 50 mg/ml hydrocortisone and 10% fetal calf serum. Cells were washed in phosphate-buffered saline, and incubated for 80 min at 37°C in Hank's balanced salt solution containing 125 I-CCK 8 (10,000 cpm, 2.9 fmol, Amersham, Bucks., UK), 16 ⁇ M PMSF and 0.1% BSA. Cells were then washed twice with PBS and lysed with NaOH. Lysates were counted in a ⁇ -counter (Packard, Downer's Grove, IL) at 77% efficiency.
  • ⁇ -counter Packard, Downer's Grove, IL
  • Gastrin/CCK-C receptors were measured by the phthalate oil centrifugation method (3) with 125 l-gastrin 17 (1.5 ⁇ 10 5 cpm, 44 fmol (Amersham, Bucks., UK)) as ligand.
  • 125 l-gastrin 17 1.5 ⁇ 10 5 cpm, 44 fmol (Amersham, Bucks., UK)
  • IC 50 values (mean ⁇ SD, calculated from at least 3 sets of data) for inhibition of cross-linking of
  • Antagonists are .
  • the non-selective antagonists proglumide and benzotript both inhibited cross-linking of 125 I-gastrin 2,17 to the GBP, as shown in Figure IB, with IC 50 values of 5.1 mM and 195 ⁇ M respectively (Table 1).
  • IC 50 values 5.1 mM and 195 ⁇ M respectively
  • gastrin and its derivatives a good correlation is observed between the IC 50 values for the 78 kDa GBP and the IC 50 values for inhibition of 125 I-gastrin 17 binding to the gastrin/CCK-C receptor, while the correlation between the IC 50 values for the 78 kDa GBP and the IC 50 values for the gastrin/CCK-B receptor is poor.
  • gastrin 17 (gastrin 1-17 G), which is transamidated by
  • the IC 50 value for glycine-extended gastrin 17 was slightly lower than the IC 50 value for gastrin 17 , as shown in Figure 5 and Table 2. Addition of a further 5 residues to the C-terminus of glycine-extended gastrin 5-17 did not alter the affinity of the resultant peptide for the GBP, while addition of 5 residues to the C-terminus of glycine-extended gastrin 12-17 actually resulted in a 25-fold
  • benzotript which we have shown in Example 1 to be the best available antagonist for the GBP, is an acylated tryptophan derivative (N-4-chlorobenzoyl-L-tryptophan), it seems likely that the tryptophan residue of either the N-terminal or C-terminal tetrapeptide, or both, makes a significant contribution to binding.
  • a cDNA fragment encoding human progastrin (18) was cut from pBR322 with Pstl and cloned into pGEM-3Z
  • pCDNA1amp (Invitrogen, San Diego, CA).
  • the corresponding antisense plasmid was constructed by cloning an XbaI/SphI fragment from pGEM-3Z into pCDNAlamp, and a XbaI/BstXI fragment thence into pCDNA1neo (Invitrogen).
  • Half-confluent YAMC cells were incubated with 20 ⁇ g/ml plasmid DNA for 4-6 hrs at 33°C in serum-free RPMI medium containing 20 ⁇ g/ml lipofectin (Gibco/BRL), 10 mM thioglycerol, 100 units/ml insulin and 50 mg/ml hydrocortisone.
  • Transfected cells were grown at 33°C in RPMI medium containing 10 mM
  • thioglycerol 100 units/ml insulin, 50 mg/ml hydrocortisone and 10% fetal calf serum for 2 days, and selected for 14 days in the same medium containing 100 ⁇ g/ml neomycin.
  • YAMC or LIM 1215 cell extracts and conditioned media were readily detected in YAMC cell extract (20 fmol/10 7 cells) and conditioned medium (66 fmol/10 6 cells) following transfection with a sense gastrin construct; however, amidated gastrin was not detected in these cells.
  • No progastrin-derived peptides were detected in YAMC or LIM 1215 cells transfected with an antisense gastrin construct.
  • COS cells were transiently transfected with a plasmid encoding sense gastrin mRNA or with plasmids encoding both sense and antisense ga ⁇ crin mRNA.
  • the levels of progastrin-derived peptides in cell extracts (760 pmol/10 7 cells) and conditioned media (1540 pmol/10 7 cells) were reduced by 37 and 48% respectively in the doubly transfected cells. Amidated gastrin was not detected in either case. Similar results were obtained in 3 separate experiments with YAMC, and two experiments with LIM 1215.
  • the total amount of 125 I-CCK 8 (10,000 cpm, 2.9 fmol) bound to gastrin/CCK-A + -B receptors on YAMC, LIM 1215 and AR4-2J cells was measured by the method of Kopin et al (24).
  • the AR4-2J cell line was derived from a rat pancreatic carcinoma, and expresses both gastrin/CCK-A + -B receptors.
  • the amount of 125 I-gastrin 17 (150,000 cpm, 44 fmol) bound to gastrin/CCK-C receptors on YAMC and LIM 1215 cells was measured by the phthalate oil centrifugation assay described by Weinstock and Baldwin (3). Background binding was measured in the presence of 1 ⁇ M (A + B) or 10 ⁇ M (C) gastrin 17 .
  • Table 3 The results are summarised in Table 3.
  • AR4-2J 10 4 10567 ⁇ 1341 1866 ⁇ 252
  • Gastrin/CCK-C receptors were readily detected on both YAMC and LIM 1215 cells using the phthalate oil centrifugation assay and 125 I-gastrin 17 as ligand.
  • benzotript 13.7 mM, 113%. Concentrations of benzotript higher than 3 mM were toxic to YAMC cells. No inhibition of proliferation was observed with the CCK-A receptor-selective antagonist L364,718 or the gastrin/CCK-B
  • LIM 2412 cells is inhibited by proglumide, with IC 50 values of 2.2 ⁇ 1.0 mM, and 2.9 ⁇ 1.3 mM respectively. No correlation was observed between the degree of inhibition by proglumide and the presence or absence of binding sites for 125 I-[Nle 15 ]-gastrin 17 determined previously. Even though gastrin 17 breakdown did not exceed 30% during a 24 hour incubation in the absence of serum and a non-saturating concentration of inhibitor was chosen to facilitate ready reversal of inhibition, no reversal of the inhibition of LIM 1215 or LIM 2412 cells by proglumide was detected with gastrin 17 concentrations as high as 50 ⁇ M. These results are shown in Figure 8.
  • NSAIDS have been shown to inhibit colorectal tumour growth in several experimental models, and have been shown to reduce the size and number of colorectal polyps in patients with familial adenomatous polyposis (26), and to reduce the death rate from cancer of the eosophagus, stomach, colon, and rectum (27). It has been widely accepted that the effect of NSAIDS on colon cancers has resulted from the ability of these agents to inhibit cyclooxygenases, which are key enzymes in the synthesis of prostaglandins, and in particular it has been proposed that the anti-tumour activity is correlated with inhibition of cyclooxygenase-2 (28).
  • cyclooxygenase I and cyclooxygenase II were measured using the colorimetric assay described above.
  • the medium was RPMI 1640 containing 10 ⁇ M thioglycerol, 25 units/ml insulin, 1 mg/ml
  • FIG. 9 shows results for indomethacin, sulindac sulphoxide and aspirin. All of these agents inhibited proliferation of LIM 1215 cells. In fact, all of the NSAIDS tested inhibited proliferation of LIM 1215 cells, with IC 50 values obtained by computer curve fitting with the programmes EBDA and LIGAND ranging from 120 ⁇ M for meclofenamic acid and sulindac sulphide to 21 mM for acetaminophen. These results are summarized in Table 4, and are also compared for some agents with IC 50 values for cyclooxygenase I and cyclooxygenase II, which have been reported in the literature.
  • Tolmetin 1900 ⁇ 170 1400 ⁇ 100
  • Figure 10 compares IC 50 values for LIM 1215 cell proliferation and for inhibition of cyclooxygenases I and II in intact bovine aortic endothelial cells and murine macrophages respectively, and shows that there is no correlation between inhibition of cell proliferation and enzyme inhibition for either enzyme.
  • the potent cyclooxygenase inhibitor indomethacin which has an IC 50 value of 30 nM for cyclooxygenase I and 1.7 ⁇ M for cyclooxygenase II, was only able to inhibit LIM 1215 cell proliferation to a moderate extent, having an IC 50 value of 200 ⁇ M.
  • acetaminophen which does not decrease the risk of gastrointestinal tract cancer (27), was even less effective in inhibiting LIM 1215 cell proliferation, having an IC 50 of 21 mM.
  • This agent is more potent than aspirin as an inhibitor of cyclooxygenase II, with an IC 50 value of 130 ⁇ M (28).
  • Our results provide further evidence that the anti-proliferative affects of NSAIDS on colon cancers are not primarily mediated by cyclooxygenase II.
  • gastrin-binding protein cross-linking assay is useful, either alone or in
  • non-selective gastrin/CCK receptor antagonists proglumide and benzotript, and the IC 50 values for inhibition of HCT 116 colon carcinoma cell proliferation by the same
  • L365,260 has any effect on cross-linking of
  • CyclooxygenaseS are involved in prostaglandin synthesis, and utilize fatty acid derivatives as
  • NSAIDS The rat mitochondrial trifunctional protein (MTP), the ⁇ -subunit of which has strong amino acid sequence homology to the porcine GBP, also utilizes fatty acid derivatives as substrates.
  • MTP mitochondrial trifunctional protein
  • NSAIDS with the exception of acetaminophen, inhibit cross-linking of iodinated gastrin to the porcine GBP, and conclude that GBP may be the target of the anti-proliferative effect of these agents on colon cancer.
  • Hereditary defects in the MTP can cause death in early childhood (29). A wide range of diagnostic symptoms has been reported, including diarrhoea, vomiting,
  • hypoketotic hypoglycaemic coma myopathy and
  • conditionally immortalised mouse colon cell line YAMC also expresses cell surface gastrin/CCK-C receptors, but not CCK-A or
  • screening may be carried out using antibody to GBP, labelled with a detectable marker, and measuring the binding of the labelled antibody to dispersed tumour cells.
  • screening may be carried out as described by Weinstock and Baldwin (3).
  • immunochemical assays of inhibition of binding of labelled antibody, or assays of inhibition of binding of labelled high-affinity antagonists provide the opportunity for automated assays which can be used for large scale screening of putative antagonists.
  • Assays of the enzyme activities intrinsic to the GBP may also be used for large-scale screening of GBP antagonists.
  • Covalent cross-linking of 125 I-gastrin 2,17 to the purified 78 kDa GBP provides a convenient assay with which to isolate more potent and selective GBP antagonists capable of blocking cell growth.
  • a colon cancer cell line (LIM 1215) derived from a patient with inherited nonpolyposis colorectal cancer

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Abstract

L'invention concerne un procédé permettant d'identifier un composé présentant l'aptitude à bloquer le récepteur de gastrine-cholécystokinine de type C, à faible affinité, comprenant l'étape consistant à mesurer l'aptitude dudit composé à bloquer la liaison à la protéine de liaison de la gastrine d'un composé sélectionné dans le groupe comprenant un peptide apparenté à la gastrine, un peptide apparenté à la cholécystokinine, un antagoniste de la gastrine ou de la cholécystokinine, un acyle CoA, un enoyle CoA, un anticorps dirigé contre la gastrine et un anticorps dirigé contre la cholécystokinine. Plusieurs façons de mettre en ÷uvre le procédé selon l'invention sont également décrites. Ainsi, l'invention offre un procédé général de criblage permettant d'identifier des antagonistes de l'interaction de la protéine de liaison de la gastrine, lesquels peuvent être utilisés pour traiter des maladies dans lesquelles sont impliquées des cellules à prolifération rapide, et pour réguler la sécrétion d'acide gastrique. Des compositions et des procédés de traitement sont également revendiqués.
PCT/US1995/001375 1994-02-02 1995-02-02 Dosage servant au criblage d'antagonistes d'un recepteur WO1995021380A1 (fr)

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AUPM3650A AUPM365094A0 (en) 1994-02-02 1994-02-02 Assay for screening of receptor antagonists
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AUPM6638A AUPM663894A0 (en) 1994-07-05 1994-07-05 Assay for screening of receptor antagonists
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AUPM7779A AUPM777994A0 (en) 1994-08-31 1994-08-31 Assay for screening of receptor antagonists

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1185303A1 (fr) * 1999-05-24 2002-03-13 George Tachas Inhibition de la production et/ou secretion de l'acide gastrique
EP1409541A2 (fr) * 2001-06-05 2004-04-21 Duke University Biocapteur monocellulaire destine a la mesure de ligands gpcr dans un echantillon pour essai
WO2006008649A1 (fr) * 2004-07-15 2006-01-26 Aphton Corporation Therapie polynucleotidique antisens contre les tumeurs favorisees par la gastrine
US11583576B2 (en) 2017-06-15 2023-02-21 Cancer Advances Inc. Compositions and methods for inducing humoral and cellular immunities against tumors and cancer

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICA ET BIOPHYSICA ACTA, Vol. 1170, issued 1993, T. MANTAMADIOTIS et al., "Nucleotide Sequence Encoding a Novel Member of the Hydratase/Dehydrogenase Family", pages 211-215. *
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY-B COMPARATIVE BIOCHEMISTRY, Vol. 104B, No. 1, issued 1993, G.S. BALDWIN, "Comparison of Sequences of the 78 kDa Gastrin-binding Protein and Some Enzymes Involved in Fatty Acid Oxidation", pages 55-61. *
EMBO JOURNAL, Vol. 8, issued 1989, D. BECKER et al., "Proliferation of Human Malignant Melanomas is Inhibited by Antisense Oligodeoxynucleotides Targeted Against Basic Fibroblast Growth Factor", pages 3685-3691. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 84, issued May 1987, F-T. MU et al., "Monoclonal Antibody to the Gastrin Receptor on Parietal Cells Recognizes a 78-kDa Protein", pages 2698-2702. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 91, issued August 1994, G.S. BALDWIN, "Antiproliferative Gastrin/cholecystokinin Receptor Antagonists Target the 78-kDa Gastrin-binding Protein", pages 7593-7597. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1185303A1 (fr) * 1999-05-24 2002-03-13 George Tachas Inhibition de la production et/ou secretion de l'acide gastrique
EP1185303A4 (fr) * 1999-05-24 2003-09-10 George Tachas Inhibition de la production et/ou secretion de l'acide gastrique
US7501400B1 (en) 1999-05-24 2009-03-10 George Tachas Inhibition of gastric acid production and/or secretion
EP1409541A2 (fr) * 2001-06-05 2004-04-21 Duke University Biocapteur monocellulaire destine a la mesure de ligands gpcr dans un echantillon pour essai
EP1409541A4 (fr) * 2001-06-05 2006-09-20 Univ Duke Biocapteur monocellulaire destine a la mesure de ligands gpcr dans un echantillon pour essai
WO2006008649A1 (fr) * 2004-07-15 2006-01-26 Aphton Corporation Therapie polynucleotidique antisens contre les tumeurs favorisees par la gastrine
US11583576B2 (en) 2017-06-15 2023-02-21 Cancer Advances Inc. Compositions and methods for inducing humoral and cellular immunities against tumors and cancer

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